Mycobacterium chimaera was found to be the dominant species in both environmental and respiratory specimens. In contrast to previous studies from the continental U.S., no Mycobacterium avium was identified. Mycobacterium intracellulare was found only in respiratory specimens and a soil sample. We conclude that Hawai’i’s household water sources contain a unique composition of Mycobacterium avium complex (MAC), increasing our appreciation of NTM organisms of pulmonary importance in tropical environments.Lung disease caused by nontuberculous mycobacteria (NTM) is an emerging infectious disease of global significance. Epidemiologic studies have shown the Hawaiian Islands have the highest prevalence of NTM lung infections in the United States. However, potential environmental reservoirs and species diversity have not been characterized. In this cross-sectional study, we describe molecular and phylogenetic comparisons of NTM isolated from 172 household plumbing biofilms and soil samples from 62 non-patient households and 15 respiratory specimens. Although non-uniform geographic sampling and availability of patient information were limitations,  Mycobacterium chimaera and several other clinically relevant NTM species and the absence of Mycobacterium avium in both the indigenous environment and clinical specimens underscore the need for further studies to define the environmental factors that drive NTM lung disease and species composition in high prevalence locations such as the Hawaiian Islands.In the U.S., the Hawaiian Islands have the highest number of nontuberculous mycobacterial (NTM) lung disease cases per capita. The tropical climate, geographical isolation of the islands, and aquifer water sources may have influence such prevalence. Previous studies suggest that NTM thrive in water biofilms and soil. To broaden our understanding of potential environmental reservoirs and species composition of NTM in the Hawaiian Islands, we sampled environmental sites and examined patient isolates. Our recovery and identification of  To date, there are over 175 species of NTM with standing in nomenclature . Potentiand soil . In the and soil –8. The mellulare . Clinicachelonae . The curchelonae . Of inteiversity .Despite the almost universal exposure to environmental NTM, pulmonary infections are relatively rare in otherwise healthy, non-bronchiectatic individuals and more common in individuals with abnormal lung architecture such as bronchiectasis and emphysema . NeverthThe high prevalence of NTM lung disease in the Hawaiian Islands provided the impetus to explore potential sources of infection and to determine the predominating NTM species in both environmental and clinical specimens. These islands are recognized for their unique island geology, flora, and fauna which are largely impacted by the tropical climate and isolation of the archipelago in the Pacific Ocean. Unlike most areas in the continental U.S. for which surface water serves as the primary public water source, underground aquifers provide water there. The Hawaiian Islands are also home to the highest number of elderly Asian-Pacific Islanders in the U.S.—a population previously recognized to be more susceptible to NTM infection . To bettIn this cross-sectional study, we use the term “Hawaiian Islands” to designate the eight islands of the State of Hawai’i; the term “Hawai’i” refers to the youngest and largest island among the eight islands. Sample collection was conducted between December 2012 and January 2013. Samples were collected from 62 non-patient households located on the islands of Oahu, Molokai, Kauai, and Hawai’i. Detailed written instructions for collecting household water biofilms and soil samples were provided to local residents who volunteered to collect samples from their home as part of this study. As NTM are most commonly found in premise plumbing biofilms, samples were obtained by swabbing with sterile cotton-tipped applicators the inner surface of showerheads, kitchen and bath faucets, kitchen sink sprayers, refrigerator water dispensers, laundry room sinks, and shower drains , 6. SampMycobacterium tuberculosis was not recovered in any of these sputum samples where NTM were isolated. As these were de-identified patient residual isolates, where only age and gender were noted from routinely ordered laboratory testing, Institutional Review Board (IRB) consent was waived. However, it was impossible to determine whether these patients met current American Thoracic Society/Infectious Disease Society of America (ATS/IDSA) diagnostic criteria for NTM pulmonary disease as private health