Sarcoptes scabiei infection and impetigo. Scabies mites can promote Streptococcus pyogenes (Group A Streptococcus) and Staphylococcus aureus infections by breaching the skin barrier and excreting molecules that inhibit host innate immune responses. However, little is known about the composition and the function of the scabies-associated microbiota. Here, high-throughput whole-metagenome sequencing was used to explore the scabies-associated microbiome. Scabies mites including their immediate microenvironments were isolated from two patients with severe scabies in Northern Australia. Two ~45–50 million paired-end reads Illumina libraries were generated of which ~2 (5.1%) and 0.7 million (1.3%) microbial reads were filtered out by mapping to human (hg19) and mite draft genomes. Taxonomic profiling revealed a microbial community dominated by the phylum Firmicutes (A: 79% and B: 59%) and genera that comprise Streptococcus, Staphylococcus, Acinetobacter, and Corynebacterium. Assembly of the metagenome reads resulted in genome bins representing reference genomes of Acinetobacter baumannii, Streptococcus dysgalactiae (Group C/G), Proteus mirablis and Staphylococcus aureus. The contigs contained genes relevant to pathogenicity and antibiotics resistance. Confocal microscopy of a patient skin sample confirmed A. baumannii, Streptococci and S. aureus in scabies mite gut and faeces and the surrounding skin. The study provides fundamental evidence for the association of opportunistic pathogens with scabies infection.Epidemiological studies link  Sarcoptes scabiei var. hominis in humans. The parasite reproduces in the skin and causes a spectrum of diseases from common scabies, with less than a dozen mites over the entire body, to severe profuse and crusted forms, where the number of infesting mites can reach millions [Scabies is a parasitic skin infection caused by the mite millions ,2. With millions ,4. Scabimillions ,4,5. Formillions . In thesmillions . Most immillions ,7.Streptococcus pyogenes  and Staphylococcus aureus [S. aureus bacteraemia [The human skin is a complex interface between the host and the environment, forming a barrier to infectious microorganisms and playing an important role between the resident skin microbiota, and the host immune response. The microbiota of healthy skin is comprised of taxonomically and functionally diverse microorganisms, most of which are benign commensals residing in epidermal and sub epidermal layers . Skin mis aureus . Once ins aureus . Post-ins aureus , which cs aureus . The pres aureus . Worldwis aureus ,14. In 2s aureus ,16. Additeraemia , leadingteraemia ,18.S. scabiei and impetigo [S. aureus [S. aureus associated with scabies.Although previous epidemiological studies have clearly identified a link between the epidermal infestation with impetigo ,24,25,26impetigo ,28,29,30impetigo ,32,33,34S. aureus could be present. In this study, high-throughput whole metagenome sequencing was employed to explore the scabies-associated microbiome in two human patients with crusted scabies. The aim of the study was to provide a first snapshot of the microbiota associated with scabies infection in humans and of the roles these bacteria play in pathogenesis.Little is known about the composition of the mite surrounding skin microbiota during a scabies infestation and its functional capabilities. Additional pathogenic bacteria other than GAS and The collection of the human patient samples was approved by the Human Research Ethics Committee of the Northern Territory Department of Health and Menzies School of Health Research and Menzies School of Health Research . Informed consent was obtained from each patient recruited into the study.S. scabiei var. hominis) were individually picked from skin samples collected by scraping the skin of each patient and were pooled. Each skin sample contained between 800 and 1000 mites of all life stages. Pooled isolated mites with their skin microenvironments were not washed to explore both the mite internal and the mite surrounding skin microbiomes.Scabies mites were isolated from skin scrapings collected from two unrelated patients with crusted scabies (Patient A and Patient B) from different regional areas of the Northern Territory, Australia. Patient A was a 26-year-old female and Patient B was a 54-year-old male. Over 20 years, both patients had presented with recurrent crusted scabies on multiple occasions. Patient B was one of the two original patients with documented clinical and in vitro ivermectin resistance following prior multiple intermittent therapy with ivermectin . Both pag for 10 min to pellet insoluble debris, the genomic DNA was isolated on the Qiagen genomic tip as instructed in the manufacturer’s protocol. Two DNA libraries were constructed and 100-base pair paired-end reads were generated on an Illumina HiSeq 2500 instrument at the Australian Genome Research Facility of Melbourne, Victoria, Australia.DNA from the mites and their skin environment was extracted from each sample using a Blood and Cell Culture DNA Kit  and a modified procedure, adapted from the manufacturer’s protocol. This protocol is detailed in Mofiz et al. ,38. BrieRaw sequencing data was processed for removal of Illumina adaptor sequences, trimmed and quality-based filtered using TRIMMOMATIC software v.0.32  (LEADING−5 to search for sequence matches in the NCBI protein reference database (downloaded 18 March 2016) and to identify the fungal taxonomic composition and abundances [The bacterial taxonomic composition and community abundances of the samples were determined using the software KRAKEN v.0.10.5 . The proundances . The reaundances . Reads wundances . Metabatundances  and checundances . Contigsundances .ftp://ftp.ncbi.nlm.nih.gov/genomes/all/GCF) for Acinetobacter baumannii (downloaded 10/01/21), S. dysgalactiae (25/02/19), S. aureus (25/02/19) and Proteus mirablis (26/02/19). PanPhlAn profiles different reference strains of a species according to their gene content, which are used as a reference to identify the strain in a metagenomic data. Multi-locus sequence typing (MLST) analysis was done using PubMLST (https://pubmlst.org).Four of the identified bacteria species, which had a predicted assembled completeness of >92%, were selected to be defined at the strain level. The tool PanPhlAn v1.2.2.5  was applemm of S. pyogenes (gb|NP_608047) using BWA-MEM [https://www.kegg.jp/) using BLAST.Microbial contigs were searched for matches to the virulence gene sequences obtained from the Virulence Factor Database (VFDB) (downloaded 18/02/18) using BLAST . In addi BWA-MEM . Antibio BWA-MEM . The deg BWA-MEM . Genes aA. baumannii polyclonal anti-rabbit antibodies , S. pyogenes polyclonal anti-goat antibodies , and S. aureus polyclonal anti-rabbit antibodies . Goat Streptococcus Group A polyclonal antibody  was used to localise both S. pyogenes and S. dysgalactiae.The bacteria of interest were searched and localised within the scabies mites and in the surrounding skin using skin sections obtained from Patient B probed with bacteria-specific antibodies, i.e., A. baumannii ATCC19606, S. pyogenes 2031 Type strain emm1, S. dysgalactiae NS3396 emm-type STG-480, and S. aureus MSSA CC75 M5) grown in Tryptic Soy Broth (Oxoid). Using cavity well slides (Proscitech), 20 µL volumes of bacterial cultures at an OD600 0.1 were incubated on the slide at 37 °C for 24 h, with A. baumannii and S. aureus incubated aerobically, and S. pyogenes and S. dysgalactiae slides incubated in 5% CO2. After incubation, the slides were heat fixed, then further fixed in 100% methanol for 1 min. After fixation, the staining protocol was carried out as previously outlined, and controls were included by staining each bacterial strain with all three primary antibodies to ensure that there was no cross-reactivity between each strain. Using the Zeiss 780-NLO  confocal microscope no cross-reactivity was seen, and staining of the appropriate bacteria with the appropriate antibody was demonstrated  glass slides [A. baumannii antibodies, 1 µg/mL of S. pyogenes antibodies, and 8 µg/mL of S. aureus antibodies. Primary antibodies were incubated overnight at 4 °C in a humidified box. The following morning, primary antibodies were decanted, and the slides were washed in 3× changes of TBS-T for 5 min. After washing the slides exposed to A. baumannii and S. aureus antibodies were incubated with secondary Alexafluor 555-labelled donkey anti-rabbit antibodies  diluted 1:1000 (2 µg/mL) in PBS, and the slides exposed to S. pyogenes antibodies were incubated with Alexafluor 488-labelled secondary donkey anti-goat antibodies (Invitrogen) diluted to 1:1000 (2 µg/mL). Secondary antibody incubation was for 2 h at RT in a dark humidified box. The secondary antibodies were decanted and the slides washed 3× in PBS for 5 min. The slides were incubated in the dark for 10 min in 1µg/mL of DAPI stain (Sigma Aldrich). The slides were mounted in fluromount aqueous mounting media (Sigma Aldrich) and kept in the dark for the coverslip to set. The slides were imaged using a Zeiss 780-NLO confocal microscope and analysed using Image J software.To localise mite gut tissue, adjacent serial sections were probed with anti-human Immunoglobulin G (IgG), which is known to be ingested by the mite . Adjacens slides . The sli2O2 for 5 min. Slides were washed with 3 rinses of TBS for 5 min, blocked with rabbit serum (1:10 dilution in PBS) for 30 min at RT and washed with 3 rinses of TBS for 5 min. Slides were incubated with horseradish peroxidase (HRP) labelled anti-human IgG  for 1 h at RT, followed by a wash with 3 washes in TBS. Nova-RED substrate  was added to initiate the chromogenic reaction and was stopped by immersion in deionised water for 3 min at RT. The slides were counterstained with haematoxylin for 1 min, dehydrated in graded ethanol, cleared in xylene and mounted with DPX histology slide mounting medium . Slides were visualised using an Aperio XT Scanscope  slide scanner at 40× magnification and analysed using eSlide manager and ImageScope viewing software .An additional serial tissue section was either standard Hematoxylin/eosin (H&E)-stained, to visualise the mite morphology, or probed with an anti-human IgG antibody to highlight the mite digestive system . For theThe bioinformatic pipeline illustrating the steps and read counts is shown in Acinetobacter (81.2%), Corynebacterium (8.1%), Staphylococcus (4.2%) and Streptococcus (4%). In contrast, the microbiome sampled from Patient B was predominantly composed of Proteus (36.7%), Staphylococcus (28.7%), followed by Pseudomonas (9.8%) and Corynebacterium (9.6%) at the genus level. In the samples from both patients, the most abundant bacteria are known skin pathobionts that have disease-causing potential. Taxonomic profiling revealed a complex bacterial community in the samples derived from both patients. At the phyla level, Proteobacteria, Firmicutes and Actinobacteria comprised about 99% of all microbial reads, with the community being dominated by the phylum Firmicutes (Patient A: 79% and Patient B: 59%). The phylum Actinobacteria (Patient A: 9.4% and Patient B: 11.9%), which is known to be most prevalent in normal skin [Malassezia. Archaea were absent. Whole-genome shotgun metagenomics allowed the exploration of viral DNA present, which were mainly phages of S. pyogenes found in the sample from Patient B and Acinetobacter phages in the samples from both patients.Eukaryotic fungi were among the rare taxa with less than 0.01% of all classified reads. The most prevalent eukaryotic genus was A. baumannii (family Moraxellaceae) dominated the microbial community in Patient A. Using hierarchical clustering of the pan genome profile indicated that the strain in Patient A was related to A. baumannii J9 (NZ_CP041587.1). Mapping of the metagenome reads obtained from Patient A to the A. baumannii J9 genome revealed a high degree of sequence identity with only four coding genes showing sequence differences (Moraxellaceae family with a high sequence completeness (100%) and no contamination or strain heterogeneity (0%). In total, the Moraxellaceae-related metagenome-assembled genome consisted of 38 contigs with 3608 predicted genes based on metagenemark. Similarity searches using BLAST to the NCBI nucleotide database confirmed that the contigs were highly similar to A. baumannii J9 as well as A. baumannii 29FS20 (NZ_CP044519.1)  .A. baumannii sequences [MLST analysis indicated that all marker genes were present in the metagenome MAG-2 in sample A and was classified as ST-49 or ST-128 based on Pasteur or Oxford marker genes schemes, respectively. A recent publication reported a similar classification of equences .S. dysgalactiae and checkM predictions estimated about 97% completeness of the metagenome-assembled genome, which was confirmed using alignment dot plots  [S. dysgalactiae subsp. equisimilis, which typically encode 1878–2223 genes.Further metagenome binning identified a metagenome-assembled genome (MAG-3) containing 149 contigs and 2003 predicted genes. Similarity analysis of the contigs indicated a close relationship to ot plots . The MLScP, xpt) . Strain Staphylococcus. Most contig sequences within this bin matched the genomes of S. aureus subsp. aureus MW2  and S. aureus strain MSSA476 . Using SCCmecFinder, two SCCmec cassettes were identified, which were predicted as SCCmec_type_I (1B) (gb|AB033763.2) and