information were delinked [Respiratory isolates of slowly-growing NTM recovered from 15 de-identified Oahu patients suspected of mycobacterial lung disease whose sputum had been submitted for mycobacterial culture were randomly selected from saved isolates at Diagnostic Laboratory Services, Inc. . delinked .rpoB) gene, also known as region 5 [rpoB type strain sequences deposited in the National Center for Biotechnology Information (NCBI) GenBank using the BLAST algorithm. Definitions of species by single genes or spacer region were those of the Clinical Laboratory Standards Institute (CLSI) [Genome identification of environmental and patient NTM isolates was conducted through the amplification and sequencing of a 723 bp segment of the RNA polymerase beta subunit (region 5 . Sequence (CLSI) . A sequee (CLSI) . The seqM. porcinum CIP 105392 T, M. abscessus subsp. abscessus ATCC 19977T, M. abscessus subsp. bolletii CIP 108541T, M. chelonae ATCC 35752T, and M. chimaera CIP 107892 T. Additionally, 33 clinical respiratory isolates of M. chimaera (one per patient) from seven other states–Maryland, Texas, Louisiana, North Carolina, Oregon, Mississippi, and Arkansas–submitted for molecular identification to the Nocardia/Mycobacteria Research Laboratory, University of Texas Health Science Center, Tyler, Texas were included. Those isolates were identified to species by partial 16S rRNA and region 5 rpoB gene sequencing. This work was approved by the Human Subjects Committee of the University of Texas Health Science Center, Tyler, Texas.To determine whether NTM isolates from the Hawaiian Islands have shared sequence similarity with isolates obtained elsewhere, NTM type strains were included in genetic analyses. Type strains are denoted by superscript “T” and include rpoB gene sequences from 166 Hawaiian Island NTM isolates and 33 M. chimaera isolates from the continental U.S. were deposited in the GenBank nucleotide database. The GenBank accession numbers for type strain and representative isolate rpoB gene sequences of M. porcinum, M. abscessus, M. chelonae, and M. chimaera from NCBI are also listed in S1 TablePartial rpoB sequences of respiratory and environmental NTM isolates (n = 166) were aligned using MUSCLE [Partial g MUSCLE  and sequg MUSCLE .rpoB sequence variant analyses, only sequences greater than 600bp and with no ambiguous base calls were included. Sequences were grouped by species and compared to selected type and non-type strain sequences from NCBI. The PopART population genetics software was used to examine intraspecies sequence variation, generate species-specific rpoB sequence variant networks, and label isolates by isolation source: i.e., kitchen, bathroom, soil, patient [M. porcinum, M. abscessus, and M. chelonae analyses, the environmental Hawaiian Island isolates and both type and non-type strains were included. For the M. chimaera analysis, environmental and clinical Hawaiian Island isolates, type, and non-type strains, as well as clinical isolates from seven states across the continental U.S. were included.For  patient . For thei.e., bathroom, kitchen, and soil) or sample type (biofilm and soil).Statistical analyses were performed using R version 2.13.2 . Fisher’, a total of 172 biofilm and soil samples were collected. The majority of the samples  were collected from Oahu and included 35 showerheads (26%), 41 kitchen faucets (31%), 6 bathroom sink faucets (4%), 2 refrigerator water taps (1%), 3 other biofilm samples from laundry room faucets (2%), and 47 soil samples (35%). The remaining 38 samples (22%) were collected from 13 households on the neighbor islands.From a total of 62 households across four islands  NTM were identified in nearly half of the samples on Oahu  and in approximately a quarter of samples from the neighbor islands . Overall, the NTM culture positivity rate for biofilms was 59% (67/113), which was significantly greater than for soil .Among all 172 biofilm and soil samples collected from the 62 households, NTM were isolated from 44% of samples (75/172) . NTM were recovered by culture from 82% of the Oahu households . For the neighboring islands, NTM were also recovered in households on Kauai, Molokai, and Hawai’i . Among the 62 collective households sampled in this study, only 14 had no NTM isolated (23%). However, the number of households with one, two, and three different NTM species isolated were 26/62 (42%), 18/62 (29%), and 4/62 (6%), respectively . Overall, the majority of households  had at least one clinically relevant species of MAC, M. abscessus subsp., or M. chelonae—.The majority of the environmental