SCCmec_type_IV (2B) (gb|AB063172.2)  was type 1 (ST-1) and spa Type t127 [Strain-level identification was carried out using PanPhlAn tool and genome coverage was assessed using alignment dot plots . Binning63172.2) . In addiP. mirabilis BB2000  and P. mirabilis strain AOUC-001 . Analysis with checkM revealed 93% completeness and 1% contamination to the class Proteobacteria.A bin of 306 contigs encoding 3456 genes in sample B was similar to A. baumannii species virulence is their capacity to form biofilm. We investigated the presence of biofilm-related genes using the VFDB and NCBI protein database. We identified several virulence factors genes, the outer membrane protein A OmpA, chaperon-usher pilus CsuE, component system BfmRS, tyrosine-protein kinase ptk, pgaB, poly-ß--N-acetyl glucosamine PNAG, extracellular exopolysaccharide EPS and partial biofilm-associated protein Bap. No contigs and reads were matching the biofilm formation genes csgA, kpsMII or fimH. We identified both integrase intA-1 and intA-2 genes. The biofilm-associated protein (Bap) was predicted on the terminal end of two contigs. Bap is a large cell surface protein, which is critical for cell–cell interaction and biofilm formation [One major factor contributing to ormation . The porormation . The Csuormation . The twoS. dysgalactiae genes lmb and gfbA  coding for adhesins, which are virulence factors related to adherence and internalisation to host cells, were present [S. dysgalactiae (WP_148848339) with an identity of 98.85%. We did not identify the complete emm (M-protein) of S. pyogenes in the skin-assembled metagenome from sample A, and only 37 reads mapped to the reference gene M-protein of S. pyogenes. Given the low read counts, no further analysis was carried out. Similarly, genes coding for streptococcal secreted factors such as Streptolysins S and O  or streptococcal exotoxins B (SpeB) were not identified with certainty in the assembled contigs and reads data.Major streptococcal virulence factors were identified which are typical and essential for these invasive pathogens . In the  present . One genStaphylococcus metagenome-assembled genome encoded various virulence factors including toxins , enzymes relevant for adherence and colonization of the bacterium , immune evasion (capsule) and iron uptake [SpA) from S. aureus was covered by the dataset from sample B. This gene encodes the bacteria surface protein SPA that binds to the Fc region of IgG which disrupts opsonisation and phagocytosis [esxA was completely recovered in the data obtained from sample B. This gene is coding for the protein EsxA, a virulence factor present in S. aureus strains recovered from lesions of psoriatic patients but absent from strains found at unaffected sites [The n uptake . Althougocytosis . SPA canocytosis ,64. The ed sites .Staphylococcus sp., tetJ (tatracycline) from Proteus sp. and ADC-3 , ADC-80 , ADC-6 , ADC-68 (carbapenem) and adeIJKLMN (multidrug classes), OXA-98 , abeS , adeFGH , ANT(3′′)-Iia (aminoglycoside antibiotic) from Acinetobacter sp.The metagenomes harboured various antibiotic resistance genes including mgrA, mecA, (methicillin-resistance), fusC (fusidic acid), tet38 (tetracycline), mepA (tetracycline), ErmC  from A. baumannii. Several antibiotic resistance proteins were identified in the metagenome-assembled genome of S. aureus using the CARD database including the antibiotic efflux pumps ArlR and MgrA, fusidic acid inactivation enzyme FusC, MecA (methicillin resistant PBP2), tetracycline resistant protein (tet38) and multidrug export protein MepA and its repressor mepR  involved in the degradation of damaged proteins. Furthermore, the sample from Patient B metagenome encoded pitrilysin, which degrades small peptides and contributes for instance to the pathogenicity of Vibrio vulnificus through the proteolytic degradation of insulin [P. mirabilis, which is an IgA-degrading metalloprotease [Using the MEROPS database as a reference, genes with proteolytic functions were identified in the above outlined microbial genomes. In the dataset obtained from the sample from Patient A, genes encoding the intramembrane protease rseP, which transmits the outer membrane environmental stress for example due to antibiotics, and S-formylglutathione hydrolase FrmB for detoxification of xenobiotic molecules in re found . In the  insulin , and mirprotease .Staphylococcus infection’ and ‘Cationic antimicrobial peptides’ , immune modulating proteins inhibiting complement activation , opsonisation  or neutrophil chemotaxis , proteins modulating immune evasion , modulating the sensitivity to cationic antimicrobial peptides (such as defensin) by increasing the positive net charge of its cytoplasmic membrane , and expression of toxins  and superantigens [Staphylococcus metagenome-assembled genome, various resistance mechanisms to CAMPs were found, comprising the capacity to sense the presence of CAMPs , to decrease the attraction or to repulse them via alteration of cell wall and membrane surface charges , to export them with membrane efflux pumps , to degrade them with secreted proteases  and finally to neutralize them by sequestration and external trapping mechanisms [Mapping of the genes and contigs revealed a good representation of three KEGG pathways, i.e., ‘Bacterial invasion’, ‘eptides’ . Genes ceptides’ . Genes fantigens  , in two human patients residing in northern Australia. By picking the parasites from unwashed and untreated skin samples, the microbial communities residing internally within the mite, and externally on the mite surface and within the skin microenvironment were captured. The microbiome analysis confirmed the presence of pathogenic bacteria of the genera A. baumannii and S. dysgalactiae subsp. equisimilis. A. baumannii is a Gram-negative bacterium that is ubiquitous in nature and a commensal of human skin, present in 20–25% of humans [A. baumannii has been identified as an important nosocomial pathogen due to its ability to form biofilms, modulate host cell signalling, induce apoptosis, develop serum resistance, and perform immune evasion [ Interestingly, genes that varied between the recently sequenced A. baumannii genome J9 and the isolate identified here are relevant in the process of attachment and invasion of human host cells. The autotransporter adhesion (Ata) protein mediates adhesion to the extracellular matrix proteins and virulence in a murine pneumonia model [A. baumannii induces host cell secretion of pro-inflammatory cytokines IL-8 during early and IL-6 during later infection stages and monocytes recruitment [A.baumannii and composed of a cytoplasmic AAA + ATPase (PilB), an integral membrane protein (PilC) and at least two pilins (PilA). A. baumannii was declared the number one priority pathogen for research by the World Health Organisation due to its pathogenicity related to a growing multidrug antibiotic resistance [From the sample of Patient A, we could assemble two bacterial genomes, classified as f humans . The bacf humans ,77,78,79 evasion . Interesia model . Experimruitment . In somesistance . A mechaS. dysgalactiae subsp. equisimilis are phylogenetically very similar to S. pyogenes. They can be skin commensals or pathogens inducing similar spectrum of illness as caused by GAS, such as wound infections, erysipelas and cellulitis. S. dysgalactiae subsp. equisimilis is also reported to be associated with life-threatening necrotizing fasciitis and streptococcal toxic shock syndrome [S. dysgalactiae subsp. Equisimilis. GAS and S. dysgalactiae subsp. Equisimilis are phylogenetically close, compete for the same resources and cause a similar disease spectrum in humans. S. dysgalatiae subsp. Equisimilis have been reported to produce antimicrobial proteins specifically active against GAS [Group C and G Streptococci such as syndrome . Notablyinst GAS  and thisS. aureus. The bacterium S.aureus is both a frequent commensal of the skin and a common human pathogen, causing skin and soft tissue infections, bacteraemia and osteomyelitis [S.aureus species are characterised by their high resistance against common antibiotics and are prominent pathogens causing persistently high mortality worldwide. The most important laboratory documented resistance is to methicillin, an anti-staphylococcal penicillin that is a surrogate used in laboratory testing for flucloxacillin resistance. Methicillin resistance in S. aureus (MRSA) is mediated by the mecA gene, which is embedded in a mobile genetic element termed staphylococcal cassette chromosome mec (SCCmec). SCCmec also includes site-specific recombinase genes  enabling the mobility of the region and is inserted within the orfX gene (RNA methyltransferase) [S. aureus strains in the sample from Patient B was identified as spa type t127, sequence Type ST-1. This strain, which is methicillin-resistant, has been frequently isolated in pigs in some countries, mostly in Europe, and is considered as the most widespread methicillin-resistant livestock-associated S. aureus [S. scabiei var. suis) to mites from humans (S. scabiei var. hominis). In a Single Nucleotide Polymorphism (SNP) analysis of mite gDNA from the same two human samples under investigation here (Patient A and B) and from four pig samples, the authors identified 16 mtDNA haplotypes in total, falling into two broad clades and six distinct haplogroups [S. aureus strain from Patient B may be zoonotically acquired, possibly from pigs. Indeed, scabies mites could have acted as a vector for transmission of this pathogenic bacterium.The data retrieved from the sample from Patient B was dominated by metagenome-assembled genomes related to myelitis ,86. S.asferase) . They no. aureus . Mofiz e. aureus  comparedlogroups . Three plogroups . This daP. mirabilis and P.aeruginosa. Both microorganisms are Gram-negative, rod-shaped bacteria widely distributed in soil and water. P. mirabilis is most frequently involved in infections of the urinary tract [P.aeruginosa can be involved in skin infections, for example after burn injuries or any cause of rupture of the skin barrier [We also found evidence of ry tract , and rar barrier .S. chromogenes) [It is generally accepted that microbiota play an important role in protecting the host from pathogens. Resident microbiota perform this protective function in a number of ways, such as colonisation resistance and modulating the host immune system. Colonisation resistance involves limiting the nutrients and space for initial colonisation of potential pathogens. In addition, the microbiota may modulate the types of immune reaction or secretion of antimicrobial peptides by the host against certain groups of microbes, thereby maintaining homeostasis. Perturbation of the resident skin microbiota  during a scabies infestation was demonstrated in vivo in a porcine experimental model . With thmogenes) . These bmogenes) . At the mogenes) ,32,33,34mogenes) ,29,30,34S. scabiei var. suis) has detected the presence of opportunistic pathogen, Klebsiella pneumonia and a probable bacterial symbiont, namely Streptomyces [In this study, we sequenced both the mite external  and the mite internal  microbiota. We therefore cannot determine here if some of the identified pathogenic bacteria were originally host skin bacteria, which were amplified due to mite infection, or if the scabies mites may even act as a vector for transmission of some of these pathogenic bacteria. We were limited to investigating samples from crusts from scabies patients and were not in the position to generate a patient-specific ‘healthy skin’ control dataset. Consequently, we are not able to establish whether the opportunistic pathogens were present as a part of the normal skin flora. Arthropods have their own, distinct microbiota, which play a role in their biology and pathogenicity. Often arthropod microbiota include symbionts, which may provide the host with specific essential nutrients, defence against external factors or which may manipulate reproduction. Disruption or removal of the internal microbiota and endosymbionts in arthropods has been reported to reduce survival, fecundity and growth of arthropods without inhibiting feeding ,94,95. Aptomyces . The finptomyces . This stA. baumannii in human head lice (Pediculus humanus capitis), present in the hair of elementary schoolchildren in Paris, France. Using molecular detection methods, the authors were able to detect A. baumannii DNA in 33% of the target population  [Pediculus humanus morphotype corporis) [Bouvresse et al. described the presence of to 2009) . Similarto 2009) ,101,102.orporis) . It willThis study is the first to describe the composition of the skin microbial community that accompanies severe scabies infection in humans. We emphasize that this exploratory pilot study provides an exemplary (not representative) picture. While limited to samples from only two patients, the data unveiled the presence of numerous pathogenic bacteria present in the mite-infested skin. This microbiota harbours complex functional capabilities that may be relevant to their invasion of the human host and to enhancing their pathogenicity. Based on these crucial fundamental findings, we are currently conducting a larger amplicon-based study including patients from four different countries with different socio-economic features and climates, to investigate the impact of scabies infection on the associated skin microbiota.