samples collected were from 49 households in seven different towns on Oahu, the most populated island  and one biofilm sample contained M. chelonae (1/7 = 14%), but the majority (5/13 = 38%) yielded other RGM species . RpoB sequences from four distinct isolates did not have NCBI database matches above 95% sequence identity, suggesting they represent novel species.To determine the diversity of NTM in non-household sites, 13 environmental samples (n = 7 biofilm and n = 6 soil) were collected from eight public areas on Oahu and Kauai Fig 2A) and 17% (13/75) grew out multiple NTM species. The most common species recovered from households were MAC organisms with M. chimaera being the predominant species  , left. The next most frequently isolated species were M. chelonae  and M. porcinum . All isolates of M. abscessus were confirmed as M. abscessus subsp. abscessus  [M. phocaicum, M. gadium, M. alvei, M. gordonae, M. paraffinicum, M. marseillense, and M. colombiense. No isolates of M. avium or M. intracellulare were recovered from household biofilm samples, though M. intracellulare was isolated from a single soil sample. While M. chimaera and M. chelonae were identified in non-household samples, the majority classified as other NTM included potentially novel species , right.Among the 75 environmental samples from the households that were NTM culture-positive, 20 different NTM species were identified  , 24. Les.M. chimaera was frequently identified from both bathroom  and kitchen  biofilms and was also identified in soil . M. porcinum was overrepresented in bathroom  compared to kitchen biofilms , while M. chelonae was significantly more common in kitchen  compared to bathroom biofilms . M. abscessus was observed in similar proportions between bathroom  and kitchen  biofilms. M. porcinum, M. chelonae, and M. abscessus were not recovered from soil. M. marseillense was recovered only from soil and not identified in any of the household biofilm samples. NTM species that showed low prevalence in our study  were primarily isolated from soil samples.To determine whether NTM were present in particular household locations, the frequencies of NTM recovery between bathroom biofilms, kitchen biofilms, and soil were compared rpoB sequences of M. porcinum, M. abscessus, and M. chelonae were analyzed . Type and non-type strain rpoB sequences were included for comparison. In the M. porcinum dataset (n = 25 sequences), a total of seven sequence variants were identified . All isolates from the bathroom, kitchen, and outside faucets were in the same sequence variant group as the M. porcinum type strain, CIP 105392T, except for one kitchen isolate that contained a single SNP difference. Among all M. abscessus sequences , six sequence variants of subsp. abscessus, four variants of subsp. massiliense, and one of subsp.bolletii were identified. Environmental M. abscessus isolates grouped with other M. abscessus subsp. abscessus and the majority of M. abscessus isolates (13/16 = 81%) shared an identical rpoB sequence with the type strain, ATCC 19977T. Three additional isolates differed by one SNP each from the ATCC 19977T type strain. Finally, M. chelonae isolates  showed the greatest rpoB sequence variation with a total of 14 rpoB sequence variants. Hawaiian Island M. chelonae isolates fell into seven rpoB sequence variant groups, but the majority (15/20 = 80%) fell into two main subgroups: one group (6/15 and 40%) sharing the M. chelonae ATCC 19237 rpoB variant and a second group (5/15 and 33%) related to the M. chelonae ATCC 35752TrpoB variant.To examine population diversity among RGM isolates from individual households, M. chimaera, 15 random respiratory SGM isolates from Oahu patients presenting to a pulmonary clinic with suspected mycobacterial lung disease were used as pilot samples to evaluate for the presence of M. chimaera in clinical specimens. As a group, the median age of the 15 patients was 75 years  and 67% (10/15) were female . Ten isolates were identified as M. chimaera  four as M. intracellulare , and one as M. marseillense . Of the ten patients with M. chimaera, 60% (6/10) were female. All four patients with M. intracellulare were female  and the patient with M. marseillense was male .M. avium was not identified from any of the Oahu clinical isolates.As the majority of the Hawaiian Island environmental NTM isolates from this study were M. chimaera, we analyzed rpoB sequence variation between the 57 Hawaiian Island environmental M. chimaera isolates and the 10 Oahu respiratory M. chimaera isolates. However, the rpoB sequence of one clinical M. chimaera isolate was excluded from these analyses due to the presence of ambiguous bases. Also included were NCBI non-type strains (n = 2), type strains (n = 2), and other M. chimaera respiratory isolates (n = 33) from seven states in the continental U.S. In total, 103 M. chimaera sequences were analyzed and only two rpoB sequence variants were observed . The larger variant subgroup comprised over 90% of the isolates including all of the Oahu respiratory and biofilm M. chimaera isolates. This group also contained the majority of continental U.S. clinical isolates and the CIP107892T type strain. The smaller variant subgroup contained continental U.S. clinical isolates, non-type strains from NCBI, and Hawaiian Island soil isolates.To measure the genetic similarity among a diverse collection of environmental and clinical M. avium subsp. “hominissuis” reported in studies from the continental U.S. and Japan [M. avium subsp. “hominissuis” has been isolated from surface water sources [M. avium detection in our samples. However, given the widespread prevalence of M. chimaera and the RGM in Hawaiian Island household biofilms, local aquifers may be a potential reservoir for M. chimaera and other NTM. Future studies are needed to examine this hypothesis.To our knowledge, this is the first assessment of environmental NTM prevalence and species composition in the Hawaiian Islands. This archipelago is approximately halfway between the continental U.S. and Asia; thus, one might speculate that the spectrum of NTM observed mirrors the results from other environmental studies from the continental U.S. or Asia. Due to the prevalence of nd Japan –27, we snd Japan  whereas  sources . Aquifer sources  which maM. fortuitum complex in Polynesian residents with suspected tuberculosis [M. intracellulare was reported as the species responsible for most lung disease cases and yet only M. avium subsp. “hominissuis”, M. kansasii, and M. abscessus isolates had a species that match between patients and their household water system [To date, species diversity assessments of environmental NTM in other tropical Pacific Islands remains scant. A recent study described the identification of the rculosis  and otherculosis , 33. On r system , 35.M. chimaera from both the environmental samples collected from bathroom, kitchen, and soil samples . In a previous U.S. study, water biofilm isolates originally reported as M. intracellulare, proved to be M. chimaera or other MAC-X [M. chimaera in the Hawaiian Islands are water biofilms and less from the soil  using partial rpoB gene sequencing [M. porcinum was identified in three patients who fulfilled ATS criteria for NTM lung disease. To our knowledge, M. porcinum infections have not yet been reported in the Hawaiian Islands, but the organism has been isolated from water supplies in other U.S. areas  [M. abscessus was recently associated with an outbreak in cystic fibrosis patients at a hospital in Hawai’i [M. chelonae infection was reported in a case study of an individual from Hawai’i after laser in situ keratomileusis (LASIK) surgery [Among our environmental samples, quencing . Of thesolynesia /87, 48% , Texas) . M. absc Hawai’i . M. chel surgery . It is iM. chelonae was observed with four major rpoB subgroups present, while most isolates of M. porcinum and M. abscessus belonged to one major genetic group per species  we were unable to consistently collect a large number of samples from the same indoor sites for each participating household, (ii) a sampling bias exists as the majority of samples were collected from Oahu (home to the majority of the state’s population) with only a few household samples collected from the less populated Molokai, Kauai, and Hawai’i and none from Kaho’olawe, Maui, Lanai, or Ni’ihau, and (iii) instead of a single person conducting all environmental sampling, household areas were sampled by local citizens, which added a layer of non-equivalency to the process of sample collection. To reduce non-uniformity in the collection process, we applied a well-accepted citizen science approach to minimize variability introduced by handling of samples by different people [M. chimaera was the most common species in both environmental and clinical isolates examined suggests the possibility of environmental exposures and clinical NTM lung disease. To determine whether NTM in the household environment contributes to clinical disease, we hope to initiate a large-scale genomic study of matched household and clinical NTM isolates from NTM-infected Hawai’i patients who fulfill ATS/IDSA criteria for lung disease. Undoubtedly, the data collectively