Coxiella burnetii. This bacterial infection has been frequently reported in both humans and animals, especially ruminants. Ticks are important ectoparasite and serve as reservoir hosts of Coxiella-like endosymbionts (CLEs). In this study, we have attempted to express chaperone-coding genes from CLEs of Rhipicephalus annulatus ticks collected fromcow path. The partial DnaK coding sequence has been amplified and expressed by Escherichia coli. Amino acid sequences have been analyzed by MS-MS spectrometry and the UniProt database. Despites nucleotide sequences indicating high nucleotide variation and diversity, many nucleotide substitutions are synonymous. In addition, amino acid substitutions compensate for the physicochemical properties of the original amino acids. Immune Epitope Database and Analysis Resource (IEDB-AR) was employed to indicate the antigenicity of the partial DnaK protein and predict the epitopes of B-and T-cells. Interestingly, some predicted HLA-A and B alleles of the MHC-I and HLA-DR alleles belonging to MHC-II were similar to T-cell responses to C. burnetii in Q fever patients. Therefore, the partial DnaK protein of CLE from R. annulatus could be considered a vaccine candidate and immunogenic marker with future prospects.Q fever is one of the most important zoonotic diseases caused by the obligate intracellular bacteria,  Coxiella burnetii which is a gram-negative intracellular bacterium phylogenetically classified as γ-proteobacteria related to the genera Legionella, Francisella and Rickettsiella  coupled with a 5600 TripleTOF mass spectrometer [Sciex]. Data from mass spectrometry analysis were interpreted to identify peptide sequences by using Mascot sequence matching software [Matrix Science] based on the UniProt database with p-value < 0.05.SDS gels were cut and subjected to in-gel tryptic digestion to identify amino acid sequences, as carried out by the Ward Medic company (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) [https://www.ncbi.nlm.nih.gov/Class/Structure/aa/aa_explorer.cgi) and by comparison of the biochemical and physiological effects of amino acid replacement  using the I-TASSER program (TASSER/) . Then, pTASSER/) . In the lacement . Then, ehttps://www.iedb.org/) [R. annulatus was used to predict the continuous B-cell epitopes, Bepipred linear epitope, Parker hydrophilicity, Emini surface accessibility, Chou & Fasman Beta-turn and Karplus & Schulz flexibility. Moreover, the ElliPro server based on the 3D structure of antigens was used to predict both continuous and discontinuous B-cell epitopes. In addition, T-cell epitope prediction of both MHC class I and class II was analyzed by IEDB. In the case of MHC-I, potential cytotoxic T lymphocyte epitopes were predicted from conserved peptides of antigens involved in MHC-I binding, transporter of antigenic peptide (TAP) transport efficiency, and proteasomal C terminal cleavage prediction before comparison of eluted ligand ability via natural processing through MHC-NP analysis. MHC class II was analyzed based on MHC-II binding, CD4 T cell immunogenicity and MHCII-NP prediction. MHC-I and MCH-II epitopes for binding and processing peptides were considered the half-maximal inhibitory concentration (IC50) values of the stabilized matrix method (SMM) and artificial neural network method (ANN), showing higher affinity of the epitopes at IC50 ≤ 250 nM.To identify immunogenic and vaccine properties, the partial DnaK protein was determined by the VaxiJen v2.0 server and Immune Epitope Database and Analysis Resource (IEDB-AR) (db.org/) . Linear R. annulatus ticks. In particular, molecular identification revealed all nucleotide sequences of R. annulatus that hit our query sequence (MW541856). Multiple sequence alignment revealed that the similarity between our sequence of R. annulatus and other sequences of R. annulatus and R. microplus from NCBI database was 99.8 to 100% and 98.8 to 99%, respectively  and 35 partial dnaK nucleotide sequences of Coxiella sp. were obtained from the NCBI database. All 774-base-pair lengths of dnaK sequences were aligned and calculated for evolutionary divergence, which was depicted through a phylogram of partial dnaK nucleotide sequences with appropriate distance values among C. burnetii and CLEs , C. burnetii (strain CbuK_Q154), C. burnetii (strain Dugway 5J108-111), C. burnetii (strain RSA 331/Henzerling II) and C. burnetii (strain RSA 493/Nine Mile phase I)  .C. burnetii and CLEs showed values ranging from 0.00 to 0.06, which were lower than those of nucleotide sequences  model with the maximum likelihood statistical method. The computation of the maximum log likelihood was -905.431, showing the occurrence of amino acid substitutions in partial DnaK amino acid sequences. The amino acid composition of  of CLEs . Each poC. burnetii Nine Mile RSA493 and CLE from R. annulatus were selected, and secondary structure was predicted based on the threading method of the I-TASSER server. The most accurate models of both C. burnetii NM RSA493 and CLE from R. annulatus were selected at C-scores of -0.84 and -0.87, respectively. The structure of the partial DnaK protein of C. burnetii and CLE displayed a few different lengths of amino acid bonding and the positions of different amino acids as shown in R. annulatus tick presented yellow spots in the same area as C. burnetii but still showed a few different areas separate from the green spot of C. burnetii  and 131, 163 and 172 (I→V) and the values of 0.30, 0.15, 0.04, and 0.13 at positions 92 (N→G), 98 (V→E), 161 (R→D) and 205 (E→N), respectively  transport efficiency and proteasomal C terminal cleavage prediction) were analyzed together with natural processing of eluted ligand derived from cleavage antigen by MHC-NP analysis. Our findings showed that 5 predicted antigen ligands indicated high affinity for processing and binding to MHC-I epitopes and presentation at infected cell surfaces . In addiolecules .50) values of the stabilized matrix method (SMM) and artificial neural network method (ANN). An IC50 value ≤ 250 nM determined the high affinity of interaction between conserved peptides of partial DnaK proteins and alleles of MHC class I and II epitopes  formed the same clade with the two dnaK sequences of R. annulatus CLEs from Israel (GenBank KY678145) and West Africa (GenBank KY678144) in phylogram [C. burnetii because they were derived from different tick species. Our finding was similar to the result described by Duron et al. [IS1111 gene showed high genetic diversity in C. burnetii and CLE, and different tick species also harbored different IS1111 haplotypes [C. burnetii. Coxiella burnetii showed 3 haplotypes and frequently harbored haplotype number 2 of the partial dnaK-encoding gene, which also indicated the low occurrence of mutations in this gene. In contrast, high mutational occurrences were found in CLEs with 7 haplotypes, while the CLE of R. annulatus showed haplotypes indicating the branch nearest to the CLE of R. microplus. Both tick species were collected from cattle, which displayed less nucleotide diversity and polymorphism than tick species collected from other vertebrate hosts. Moreover, a partial sequence of the dnaK-encoding gene exhibited nucleotide variation from entropy analysis and showed greater evolutionary distances, but no sequences had free gaps derived from either deletion or insertion inside these genes. Hence, dnaK-encoding genes may play a crucial role in biological and pathogenic functions such as protein folding. Similarly, Jasinskas et al. [C. burnetii and CLE showed the same gene order: smpB, fur, grpE, dnaK, dnaJ and carA. Furthermore, cDNA sequences of the dnaK-encoding gene, FusA (elongation factor G), RpsF , RpsG , and 16S rRNA gene of CLE from A. americanum closely matched those of C. burnetii [Similarity analysis of partial  genomes . Base su genomes . Howeveremselves , but CLEhylogram . Furthern et al. , who preplotypes . In thiss et al.  reportedburnetii .dnaK-encoding genes was commonly synonymous substitution. The Ka/Ks ratio derived from the frequency of non-synonymous and synonymous substitutions was calculated and determined to be less than 1 (= 0.0399). Similarly, Tsementzi et al. [R. sanguineus s.l., R. turanicus and Candidatus C. mudrowiae, which showed Ka/Ks < 1 for several genes encoding molecular chaperones such as DnaK, DnaJ, HtpG and GrpE. The identification of low ratio values of Ka/Ks indicated strong purifying selection (negative selection), which is typically formed by natural selection and is likely to play potentially functional roles in C. burnetii and CLEs. In addition, both radical and conservative non-synonymous substitutions were observed, and there were fewer radical non-synonymous substitutions than conservative non-synonymous substitutions determining the purifying selection of these genes. However, partial dnaK-encoding genes of C. burnetii and CLE of R. annulatus indicated the same number of radical and conservative non-synonymous substitutions due to the same amount of selective pressure [In this study, it was found that base substitution of partial i et al.  reportedpressure .R. annulatus were selected for amplification and expression, but the limitation of DNA sequences from partial dnaK-encoding genes from the NCBI database was the main obstacle to obtaining complete genes. Moreover, the perplexing nature and plasticity of CLE genomes influenced the amplification and expression of several gene candidates, and some gene amplicons were unfortunately not amplified and possibly indicated to be junk genes. However, the dnaK-encoding gene was selected as a protein candidate based on the main functional role of multiple biological processes involving protein folding, protein degradation, and cell survival under stress conditions and served as a prokaryotic ortholog of Hsp70 [C. burnetii in dairy cattle, and Coxiella sp. was also detected in R. microplus ticks and was later commonly found to be CLEs [C. burnetii according to enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay (IFA) and real-time PCR analyses targeting the IS1111 gene [R. annulatus ticks collected from cow path in Chiang Mai Province were selected for consideration as immunogenic markers and vaccine candidates. Interestingly, Duron et al. [C. burnetii, and they recently reported C. burnetii in ticks including Dermacentor steini, H. hystricis, R. microplus, R. sanguineus s.l., Amblyomma varigatum, R. annulatus, Hyalomma impeltatum, Rhipicephalus evertsi, Ixodes ricinus and Haemaphysalis punctata from Nigeria, Malaysia, the Philippines and Slovenia [Neglected proteins from CLE of of Hsp70 . In addi be CLEs , 10, 15.111 gene –11, 41. 