presented in this study will be valuable in guiding the design of a more comprehensive study.This study has some limitations including the following in methodology: t people . AlthougM. chimaera was the most common NTM species identified in both our Hawai’i environmental samples as well as in a small sampling of respiratory specimens from patients with suspected mycobacterial lung disease suggests that M. chimaera may be an important environmentally acquired respiratory pathogen. Furthermore, M. chimaera may be unique in prevalence in tropical climates such as Hawai’i. Additional studies with systematic collection of matched environmental and respiratory specimens, high-resolution genotyping methods, and correlation with demographic and epidemiological data  will be necessary to further characterize this observation and the important clinical implications.In summary, this study describes environmental sampling, microbiological selection, and molecular identification to determine the NTM species diversity in the Hawaiian Island environment. The observation that S1 Checklist(DOC)Click here for additional data file.S1 TableMycobacteria isolates derived from Hawaiian Island and Continental U.S.A. clinical and household specimens. Species identification and NCBI GenBank accession numbers are provided for each isolate.(PDF)Click here for additional data file.


Aspergillus fumigatus and multiple other Aspergillus species cause a wide range of lung infections, collectively termed aspergillosis. Aspergilli are ubiquitous in environment with healthy immune systems routinely eliminating inhaled conidia, however, Aspergilli can become an opportunistic pathogen in immune-compromised patients. The aspergillosis mortality rate and emergence of drug-resistance reveals an urgent need to identify novel targets. Secreted and cell membrane proteins play a critical role in fungal-host interactions and pathogenesis. Using a computational pipeline integrating data from high-throughput experiments and bioinformatic predictions, we have identified secreted and cell membrane proteins in ten Aspergillus species known to cause aspergillosis. Small secreted and effector-like proteins similar to agents of fungal-plant pathogenesis were also identified within each secretome. A comparison with humans revealed that at least 70% of Aspergillus secretomes have no sequence similarity with the human proteome. An analysis of antigenic qualities of Aspergillus proteins revealed that the secretome is significantly more antigenic than cell membrane proteins or the complete proteome. Finally, overlaying an expression dataset, four A. fumigatus proteins upregulated during infection and with available structures, were found to be structurally similar to known drug target proteins in other organisms, and were able to dock in silico with the respective drug. Aspergillus species. The majority of reported aspergillosis cases originate from ten species: Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus terreus, Aspergillus versicolor, Aspergillus lentulus, Aspergillus nidulans, Aspergillus glaucus, Aspergillus oryzae, and Aspergillus ustus3. A. fumigatus alone is responsible for over 90% of the reported cases, followed by A. flavus, A. niger, A. terreus and A. versicolor5. The disease is an increasing concern for immune-compromised individuals, putting at risk patients with neutropenia, allogenic stem cell transplantation, organ transplantation, and acquired immunodeficiency syndrome (AIDS), among others6. High-risk patients can see mortality rates between 40–90%7. Compounding the issue is the limited number of drugs available to treat aspergillosis, and their widespread use has led to the emergence of drug-resistant strains, creating the pressing need for novel drugs, biomarkers and preventive therapies such as vaccines8.Aspergillosis is an umbrella term for a wide array of infections caused by multiple Aspergilli are saprotrophic fungi which are present in soil and decaying organic matter, thereby, playing a fundamental role in nitrogen and carbon recycling. They have a broad geographical range with colonies typically spread through microscopic airborne conidia. As Aspergillus conidia are ubiquitous in the atmosphere, humans often inhale several hundred spores daily. While these spores are quickly eliminated by a healthy immune system9, in an immune-compromised host, the fungus becomes an opportunistic pathogen and overwhelms the weakened defences. The underlying mechanisms behind the successful initiation of pathogenesis by Aspergilli remain unclear. However, studies have shown that