111 gene . Therefo111 gene –15. For n et al.  reportedSlovenia –44.C. burnetii. Thus, the denatured form of the partial DnaK protein of CLE of R. annulatus may be used to induce immunized antibodies and avoid hazardous processes to achieve direct exposure to causative agents such as C. burnetii in the future. However, proteomic analysis and microarray approaches used to investigate C. burnetii indicated that the DnaK protein was extracellular and secreted into the cytoplasm, and it mediated the post-translational modification of multiple proteins that tended to be immunogenic proteins [MS-MS analysis identified amino acid sequences that were hit for the DnaK and HSP70 proteins of both CLEs and proteins . Moreoveproteins  reportedproteins . Therefoproteins .R. annulatus CLE from Thailand (MW288022) also formed the same clade with CLEs from Israel (GenBank KY678145) and West Africa (GenBank KY678144) [C. burnetii and CLE of R. annulatus showed a few different of amino acid interactions in the 3D structure, which were possibly affected by amino acid substitutions, they also showed neutral effects based on protein functional assessment [C. burnetii and CLE of R. annulatus had pI values similar to those of the complete DnaK protein of C. burnetii with a value of 5.14, which could survive in the acidic environment of parasitophorous vacuoles (PVs) [C. burnetii RSA 493/331 contained a region similar to that of the partial DnaK protein of CLE from R. annulatus. Even the partial DnaK protein of C. burnetii had a larger size (45.42 kDa) than the partial DnaK protein of CLE from R. annulatus (27 kDa), and a similar region in the partial DnaK protein was recognized by acute Q fever-infected patient and guinea pig sera but was not recognized by mouse sera [C. burnetii was considered a seroreactive antigen for Q fever diagnosis, and the partial DnaK protein of CLE from R. annulatus was also recommended as an immunogen.Given the non-synonymous substitutions of nucleotides, amino acid substitutions were investigated and showed less biased amino acid usage in the amino acid composition. Consistently, multiple sequence alignment comparison of amino acid sequences determined a few variations of amino acid position among 10 CLE sequences. While, amino acid sequence of DnaK of Y678144) . Each posessment . In addies (PVs) . InteresR. annulatus showed high antigenicity along conserved sequences that could interact with the recognition region of both continuous and discontinuous B-cell epitopes. However, attractive vaccine candidates are necessary to activate the cell-mediated immune responses of both B- and T-cells [R. annulatus while interacting with the alleles of MHC-I and MHC-II epitopes was determination of the half-maximal inhibitory concentration (IC50 ≤ 250) values by using the stabilized matrix method (SMM) and artificial neural network method (ANN). Our findings showed that some of the HLA-A and B supertype alleles from MHC-I and the HLA-DR supertype alleles of MHC-II were also present during the T-cell response in chronic Q fever patients. We also assessed the T-cell response to the C. burnetii vaccine, including HLA-A*02:06, B*35:01, and B*44:03 from the MHC-I allele and HLA-DRB1*01:01, DRB1*03:01, DRB1*04:01, DRB1*07:01, and DRB1*08:02 from the MHC-II allele. Additionally, HLA-DR3 of the MHC-II epitope was reported to recall long-lived memory to C. burnetii exposure through the release of IFNγ [R. annulatus. Computational analysis showed that the partial DnaK protein of CLE from R. annulatus or other expressed proteins from CLEs may be vaccine candidates or immunogenic markers with future prospects. Even though CLE genomes still require clarity, and it is difficult to amplify and express several genes from CLEs, investigation of neglected proteins from CLEs may be an alternative way to study and provide clues to understand CLE and C. burnetii evolution in the future. Additionally, the consideration of other immunogenic markers is still important because C. burnetii infection inactivated phase I and attenuated phase II; for example, commercial Q-VAX® is a cause of unwanted side effects in some people. Therefore, recombinant proteins from C. burnetii and even CLEs could be new vaccine candidates instead of direct exposure to virulent C. burnetii.In the present study, the IEDB-AR database was used to predict the antigenicity of the protein. The partial DnaK protein of CLE from  T-cells . Both MHration IC ≤ 250 va of IFNγ , which wR. annulatus exhibited similarity of both bioinformatics and MS-MS analysis to C. burnetii. Even though they have dissimilarity of nucleotide sequences, they indicated a high number of synonymous substitutions and indicated purifying selection of these genes. Interestingly, partial DnaK protein of CLE from R. annulatus could be considered an immunogen to immunize humoral immunity as analyzed by computational approach, which may further develop to be vaccine candidate and serodiagnostic markers in the further prospects.In conclusion, partial DnaK amino acid sequences of CLE from S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.S7 Fig(TIF)Click here for additional data file.S8 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S1 Raw image(TIF)Click here for additional data file.S2 Raw image(TIF)Click here for additional data file.


Rickettsia felis genome revealed an unexpected plasmid carrying several genes usually associated with DNA transfer, suggesting that ancestral rickettsiae might have been endowed with a conjugation apparatus. Here we present the genome sequence of Rickettsia bellii, the earliest diverging species of known rickettsiae. The 1,552,076 base pair–long chromosome does not exhibit the colinearity observed between other rickettsia genomes, and encodes a complete set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25, an obligate symbiont of amoebae. The genome exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. We sought and observed sex pili-like cell surface appendages for R. bellii. We also found that R. bellii very efficiently multiplies in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic “melting pot” where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to the intracellular lifestyle within eukaryotic cells.The recently sequenced  Rickettsia bellii is the earliest diverging species of Rickettsia, a group of obligate intracellular bacteria causing human diseases such as epidemic typhus. Sequencing and computer analysis of its genome now provides evidence that many genes were transferred between the ancestors of Rickettsia and other microbes living inside amoebae, including the human pathogen Legionella pneumophila and the chlamydia-related symbiont Protochlamydia amoebophila. Remarkably, R. bellii exhibits a complete set of predicted genes for conjugative DNA transfer. Microscopic analyses confirmed this prediction by revealing bacterial mating through sex pili-like surface appendages. Furthermore, the authors demonstrate that R. bellii can survive in an amoeba for a long period of time. Taken together, these results suggest that amoeba-like ancestral protozoa acted as a “breeding ground” for different bacteria, promoting acquisition of the gene repertoire required to infect the cells of higher eukaryotes.Obligate intracellular pathogens, by their isolated way of life, are thought to have little opportunity to exchange DNA with other microbes, making their genome relatively immune to horizontal gene transfer.  R. prowazekii (the agent of epidemic typhus) and the flea-borne R. typhi (the agent of endemic typhus). The spotted fever group (SFG) includes tick-borne R. rickettsii (the agent of Rocky Mountain spotted fever), R. conorii (the agent of Mediterranean spotted fever), R. africae (the agent of African tick bite fever), R. sibirica (the agent of North Asian tick-borne fever), mite-borne R. akari , and flea-borne R. felis (the agent of flea-borne spotted fever). R. bellii is the only known species representing a third group that diverged prior to the separation of the other two groups  with a G + C content of 31.7% , 34 tRNA genes, one set of genes for rRNA, and three other structural RNA genes (R. felis exhibits a comparable level of repeat content (4.3%) . The. TheR. bR. bellii . For TraDF and TraHF, the grouping is supported by high bootstrap values . For the remaining seven ORFs, no significantly supported clade containing R. bellii was obtained, except that the tree for TrbCF indicates an evolutionary relationship between R. bellii TrbCF and its homologues found in Serratia marcescens and Salmonella typhi plasmids . We also generated phylogenetic trees for the two ORFs encoded in the Ti-like region. The tree for the TraATi supports a phylogenetic affinity of the R. bellii ORF, with homologues found in Legionella pneumophila Paris and Vibrio vulnificus, while the phylogeny for TraDTi is not informative regarding the position of the R. bellii ORF.We conducted phylogenetic analyses for every ORF of the F-like region  except TR. bellii and P. amoebophila suggested for several ORFs of the F-like region, we concatenated the alignments of the six longest ORFs  and conducted phylogenetic reconstructions. The maximum likelihood tree supports a clustering of R. bellii and P. amoebophila sequences with a 100% bootstrap value , suggesting a common origin for the chromosome-encoded tra gene cluster of R. bellii and the plasmid-encoded tra cluster of R. felis. The grouping of the rickettsial homologues and P. amoebophila homologues was also supported by a high bootstrap value (95%). We applied three additional tree inference approaches  for the two concatenated alignments. We obtained significant bootstrap supports for the sister grouping of Rickettsia and P. amoebophila by all the approaches for the two datasets, except for the TraDF/TraGF tree obtained by the maximum parsimony method .To further examine the phylogenetic affinity between ap value . We also plasmid . In thisy method . OverallR. bellii ORFs exhibit a high level of sequence similarity to homologues found in two intracellular bacteria of amoebae: Legionella pneumophila, a facultative intracellular bacterium of amoebae, and P. amoebophila. BLAST homology searches  against the SWISS-PROT/TrEMBL database (excluding sequences of Rickettsia spp.) detected 46 and 17 R. bellii ORFs best matching to the sequences of L. pneumophila and P. amoebophila, respectively.We noticed that many Legionella-like or Protochlamydia-like sequences in R. bellii, we performed BLAST searches  against the database now excluding all the sequences of Alphaproteobacteria, and counted the number of best hits against Legionellaceae or Parachlamydiaceae. Among 1,065 ORFs with database hits , 94 ORFs (8.8%) exhibited their highest scores to Legionellaceae (72 ORFs) or Parachlamydiaceae (22 ORFs). We performed the same analyses for other genomes of Alphaproteobacteria , and four Alphaproteobacteria outside Rickettsiales: Pelagibacter ubique , Brucella melitensis , Caulobacter crescentus , and Mesorhizobium loti . The latter four species were sampled to cover the wide range of ecological niches and lifestyles found among Alphaproteobacteria. The result (Legionella-like or Protochlamydia-like sequences of R. bellii (8.8%) is significantly higher than those for the four Alphaproteobacteria outside Rickettsiales . For the eight Rickettsiales genomes, the corresponding proportions of Legionellaceae or Parachlamydiaceae best hits are from 3.1%  to 8.2% (R. felis), which are comparable to or slightly lower than that for R. bellii.To assess the significance of the enrichment of bacteria . We usede result  shows thR. bellii ORFs similar to homologues of the two groups of intra-amoeba bacteria could be at least partially due to horizontal gene transfers (HGTs) between the ancestor of R. bellii and these amoebal parasites. In order to identify potential candidate genes for such HGTs, we prepared a database by removing sequences of Rickettsiales from the SWISS-PROT/TrEMBL database and performed bidirectional BLAST searches . We first carried out BLAST searches for R. bellii ORFs against this database , and extracted the sequences of Legionellaceae that got best matches from R. bellii ORFs. Next, each of the Legionellaceae sequences was searched against the same database plus all the R. bellii ORFs. When the Legionellaceae sequence exhibited its best BLAST score (omitting hits against Legionellaceae) to the R. bellii ORF that detected the Legionellaceae sequence in the first BLAST search, we recorded such a R. bellii–Legionellaceae ORF pair. With this approach we identified 15 candidates for HGT between R. bellii and Legionellaceae. We performed the same type of analysis for the R. bellii and Parachlamydiaceae pair, and found seven candidates for HGT between these bacteria. The list of these 22 HGT-candidate ORFs is given in Rickettsiales genomes (R. felis (17 ORFs) and Wolbachia pipientis wMel (eight ORFs), and less than six for other Rickettsiales. We conducted phylogenetic analyses for all 22 R. bellii HGT candidates. We found nine cases of well-supported clustering  of rickettsial sequences exclusively with the sequences of Legionellaceae (six cases) or Parachlamydiaceae (three cases) (R. bellii), which are homologous to the L. pneumophila RalF protein. RalF is secreted into host cytosol, and used to recruit ADP-ribosylation factor to Legionella-containing phagosomes in order to establish a replicative organelle  curve.(1.1 MB PPT)Click here for additional data file.Figure S2R. bellii transposases were searched in other bacteria by screening the Genpept database using BLAST. Multiple alignments were carried out using the MUSCLE program. (A), (B), (C), and (D) correspond to families 1, 2, 3, and 4 in the text. Some rickettsial fragmented transposases were not included in the trees.The phylogenetic trees were generated using the neighbor-joining method and the JTT substitution matrix. Proteins homologous to the (80 KB PPT)Click here for additional data file.Figure S3F, (B) TraCF, (C) TraNF, (D) TraDF, (E) TraBF, (F) TraHF, (G) TraUF, (H) TraFF, (I) TraWF, (J) TraEF, (K) TrbCF, (L) TraATi, (M) TraDTi. The tree was generated using the neighbor-joining method with the JTT model and midpoint rooting.(A) TraG(149 KB PPT)Click here for additional data file.Figure S4F, TraCF, TraDF, TraGF, TraHF, and TraNF. Plasmid-encoded sequences are indicated by asterisks. Other sequences are encoded in chromosomes.The tree was built using a maximum likelihood method with JTT model and midpoint rooting from the concatenated sequence alignment of TraB(34 KB PPT)Click here for additional data file.Figure S5(A) RBE_0090, (B) RBE_0387, (C) RBE_0461, (D) RBE_0462, (E) RBE_0486, (F) RBE_0692, (G) RBE_0860, (H) RBE_0868, (I) RBE_1344. The trees were generated using the neighbor-joining method with the JTT model and midpoint rooting.(153 KB PPT)Click here for additional data file.Figure S6R. bellii showing actin  and bacteria . Nucleic acids were stained with DAPI (blue). White arrowheads point to actin tails.Vero cells infected for 6 h with (1.6 MB PPT)Click here for additional data file.Figure S7R. bellii and gently permeabilized with Triton before incubation with a rabbit polyclonal anti–R. bellii Ab  revealed by an Alexa 546–coupled anti-rabbit (1:300) as secondary Ab. Magnification 100×.Bacteria were detected within Vero cells infected for 5 d with (896 KB PPT)Click here for additional data file.Figure S8L. pneumophila are stained red .Amoeba nucleotides (mostly in the nucleus) are stained blue by DAPI, and (1.3 MB PPT)Click here for additional data file.Figure S9R. bellii  and R. conorii (large eschar) suspensions. The same dilutions of R. montanensis induced a slight inflammation only with the highest dose. This picture was taken 6 d after inoculation. The numbers of inoculated bacteria are indicated. (B) Immunohistochemistry of the escarotic lesion using anti–R. bellii antibodies.(A) Inoculation eschars on a guinea pig injected intradermally with increasing dilutions of (8.0 MB PDF)Click here for additional data file.Figure S10R. bellii  and specific mouse polyclonal antibodies against L. pneumophila  shows colocalization within a vacuole of A. polyphaga. Amoeba nucleotides (mostly in the nucleus) are stained blue by DAPI.Immunofluorescence detection using specific rabbit polyclonal antibodies against (1.8 MB PPT)Click here for additional data file.Table S1(39 KB DOC)Click here for additional data file.Table S2(22 KB DOC)Click here for additional data file.Table S3(45 KB DOC)Click here for additional data file.Table S4(23 KB DOC)Click here for additional data file.Table S5(25 KB DOC)Click here for additional data file.Table S6(25 KB DOC)Click here for additional data file.Table S7(27 KB DOC)Click here for additional data file.Table S8(26 KB DOC)Click here for additional data file.http://www.ncbi.nlm.nih.gov/Genbank) accession number for the R. bellii genome is CP000087.The GenBank (


Stenotrophomonas maltophilia.The aim of this study was to determine incidence, risk factors, and impact on outcome of intensive care unit (ICU)-acquired S. maltophilia who developed an ICU-acquired NF-GNB other than S. maltophilia were also excluded. Screening  for NF-GNB was performed in all patients at ICU admission and weekly. Univariate and multivariate analyses were performed to determine risk factors for ICU-acquired S. maltophilia and for ICU mortality.This prospective observational case-control study, which was a part of a cohort study, was conducted in a 30-bed ICU during a three year period. All immunocompetent patients hospitalised >48 hours were eligible. Patients with non-fermenting Gram-negative bacilli (NF-GNB) at ICU admission were excluded. Patients without ICU-acquired S. maltophilia ICU-acquired colonisation and/or infection and were all successfully matched with 76 controls. Chronic obstructive pulmonary disease (COPD) and duration of antibiotic treatment ] = 9.4 [3 to 29], p < 0.001, and 1.4 [1 to 2.3], p = 0.001, respectively) were independently associated with ICU-acquired S. maltophilia. Mortality rate , duration of mechanical ventilation , and duration of ICU stay  were significantly higher in cases than in controls. In addition, ICU-acquired infection related to S. maltophilia was independently associated with ICU mortality .Thirty-eight (2%) patients developed an S. maltophilia. ICU-acquired S. maltophilia is associated with increased morbidity and mortality rates. ICU-acquired infection related to S. maltophilia is an independent risk factor for ICU mortality.COPD and duration of antibiotic treatment are independent risk factors for ICU-acquired  Pseudomonas aeruginosa and Acinetobacter baumannii colonisation and infection. However, few studies have investigated patients with colonisation and/or infection related to Stenotrophomonas maltophilia.Non-fermenting Gram-negative bacilli (NF-GNB) colonisation and infection are frequent among critically ill patients -3. AntibS. maltophilia have been increasing since the early 1970s, according to reports from several centres  was calculated for all significant (p < 0.05) qualitative variables in univariate analysis and for all significant variables in multivariate analysis.SPSS software  was used for data analysis. Proportions were compared using the χS. maltophilia occurrence (controls ≥ cases), (b) Simplified Acute Physiology Score (SAPS) II at ICU admission ± 5 points, (c) age ± 5 years, (d) admission category , and (e) date of ICU admission when more than two control patients were well matched to a case. The controls chosen were the ones with the closest date of admission to that of case patients.Every case patient was matched to two control patients according to all the following criteria: (a) duration of ICU stay before ICU-acquired S. maltophilia. The following variables were included in univariate analysis: age, gender, SAPS II and organ failures on ICU admission, transfer from other wards, admission category , diabetes mellitus, chronic obstructive pulmonary disease (COPD) [S. maltophilia, only exposure to potential risk factors before S. maltophilia acquisition was taken into account.Univariate and multivariate analyses were used to determine variables associated with ICU-acquired e (COPD) , prior aS. maltophilia, cases with only colonisation and their controls were excluded from risk factor analysis. Cases with an ICU-acquired infection and their controls were compared using univariate and multivariate analyses. All the above-cited variables were included in these analyses.To determine risk factors for ICU-acquired infection related to S. maltophilia and ICU-acquired infection related to S. maltophilia were included in risk factor analyses of ICU mortality. Variables with p < 0.2 by univariate analysis were included in stepwise logistic regression models. Prior antibiotic use as well as antibiotic treatment in the ICU and its duration were compared between patients with COPD and patients without COPD.Univariate and multivariate analyses were performed to determine risk factors for ICU mortality among cases and controls. All the above-cited variables were included in univariate analysis. In addition, ICU-acquired S. maltophilia were excluded for ICU-acquired colonisation and/or infection related to NF-GNB other than S. maltophilia. Thirty-eight (2%) patients developed an ICU-acquired colonisation and/or infection related to S. maltophilia, representing two patients with ICU-acquired S. maltophilia per 1,000 ICU days. These patients were all successfully matched with 76 control patients  patients had prior colonisation related to S. maltophilia.One thousand eight hundred and eighty-five patients were eligible, 25 1%) patients were excluded for NF-GNB colonisation and/or infection at ICU admission, and 20 (1%) patients for absence of NF-GNB screening at ICU admission and more than 48 hours after ICU admission. Among the 1,840 remaining patients, 324 (17%) patients developed an ICU-acquired NF-GNB. Two hundred and eighty-six (15%) patients without ICU-acquired % patientS. maltophilia was 14 ± 11 days. Thirty of 38 (78%) patients developed 36 ICU-acquired infections related to S. maltophilia, including 22 VAP, eight ventilator-associated tracheobronchitis, four urinary infections, and two bacteremias. Patient characteristics are presented in Table The mean time between ICU admission and first ICU-acquired S. maltophilia and for ICU-acquired infection related to S. maltophilia which were determined by univariate analysis are presented in Tables p < 0.001) and duration of antibiotic treatment  were independently associated with ICU-acquired S. maltophilia. Antecedent COPD and duration of antibiotic treatment were also independently associated with ICU-acquired infection related to S. maltophilia .Risk factors for ICU-acquired p = 0.101) and antibiotic use during ICU stay  were similar in patients with COPD as compared with patients without COPD, respectively. Duration of antibiotic treatment during ICU stay was similar in patients with COPD and patients without COPD .Rates of prior antibiotic use , duration of mechanical ventilation  and duration of ICU stay  were similar in the two groups.Although mortality rate was significantly higher in cases with S. maltophilia ICU-acquired infection, mortality rate was significantly higher in patients who received inappropriate initial antibiotic treatment than in patients who received appropriate initial antibiotic treatment .In cases with p = 0.001), S. maltophilia ICU-acquired infection , and respiratory failure  as independent risk factors for ICU mortality.Risk factors for ICU mortality which wer determined by univariate analysis are presented in Tables S. maltophilia colonisation and/or infection is not common in this population of immunocompetent ICU patients. COPD and duration of antibiotic treatment are independently associated with ICU-acquired S. maltophilia. ICU-acquired S. maltophilia is associated with high ICU mortality and morbidity rates. In addition, ICU-acquired infection related to S. maltophilia is an independent risk factor for ICU mortality.Our results suggest that S. maltophilia represented 0.6% to 0.9% of all isolates collected during a three year period [S. maltophilia site of infection in all geographic regions. Incidence of ICU-acquired S. maltophilia found by our study (2%) is higher than previously reported. This is probably related to the fact that screening of patients with S. maltophilia was not performed in previous studies. Our study differs from previous studies in its methodology. In previous studies, patients with infection related to S. maltophilia were compared with patients without S. maltophilia infection. However, in our study immunocompetent patients with ICU-acquired S. maltophilia were compared with immunocompetent patients without NF-GNB colonisation and/or infection. Risk factors for S. maltophilia and other NF-GNB are probably similar. Therefore, exclusion of patients with colonisation and/or