in immune-compromised hosts, A. fumigatus can reach the respiratory epithelia upon inhalation where it secretes proteases and secondary metabolites, specifically gliotoxin, which aids in colonizing healthy lung tissue11. Proteases secreted by A. fumigatus have also been implicated in establishing infection by morphologically altering respiratory epithelial cells12. In addition, A. fumigatus secretes a diverse array of catabolic enzymes that enable degradation of macromolecular biopolymers like elastin and collagen, which are present in large quantities in lung tissue, for uptake of nutrients13. Apart from secreted proteins, cell membrane and cell wall proteins also play a crucial role in interactions with the host immune system and establishing infection10. Thus, the diverse set of secreted and cell membrane proteins of A. fumigatus and other Aspergillus species play a vital role in pathogenesis.Aspergillosis is caused by an opportunistic pathogen. Under normal conditions, Aspergillus species during human pathogenesis, they are potential candidates for identifying new biomarkers, vaccines and druggable targets. This is especially urgent as prophylactic use of antifungal therapies has led to the emergence of resistance in many Aspergillus species to antifungal drugs15. Prospecting the secretome and cell membrane proteins of Aspergillus species for druggable targets is advantageous, since any identified target will be exposed to the extracellular space. In addition, targeting extracellular rather than intracellular proteins will provide fewer mechanisms for resistance to develop in the pathogen.Given the central role played by the secretome and cell membrane proteins of A. fumigatus and nine other Aspergillus species known to cause aspergillosis, namely, A. flavus, A. niger, A. terreus, A. versicolor, A. lentulus, A. nidulans, A. glaucus, A. oryzae, and A. ustus. To our knowledge, this is the first dedicated effort to characterize the secretome and cell membrane proteins of Aspergillus species with an aim to understand their role in pathogenesis and to analyze them as potential druggable proteins. While previous studies such as FSD16, FunSecKB217 and SECRETOOL18 have developed computational pipelines for fungal secretome prediction, they rely solely on bioinformatic predictions even though many experimental high-throughput proteomic datasets in Aspergillus species are available. Thus, we have designed a computational pipeline which systematically integrates experimental high-throughput proteomic datasets, UniProt19 annotations with experimental evidence, and predictions from bioinformatic tools to identify a comprehensive set of secreted extracellular proteins and cell membrane proteins in Aspergillus species. Furthermore, small secreted and effector-like proteins23 were identified in Aspergillus species, and the set of secreted and cell membrane proteins were analyzed for the abundance of antigenic regions (AAR)24 and similarity to known drug target proteins from DrugBank25. Finally, analysis of a published gene expression dataset26 for A. fumigatus, led to identification of secreted and cell membrane proteins which are upregulated under pathogenesis and have no human homologs, and such candidates were taken for further druggability analysis by computational docking experiments, thereby identifying pre-existing drugs used against other pathogens as candidates for repurposing against aspergillosis.This study aims to identify and analyze the secretome and cell membrane proteins of Aspergillus species. A systematic search led to a comprehensive list of 46 high-throughput proteomic studies73 on the secretome of 6 Aspergillus species analyzed here. Note that many high-throughput studies report proteins with transmembrane (TM) domains as part of the secretome but a few studies have specifically identified cell membrane and cell wall associated proteins along with the secretome36. Our compilation of high-throughput proteomic studies led to experimentally identified lists of 437, 364, 454, 96, 469 and 171 secreted proteins in A. fumigatus41, A. flavus45, A. niger58, A. terreus60, A. nidulans65 and A. oryzae73, respectively  and those that exit the cell boundary through a non-classical secretion pathway. Figure 19 annotation with experimental evidence were removed from later analysis. Subsequently, the remaining proteins without experimental evidence for intracellular localization were classified into two mutually exclusive categories. The first category contained secreted or cell membrane proteins with experimental evidence from a compiled list of