infection related to NF-GNB other than S. maltophilia probably allowed a more accurate evaluation of risk factors for ICU-acquired S. maltophilia.In the SENTRY Antimicrobial Surveillance Program, r period . In thatS. maltophilia. Previous studies identified COPD as a risk factor for VAP and for respiratory tract colonisation by GNB [P. aeruginosa was isolated more frequently in patients with low FEV1 (forced expiratory volume in one second). Unfortunately, information on severity of COPD was not available in our study. Another potential explanation for the association between COPD and ICU-acquired S. maltophilia is the frequent antibiotic use in patients with COPD. Patients with severe COPD are prone to frequent acute exacerbations requiring antimicrobial therapy [COPD was identified as an independent risk factor for ICU-acquired n by GNB ,21. Factn by GNB . A multin by GNB . P. aeru therapy . In our S. maltophilia. Two recent randomised trials demonstrated that shorter duration of antibiotic treatment is effective and safe in ICU patients with VAP [Prolonged duration of antibiotic treatment is a well-known risk factor for emergence of MDR bacteria ,26. Shorwith VAP . In addiwith VAP . Based oS. maltophilia, in patients with cystic fibrosis [S. maltophilia acquisition. However, few patients with cystic fibrosis were included in this study. Marchac et al. [S. maltophilia, in respiratory tract of patients with cystic fibrosis, increased from 3.3% to 15% during the last decade. Antibiotic treatment, isolation of Aspergillus fumigatus in sputum, and oral corticosteroid use were significantly associated with S. maltophilia [S. maltophilia acquisition in patients with cystic fibrosis, suggesting that S. maltophilia is rather a marker of worse lung function in these patients [Recent reports documented an increasing incidence of NF-GNB, including fibrosis ,30. In oc et al.  reportedtophilia . Anotherpatients .S. maltophilia. However, previous studies identified carbapenem use as an independent risk factor for S. maltophilia acquisition [S. maltophilia acquisition, whereas agents other than carbapenems were identified as risk factors for the acquisition of S. maltophilia [S. maltophilia acquisition [Carbapenem use was not significantly associated with ICU-acquired uisition ,34. Thestophilia ,36. Thesuisition ,37. Althuisition .S. maltophilia were similar, outcomes of patients with colonisation or infection related to S. maltophilia were clearly different. A recent meta-analysis demonstrated that risk factors for colonisation and infection related to MDR bacteria were similar [Although risk factors for colonisation and infection related to  similar .S. maltophilia was associated with significantly increased ICU mortality and longer duration of mechanical ventilation and ICU stay. However, S. maltophilia was previously considered to have limited pathogenicity. In a recent retrospective study, S. matophilia was isolated in the respiratory tract specimens of 64 patients without pneumonia [S. maltophilia VAP were compared with patients with late-onset VAP related to other GNB [S. maltophilia VAP and patients with late-onset VAP related to other GNB, S. maltophilia was associated with increased patient morbidity. Recent studies indicated that infection with this organism was associated with significant morbidity and mortality, particularly in severely compromised patients [S. maltophilia, has evaluated risk factors for mortality [S. maltophilia has an indirect impact on mortality. In contrast, our results suggest a direct impact of S. maltophilia infection on ICU mortality given that this infection was found to be an independent risk factor for ICU mortality. In cases with ICU-acquired infection related to S. maltophilia, inappropriate initial antibiotic treatment was associated with increased ICU mortality rate. Inappropriate initial antibiotic treatment is a well-known risk factor for mortality in patients with infection related to NF-GNB [In our study, neumonia . No signther GNB . Althougpatients ,13,15. Aortality . McCabe S. maltophilia isolates. Therefore, the impact of patient-to-patient transmission could not be determined. Previous studies using molecular typing in newborn and pediatric ICUs have demonstrated that cross-transmission of S. maltophilia could be identified using molecular typing [S. maltophilia outbreak occurred during the study period. Finally, the number of patients with ICU-acquired S. maltophilia was small. Therefore, some of the trends observed in this study could have reached statistical significance if the study sample had been larger.Our study has several limitations. First, it was performed in a single ICU. Therefore, our results may not be applicable to other ICU patients. Similarly, only immunocompetent patients were included. This precludes application of our results to patients with immunosuppression. Second, during the study period, no molecular typing was performed on r typing ,42. HoweS. maltophilia colonisation and/or infection is not frequent in this cohort of immunocompetent ICU patients. COPD and duration of antibiotic treatment are independently associated with ICU-acquired S. maltophilia. ICU-acquired S. maltophilia is associated with high mortality and morbidity rates. In addition, ICU-acquired infection related to S. maltophilia is an independent risk factor for ICU mortality.S. maltophilia is not common among this cohort of immunocompetent ICU patients.• ICU-acquired S. maltophilia and with ICU-acquired infection related to S. maltophilia.• COPD and duration of antibiotic treatment are independently associated with ICU-acquired S. maltophilia is associated with high mortality and morbidity rates. In addition, ICU-acquired infection related to S. maltophilia is an independent risk factor for ICU mortality.• ICU-acquired CI = confidence interval; COPD = chronic obstructive pulmonary disease; ICU = intensive care unit; MDR = multi-drug-resistant; NF-GNB = non-fermenting Gram-negative bacilli; OR = odds ratio; SAPS = Simplified Acute Physiology Score; VAP = ventilator-associated pneumonia.The authors declare that they have no competing interests.SN and AD designed the study. All authors contributed to data collection. CDP performed statistical analyses. SN wrote the manuscript, all authors participated in its critical revision. SN had full access to all data in the study and had final response ability for the decision to submit for publication. All authors read and approved the final manuscript.


Escherichia coli, ompF, ompC, and ompA respond to stress factors. Transcripts from these genes are regulated by the small non-coding RNAs micF, micC, and micA, respectively. Here we examine Photorhabdus luminescens, an organism that has a different habitat from E. coli for outer membrane protein genes and their regulatory RNA genes.Three major outer membrane protein genes of ompF -like porin gene in P. luminescens as well as a duplication of this gene have been predicted. Gene loci for micF RNA, as well as OmpC protein and its associated regulatory micC RNA, were not found. Significantly, a sequence bearing the appropriate signatures of the E. coli micA RNA was located. The ompA homolog was previously annotated in P. luminescens.By bioinformatics analysis of conserved genetic loci, mRNA 5'UTR sequences, RNA secondary structure motifs, upstream promoter regions and protein sequence homologies, an ompF-like porin in P. luminescens is in keeping with the necessity to allow for passage of small molecules into the cell. The apparent lack of ompC, micC and micF suggests that these genes are not essential to P. luminescens and ompC and micF in particular may have been lost when the organism entered its defined life cycle and partially protected habitat. Control of porin gene expression by RNA may be more prevalent in free- living cells where survival is dependent on the ability to make rapid adjustments in response to environmental stress. Regulation of ompA by micA may have been retained due to a necessity for ompA control during one or both stages of the P. luminescens life cycle.Presence of an This article was reviewed by Tal Dagan (nominated by Dan Graur), Mikhail Gelfand and Anna Gerasimova (nominated by Mikhail Gelfand) and J Peter Gogarten.Reviewed by Tal Dagan (nominated by Dan Graur), Mikhail Gelfand and Anna Gerasimova (nominated by Mikhail Gelfand) and J Peter Gogarten. For the full reviews, please go to the Reviewers' comments section. E. coli and related gamma-proteobacteria, the major outer membrane porin proteins are OmpF and OmpC  and the have authors satisfactorily addressed these.National Center for Biotechnology Information (NCBI) GenBank sites:BioCyc Database Collectionmfold server : 1995-2006, Michael Zuker, Rensselaer Polytechnic Institute:Donald Danforth Plant Science Center:The Welcome Trust Sanger InstituteDNASTAR, IncNC_004061Buchnera aphidicola str. SgNC_00443Escherichia coli CFT073NC_000913Escherichia coli K12NC_002655Escherichia coli O157:H7 EDL933NC_005126Photorhabdus luminescens subsp. laumondii TTO1NC_003197Salmonella typhimurium LT2,NC_003198Salmonella enterica (subsp. enterica serovar Typhi str. CT18)NC_004337Shigella flexneri 2a str. 301,NC 004741Shigella flexneri 2a str. 2457T,NC_003143Yersinia pestis CO92NC_006155Yersinia pseudotuberculosis IP 32953P. luminescens micF RNA c. Promoter search methods d. Figures: ompA mRNA 5' UTR and micA RNA e. Non-coding RNAs in E. coli-related bacteriaa. OmpF Loop 3 b. Search for a putative Click here for file


P. falciparum, P. y. yoelii and P. vivax have revealed some unusual features. P. falciparum contains the most AT rich genome sequenced so far – over 90% in some regions. In comparison, P. y. yoelii is ~77% and P. vivax is ~55% AT rich. The evolutionary reasons for these findings are unknown. Mobile genetic elements have a considerable impact on genome evolution but a thorough investigation of these elements in Plasmodium has not been undertaken. We therefore performed a comprehensive genome analysis of these elements and their derivatives in the three Plasmodium species.The completed genome sequences of the malaria parasites P. vivax, eight in P. y. yoelii and only six in P. falciparum. Further investigation of the six open reading frames in P. falciparum revealed that only one is potentially an active mobile genetic element. Most of the open reading frames identified in all three species are hypothetical proteins. Some represent annotated host proteins such as the putative telomerase reverse transcriptase genes in P. y. yoelii and P. falciparum. One of the P. vivax open reading frames identified in this study demonstrates similarity to telomerase reverse transcriptase and we conclude it to be the orthologue of this gene.Whole genome analysis was performed using bioinformatic methods. Forty potential protein encoding sequences with features of transposable elements were identified in Plasmodium species investigated. Despite the limitations of whole genome analytical methods, it is tempting to speculate that mobile genetic elements might have been a driving force behind the compositional bias of the P. falciparum genome.There is a divergence in the frequencies of mobile genetic elements in the three  Neurospora crassa, which silences repetitive elements by methylation and subsequent mutation of cytidine to thymidine nucleotides [N. crassa where very few, if any, active MGEs have been detected.Mobile genetic elements (MGEs) play a fundamental role as drivers of genome evolution, shaping both genes and genomes and often constitute a large fraction of the genome . The leotides . This exMGEs are usually classified according to their mechanism of replication and whether they are autonomous (self-replicating) or non-autonomous (dependant on related elements for replication or mobility) . The twoAnother intriguing group of mobile elements are the mobile