high-throughput proteomic studies or UniProt, and the second category contained proteins without experimental evidence of secretion to the extracellular matrix or localization to the cell membrane.Our pipeline starts from the complete proteome of a fungus. Initially, intracellular proteins based on UniProtThe first category of proteins with experimental evidence were assessed for signal peptide, Glycosylphosphatidylinositol (GPI) anchor or TM domain, confirming passage through the classical pathway , alcohol degradation and gliotoxin metabolism. Many of the GO processes annotated with A. fumigatus proteins secreted by non-classical pathway are associated with virulence. Especially, gliotoxin has immunosuppressive properties and is suspected to be an important virulence factor in Aspergillus pathogenesis79. This suggests that non-classical pathways may be important for the secretion of virulence factors in A. fumigatus.Using this pipeline, we identified comprehensive sets of secreted and cell membrane proteins in ten Aspergillus species from Branch B of our computational pipeline shown in Fig. Aspergillus species of which 3 are in A. fumigatus81, 13 in A. nidulans94 and 1 in A. oryzae95 . We found that SSPs and effector-like proteins account for more than 30% and 10%, respectively, of the secretomes in Aspergillus species  with sequence length less than 300 amino acids have been widely studied for their role in fungal-plant pathogenesisA. fumigatus within the context of aspergillosis, we overlaid a previously published gene expression dataset26. Significantly differentially expressed and upregulated genes coding for secreted and cell membrane proteins may suggest a vital path or key towards pathogenesis. Note that several gene expression studies in A. fumigatus have been published on various cell cultures. However, due to the difficulty in obtaining sufficient high-quality RNA directly from sites of infection, gene expression studies of A. fumigatus in live animal models are limited. Thus, we selected for our analysis a published microarray dataset26 from a murine lung model which provided one of the first transcriptional snapshots of A. fumigatus during initiation of mammalian infection.To place the identified secretome and cell membrane proteins of 26, A. fumigatus genes significantly upregulated over 2-fold during pathogenesis when compared to control conditions were selected for further analysis, totaling, 1264 proteins (12.9%) of the proteome. Within the predicted A. fumigatus secretome, 121 out of 662 proteins (18.3%) were upregulated over 2-fold during pathogenesis, indicating that a larger fraction of secretome is employed during pathogenesis. Moreover, 113 out of the 121 upregulated proteins in the A. fumigatus secretome are secreted via classical pathway and their GO analysis revealed involvement in carbohydrate metabolic processes and regulation of host immune response, while the remaining 8 are secreted via a non-classical pathway and their GO analysis revealed involvement in response to mycotoxin, synthesis of mycotoxin and gliotoxin, and response to oxidative stress apart from carbohydrate metabolic processes. Within the cell membrane proteins of A. fumigatus, 151 out of 1129 proteins (13.3%) were upregulated during pathogenesis, and GO analysis revealed that many of them are involved in transmembrane transport processes. Moreover, 45 SSPs in the A. fumigatus secretome, of which 21 have effector-like features, were upregulated over 2-fold during pathogenesis. For example, Afu3g14940 a predicted peptidase inhibitor I78 family protein which has a known conserved effector motif ([KRHQSA][DENQ]EL) and Afu1g01210 with the adhesion fasciclin domain, are among the 21 effector-like SSPs with over 2-fold upregulation during pathogenesis.In this datasetAspergilli to switch into an opportunistic pathogen may be derived from conserved proteins. Conversely, species-specific proteins may explain the observed differences in virulence between Aspergilli. To determine the unique and conserved proteins across Aspergillus species, OrthoMCL100 was used to identify orthologs between the proteins of nine Aspergillus species. As A. ustus has an incomplete genome, it was omitted from this comparative analysis. Figure Aspergillus species. In comparison to the cell membrane proteins, the secretomes of Aspergillus species have a larger set of unique proteins and a smaller set of conserved proteins across the nine Aspergillus species. Interestingly, 3.6% of the secretome of A. fumigatus is unique, while 27.3% and 26.9%, respectively, of the secretomes of A. niger