introns. Divided into three groups based on their mechanisms of mobility (for a review of mobile introns see ), these Plasmodium species and the role they may have played in the evolution of the parasites' genomes. Initial reports after sequencing of the P. falciparum and P. y. yoelii genomes suggested that there may be a complete absence of MGEs in P. falciparum and no comment was made about MGEs in P. y. yoelii [P. falciparum and P. y. yoelii [Plasmodium spp. are the Alu elements (non autonomous non-LTR retrotransposons) located within the antigen coding genes of P. vivax [var, rif and stevor in P. falciparum [Very little is known about the occurrence of MGEs in . yoelii ,9. There. yoelii -12. The P. vivax  and the lciparum . The funP. falciparum, P. y. yoelii and P. vivax. This provides further insight into Plasmodium genome evolution with particular reference to the compositional bias in P. falciparum.We report here a comprehensive genome analysis of potential MGEs and their derivatives in P. falciparum and P. y. yoelii entries with MGE domains in InterPro [P. y. yoelii and four P. falciparum ORFs were identified. The four P. falciparum ORFs include one with similarity to a phage integrase, one with similarity to an intron-encoded nuclease and two domesticated derivatives. InterPro (and Pfam) domains that did not yield any hits in the Plasmodium database PlasmoDB [Table InterPro  and PfamInterPro  databasePlasmoDB  were:IPR000477 (PF00078) Reverse transcriptase; IPR001584 (PF0065) Integrase catalytic domain; IPR002156 (PF00075) Ribonuclease H fold; IPR001037 (PF00552) Retroviral integrase C terminal; IPR003308 (PF02022) Integrase N terminal Zn binding domain; IPR002050 (PF00429) ENV polyprotein; IPR004875 (PF03184) Endonuclease CENP-B protein; IPR008906 (PF05699) hAT element; IPR003322 (PF02337) retroviral GAG protein; IPR005162 (PF03732) retrotransposon GAG protein; IPR000442 (PF01348) Group II intron-encoded maturase; IPR002492 (PF01498) DNA-binding part of catalytic domain of Tc3 transposase; IPR008042 (PF05380) Aspartic protease from PAO retrotransposon family; IPR000305 (PF01541) Group I excinuclease; IPR003545 Telomerase reverse transcriptase; IPR001162 Excinuclease; IPR006350 Intron endonuclease group I; IPR008180 (PF00692) DeoxyUTP pyrophosphatase; IPR001995 (PF00077) Peptidase A2A, retrovirus; IPR000721 (PF00607) retroviral nucleocapsid protein gag (p24).P. falciparum organellar genomes (ORFs>50 amino acids) in PlasmoDB with E values less than 100 that demonstrated significant similarity to either of the two group II intron HMMs. Where significant similarity did arise, this was due to upstream exons being fused to the mitochondrial group II introns used in generating the HMM, e.g. PFB0795w is the Plasmodium gene orthologue of the ATP synthase gene and corresponds to a fused exon located upstream of several mitochondrial group II introns.There were no hits identified in the P. y. yoelii (PY00479 and PY03683) and four P. falciparum  ORFs with similar E values (less than 10). Of the two P. y. yoelii genes, PY00479, which is annotated as a putative telomerase reverse transcriptase in PlasmoDB, was not detected by the InterPro TERT HMM. PY03683 is a hypothetical protein of 48 amino acids, making it too short for any meaningful comparison, and was not investigated further. Of the four P. falciparum genes only PF13_0080 and PFE1555c appear to have significant non-random homology in the most conserved residues, despite the E values of all four being similar. PF13_0080 is annotated as a putative telomerase reverse transcriptase in PlasmoDB and bioinformatic evidence for this has already been published [The WashU-BLASTP 2.0 search of PlasmoDB using the RT consensus sequence developed in this study, detected two ublished ,12. PFE1P. vivax genome available from TIGR [P. falciparum and P. y. yoelii. Figure P. vivax TERT (PvTERT) which is reported here for the first time.The RT consensus sequence was also used to probe the rom TIGR . A WashUP. vivax genome.An orthologue of the PFE1555c gene, Pv079710, was identified by a simple BLASTP search of the P. vivax ORFs were identified by a BLASTP homology search as orthologues of the P. y. yoelii and P. falciparum genes located in InterPro (without a corresponding match in Pfam). HMMER searches of the P. vivax genome using Pfam_fs HMMs identified 28 ORFs containing MGE domains. In total therefore, 40 P. vivax ORFs containing MGE domains were identified and these are listed in Table Ten Plasmodium species: six in P. falciparum, eight in P. y. yoelii and 40 in P. vivax. In each species there are three potential domesticated derivatives of MGEs. They are meiotic recombinase, DNA repair protein rad51 and TERT. Although these three proteins are common to all eukaryotes and are no longer active MGEs, they have clearly defined MGE domains and are included for completeness. Recently, identification of the putative TERT genes in P. falciparum and P. y. yoelii has been of interest and bioinformatic evidence of this has been published [P. vivax using the conserved RT sequence developed in this study . The yir family in P. y. yoelii is analogous to the rif, stevor and var superfamilies in P. falciparum where recombination events play an important role in antigen switching. Analysis of the P. vivax genome yielded a relatively large number of RT domains, 23 in total. The reasons for this are not clear and require further investigation.There were seven ORFs identified in P. falciparum and P. y. yoelii, probably due to the stringency of the HMM search. This highlights the pitfalls of using generic bioinformatic programmes for studying a genome as exceptional as that of Plasmodium. The problems associated with using bioinformatic methods to compare divergent sequences have been discussed before [P. y. yoelii and P. falciparum TERTs and the PFE1555c ORF discussed above. Secondly, less stringent statistics were used in the scanning process. While it is more common to use E values of 10-5, we used values of 1 or 10. Despite the relatively high E values there were few positive hits (see Results) and we eliminated potential false positives by excluding sequences that demonstrated random homology patterns or which demonstrated no homology in the conserved domains.The InterPro entries for TERT failed to detect the two TERT orthologues in d before . It mustSaimiri boliviensis boliviensis sequences and the related Mucaca mulatta genome were searched for ORFs with similarity to the potential P. vivax MGEs identified in this study. Similarly, the Mus musculus and the Homo sapiens genomes were scanned for ORFs with similarity to the potential MGE domains detected in P. y. yoelii and P. falciparum respectively (see Methods). No potential contaminating vertebrate host DNA was identified amongst the potential P. vivax and P. falciparum MGE domains. However, several P. y. yoelii sequences demonstrated >80% identity to ORFs in the M. musculus genome and were rejected from the data as being contaminating sequences. These were PY06984, PY07613, PY07841, PY07669, PY07288, PY06363 and PY07014. The fact that these ORFs have homologues in other rodents e.g. Rattus norvegicus, confirms they are contaminating sequences.To further refine and validate our data we excluded ORFs that could potentially represent contaminating DNA. This was done by scanning the genomes of the vertebrate hosts that were used in the respective sequencing projects. All available P. y. yoelii and P. vivax, P. falciparum appears to contain very few. MGEs are ubiquitous in nature and are considered important catalysts for genome evolution. An interesting exception is the fungal genus Neurospora, which has the widest array of genome defence mechanisms found in eukaryotes, including the repeat-induced point mutation (RIP) mechanism whereby cytidine is mutated to thymidine [Neurospora, this mechanism has been demonstrated in all kingdoms of life. Most recently, Kuhlmann et al have demonstrated that a RIP-like mechanism is capable of silencing retrotransposons in Dictyostelium [P. falciparum. In addition, two putative DNA methyltransferases (PF11_0284 and PF13_0286) and a putative cytidine deaminase (PF13_0259) have been identified in P. falciparum, suggesting that the potential exists for methylation and deamination of cytidine residues in the genome.Given the above limitations and that the evidence presented here would need to be confirmed experimentally, these findings raise some interesting questions. While there are potentially several MGEs of different classes in hymidine ,21. Thishymidine . Since tostelium . This noP. falciparum. Since the initial effect of MGE invasion on the host genome is almost always negative [P. falciparum genome:The evolutionary forces that drive genomes to become compositionally biased are complex and largely theoretical. The arguments put forward depend upon the ecological niche of the individual organism and proposed hypotheses are therefore species specific. No hypothesis has been forthcoming for the AT richness found in negative , a selecP. falciparum genome.i) A genome protective mechanism, such as the RIP-induced silencing of transposable elements, has been a potential driving force behind the compositional bias of the P. falciparum has been selected for as a result of its inherent resistance to MGE invasion.ii) The AT-rich genome of P. falciparum genome lends support to the first hypothesis. The second hypothesis is based on findings that various MGEs may require specific recognition sequences as potential sites for integration e.g. the mariner element from Drosophila mauritiana appears to recognize specific nucleotide patterns as potential sites for integration [The finding that the putative RIP machinery is represented in the egration .P. falciparum, P. y. yoelii and P. vivax revealed that a decrease in the frequency of transposable elements coincides with an increase in AT bias. A causal relationship between these two parameters remains to be established since there are limitations in the currently available bioinformatic screening methods. However, the hypothesis that a genoprotective mechanism exists in P. falciparum and that this may contribute to the compositional bias should be considered. Whole genome analytical methods that are particularly suited to compositionally biased genomes would be of value in further understanding the P. falciparum genome.A comprehensive genome analysis of the three complete sequences of Protein sequences, as opposed to nucleic acid sequences, were used as they tend to be more sensitive and specific and the signature domains used in this study are all protein coding. Protein domain signatures characteristic of MGEs and their derivatives were identified based on their annotation in InterPro (release 12)  and Pfam(i) for signature domains : transposase, reverse transcriptase, ribonuclease, integrase, protease, endonuclease, zinc finger, GAG polyprotein, maturase and recombinase.(ii) for classes of MGEs: transposon, retroelement, retrovirus, non-LTR retrotransposon, LTR retrotransposon and group II introns.The signature domains of group II introns are not well represented in InterPro or Pfam. Hidden Markov models (HMMs) were therefore generated for mitochondrial and chloroplast forms of this MGE (see  for origX04826 (trnK I1), Z00044 (trnK I1), Z11741 (trnK I1), JN0302 (trnK I1), X52765 (trnK I1), X15901 (trnK I1), X57097 (trnK I1), D11467 (trnK I1), X04465 (trnK I1), P19593 (petD I1), X55877 (rbcL I1), X70810 .Chloroplast: Mitochondrial: X57968 (nad1 I4), M63034 (nad1 I4), M30176 (nad1 I4), M68929 , X14669 (coI I1), X55026 , M62622 , X57546 (cox1 I1), X54421 (cob I1).P. falciparum organellar genomes were investigated.The software package HMMER 2.3.2  was usedThe RT domain is common to all MGEs (including group II introns), except DNA transposons, and has been the most intensively studied MGE domain. However, the divergent and abundant nature of RT containing sequences makes it difficult to construct a meaningful HMM or consensus sequence for use in BLAST searches and alignments. Based on biochemical knowledge of the RT domain such as its crystal structure and point mutation data ,30, we gin vitro, using the EMBOSS:cons software from the European Bioinformatics Institute [X54421), chloroplast group II intron psbA (AY290861), HIV1-RT (AAY66384), T. thermophila telomerase reverse transcriptase TtTERT (AF062652), LINE element DONG (L08889) and D. melanogaster telomere associated retrotransposon DmTART (U14101). Accession numbers are those in GenBank. This initial consensus sequence was refined manually in the following way:An initial consensus sequence of the RT domain was generated that was representative of RT-encoding MGE classes  and two related domesticated MGEs (a telomerase and a telomere associated non-LTR retrotransposon). The consensus sequence was generated from six sequences, all of which had laboratory evidence of RT activity nstitute  with the2+ cofactor [i) Residues that were determined by crystal structure of a retrovirus RT to have sidechain or mainchain contact to either nucleic acid, dNTPs or Mgcofactor  and thatin vitro [ii) Amino acid residues that have been demonstrated to be essential for RT activity in vitro  were insiii) A ClustalW alignment   has not been sequenced. We therefore screened the available S. b. boliviensis sequences as well as the complete genome of a related primate, Mucaca mulatta, for any of the P. vivax MGE domains identified. The P. falciparum MGE domains were screened against the human genome. The genomes of M. musculus, M. mulatta and H. sapiens are available at [projects ,9,34. MGlable at .P. vivax in the study, helped draft the manuscript and gave considerable intellectual input in analyzing the results. All authors read and approved the final manuscript.PD conceptualized and designed the study, carried out the analysis and drafted the manuscript. AO assisted with and critically reviewed the bioinformatic methods. TC conceptualized the inclusion of


Rickettsia conorii strain, include Indian tick typhus rickettsia (ITTR), Israeli spotted fever rickettsia (ISFR), and Astrakhan fever rickettsia (AFR). Although closely related genotypically, they are distinct serotypically. Using multilocus sequence typing (MLST), we have recently found that distinct serotypes may not always represent distinct species within the Rickettsia genus. We investigated the possibility of classifying rickettsiae closely related to R. conorii as R. conorii subspecies as proposed by the ad hoc committee on reconciliation of approaches to bacterial systematics. For this, we first estimated their genotypic variability by using MLST including the sequencing of 5 genes, of 31 rickettsial isolates closely related to R. conorii strain Malish, 1 ITTR isolate, 2 isolates and 3 tick amplicons of AFR, and 2 ISFR isolates. Then, we selected a representative of each MLST genotype and used multi-spacer typing (MST) and mouse serotyping to estimate their degree of taxonomic relatedness.Rickettsiae closely related to the Malish strain, the reference gltA, ompA, ompB, and sca4 genes, respectively. Representatives of the 4 MLST types were also classified within four types using MST genotyping as well as mouse serotyping.Among the 39 isolates or tick amplicons studied, four MLST genotypes were identified: i) the Malish type; ii) the ITTR type; iii) the AFR type; and iv) the ISFR type. Among these four MLST genotypes, the pairwise similarity in nucleotide sequence varied from 99.8 to 100%, 99.4 to 100%, 98.2 to 99.8%, 98.4 to 99.8%, and 99.2 to 99.9% for 16S rDNA, R. conorii species show MST genotypic, serotypic, and epidemio-clinical dissimilarities. We, therefore, propose to modify the nomenclature of the R. conorii species through the creation of subspecies. We propose the names R. conorii subsp. conorii subsp. nov. , R. conorii subspecies indica subsp. nov. (type strain = ATCC VR-597), R. conorii subspecies caspia subsp. nov. (type strain = A-167), and R. conorii subspecies israelensis subsp. nov. (type strain = ISTT CDC1). The description of R. conorii is emended to accomodate the four subspecies.Although homogeneous genotypically, strains within the  Rickettsiales was originally based on relatively few phenotypic criteria .-22.R. cos Figure . One of n Figure . SerotypR. conorii species ). PCR products were visualized after electrophoresis on a 1% agarose gel. Each amplicon was purified for sequencing using the QIAquick Spin PCR Purification Kit (Qiagen) according to the manufacturer's instructions. Sequencing reactions were carried out using PCR primers and the D-rhodamine terminator cycle DNA sequencing Kit  according to the manufacturer' instructions. Sequencing reaction products were resolved by electrophoresis with an ABI Prism 3100 Automated Sequencer (Applied Bioystems). Each base position was established twice in both the forward and reverse directions.When available, we used gene sequences present in GenBank. The accession numbers for these sequences are reported at the end of the manuscript. Missing sequences were obtained in this study. Genomic DNA was extracted from 200 μl of rickettsial suspension and from 57 strakhan  using thstrakhan , gltA [3an [gltA , ompA [3tA [ompA , ompB [2pA [ompB  and sca4For each gene, the pairwise sequence similarity among studied isolates and tick amplicons was estimated using the multisequence alignment program CLUSTAL W . Each isDistance matrices generated by DNADIST were determined under the assumptions of Kimura and were used to infer dendrograms by the neighbor-joining method  using thdksA / xerC, mppA / purC, and rpmE / tRNA-fMet intergenic spacers for each of R. conorii isolate Malish, ITTR, ISFR, and AFR isolates A-167 and Chad. Primers and methods were those described previously [We amplified and sequenced the eviously . Subsequeviously  as descrR. conorii isolate Malish, ITTR, ISFR, and AFR isolates A-167 and Chad, five BALB/c mice were inoculated intravenously (tail vein) with purified bacterial suspension (0.1 ml containing 104 bacteria). The concentration of rickettsial suspensions was estimated using a plaque assay as previously described [For each escribed . On day escribed . Sera frThe MIF test was performed and the SPD was calculated using the method of Philip et al. . Each ofThe individual parts of the work presented in the paper were conducted as follows: YZ and PEF carried out the molecular genetic studies, analyzed the sequences and drafted the manuscript. ME participated in the design of the study and helped to draft the manuscript. DR conceived the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.


Trypanosoma brucei and Leishmania major reveal that these unicellular kinetoplastids possess arrays of tandem-duplicated tubulin genes, but with differences in organisation. While L. major possesses monotypic α and β arrays in trans, an array of alternating α- and β tubulin genes occurs in T. brucei. Polycistronic transcription in these organisms makes the chromosomal arrangement of tubulin genes important with respect to gene expression.α- and β-tubulin are fundamental components of the eukaryotic cytoskeleton and cell division machinery. While overall tubulin expression is carefully controlled, most eukaryotes express multiple tubulin genes in specific regulatory or developmental contexts. The genomes of the human parasites T. brucei and L. major were compared to examine the relationship between the two character states. Intergenic regions between tubulin genes were sequenced from several trypanosomatids and related, non-parasitic bodonids to identify the ancestral state. Evidence of alternating arrays was found among non-parasitic kinetoplastids and all Trypanosoma spp.; monotypic arrays were confirmed in all Leishmania spp. and close relatives.We investigated the genomic architecture of tubulin tandem arrays among these parasites, establishing which character state is derived, and the timing of character transition. Tubulin loci in Leishmania. This fundamental reorganisation accounted for the dissimilar genomic architectures of T. brucei and L. major tubulin repertoires.Alternating and monotypic tubulin arrays were found to be mutually exclusive through comparison of genome sequences. The presence of alternating gene arrays in non-parasitic kinetoplastids confirmed that separate, monotypic arrays are the derived state and evolved through genomic restructuring in the lineage leading to  Trypanosoma spp. and Leishmania spp., with a comparative genomic approach. These unicellular flagellates are responsible for various human diseases around the world, namely African sleeping sickness, leishmaniasis and Chagas' disease.Gene expression in kinetoplastids (Euglenozoa) takes a peculiar form, quite unlike the system of promoter and terminator signals typical of other eukaryotes. Expression of several, contiguous loci occurs simultaneously through polycistronic transcription -3 and reTrypanosoma and Leishmania spp., possibly as the relative demand for these structures fluctuates  and [Genbank: M97956], among others). Otherwise, the genome sequence contained three contigs that include tubulin. The first (Tc00.1047053506563.40) showed a single β-tubulin and three other loci; together, they were colinear with chromosome 1 in T. brucei, and indicate that this single β-tubulin gene occupies a homoeologous position to the alternating array  was a duplicate of the first, although the β-tubulin is annotated as a pseudogene and apparently lacks 700 bp from the 3' end. The third contig (Tc00.1047053411235.9) included a lone α-tubulin without any contextual information. In summary, these three Trypanosoma genome sequences concurred with the arrangement in T. brucei, showing an α-β alternating array, with conservation of surrounding gene order. IGSs following the two isotypes were always dissimilar, but each class of CDS and internal IGS were consistently concerted. There was no evidence for further tubulin loci.No array is evident from the current release of the sequence . Howeversequence ,41, and ce AF09186 and , T. pestanai [GenBank: AJ009159], T. congolense [GenBank: AJ223563], T. brucei [GenBank: AJ009142], T. grayi [GenBank: AJ005278], T. cyclops [GenBank: AJ131958], T. vivax [GenBank: U22316], L. major [GenBank: X53915], L. mexicana [GenBank: X53912], H. megaseliae [GenBank: U01014], C. fasciculata [GenBank: Y00055], L. infantum [GenBank: X07773], L. braziliensis [GenBank: M80292], C. oncopelti [GenBank: AF038025], B. saltans [GenBank: AF208889], N. designis [GenBank: AY998646], P. caudatus [GenBank: AY490218] and L. seymouri [GenBank: AF153040]. Note that C. oncopelti, a close relative of C. deanei, was used as a surrogate in the rRNA alignment, given the absence of any SSU rRNA sequence for C. deanei. Sequences were aligned by eye and maximum likelihood phylogenetic estimation was carried out using PHYML [A comparative approach to the evolution of genomic characters requires a species phylogeny. This was obtained through phylogenetic analysis of small subunit ribosomal RNA sequences for the species concerned. These were selected from depositions to GenBank: ng PHYML ,69. A geng PHYML ) model wPCR Polymerase chain reactionBLAST Basic local alignment search toolGTR General-time reversibleCDS Coding sequenceIGS Intergenic sequenceUTR Untranscribed regionAPJ carried out cell culture, DNA preparation and molecular screening of kinetoplastids, as well as bioinformatic comparisons of genome sequences and drafting of the manuscript. SV produced preliminary bioinformatic analyses and gave assistance in experimental design and manuscript preparation. KG supervised the study design, concept and execution, and contributed to manuscript preparation. All authors read and approved the final manuscript.