and A. glaucus is unique, when compared across the nine Aspergillus species. Thus, the secretome of A. fumigatus has among the smallest fraction of unique proteins which is in contrast to its position as the most prevalent agent of aspergillosis. The unique and conserved proteins identified here across the secretomes of Aspergillus species could lead to a better understanding of the important players for virulence and provide potential targets for the development of broad spectrum antifungals.The ability of multiple Aspergillus proteins without a close homolog in the human proteome. We find that more than 70% of the secretomes in Aspergillus species is unique, without a homolog in humans 24 value of proteins was calculated using two methods, Kolaskar-Tongaonkar106 and BepiPred 2.0107 (Methods). The lower the AAR value the more antigenic the protein24. Interestingly, in each Aspergillus species, the average AAR of the secretome is always significantly lower than cell membrane proteins . Experimentally identified protein structure was available only for Afu6g09740, and it was downloaded from the protein data bank111 (PDB). Since a good-quality structure model was also unavailable for Afu2g15160, it was omitted from later analysis. For the remaining five secreted proteins and one cell membrane protein, structure models were obtained from ModBase112 and SWISS-MODEL repository113 and were subsequently checked for their quality (Methods). Thereafter, the compiled protein structures were compared to their DrugBank counterparts for structural similarity. Four secreted proteins, Afu5g14380, Afu8g01670, Afu1g09900 and Afu6g09740, had significant structural similarity with TM scores greater than 0.8 and root-mean-square deviation (RMSD) values lower than 3Å2 . The proteomes of A. flavus NRRL3357 and A. ustus 3.3904 were retrieved from FungiDB120 (http://fungidb.org/fungidb/) and Ensembl Genomes121 (http://ensemblgenomes.org/), respectively. Note that the A. ustus 3.3904 genome sequence is still incomplete, and thus, its incomplete proteome was used here for predictions.The proteomes of 73 which have characterized the secretome of 6 Aspergillus species studied here (Supplementary Table Aspergillus species were mapped to standard identifiers in the reference sequence using OrthoMCL100 (http://orthomcl.org/orthomcl/) with inflation option set at 1.5. In addition to high-throughput studies, experimentally verified secreted or cell membrane proteins in Aspergillus species were retrieved from UniProt19 (http://www.uniprot.org/) by filtering proteins with subcellular localization as extracellular or cell membrane, respectively, with evidence code of ECO:0000269. Note that ECO:0000269 corresponds to manually curated information with published experimental evidence. Similarly, experimentally verified secreted proteins in all other fungal species were retrieved from UniProt19 by filtering proteins with subcellular localization as extracellular with evidence code of ECO:0000269. OrthoMCL100 with inflation option set at 1.5 was used to identify orthologs of experimentally verified secreted proteins in other fungi in Aspergillus species.We performed an extensive literature search to compile 46 published high-throughput experimental studies122  and Phobius123 (http://phobius.sbc.su.se/). The presence of GPI anchor was predicted using PredGPI124 (http://gpcr.biocomp.unibo.it/predgpi/) and big-PI125 (http://mendel.imp.ac.at/gpi/fungi_server.html). The presence of TM domain was predicted using TMHMM 2.0126 (http://www.cbs.dtu.dk/services/TMHMM/) and Phobius123. Note that TM domain predictions by TMHMM within the last 70 amino acid residues of the N-terminus of a protein sequence were not considered as the tool can sometimes predict signal peptides as false-positive TM domains. The presence of ER retention signal was predicted using PS SCAN127 with PROSITE (https://prosite.expasy.org/) pattern PS00014128. The subcellular localization of proteins was predicted using WoLF PSORT 0.2129 (https://psort.hgc.jp/), TargetP 1.1130 (http://www.cbs.dtu.dk/services/TargetP/) and ProtComp 9131 (http://www.softberry.com/berry.phtml?topic=protcomp&group=help&subgroup=proloc). Importantly, UniProt19 annotations with published experimental evidence (ECO:0000269) for the protein-based features were also retrieved for Aspergillus proteins. UniProt identifiers for Aspergillus proteins were mapped to reference sequence identifiers using pre-existing maps from AspGD119, UniProt19, and in-house python scripts.The following computational tools were employed to predict protein-based features in our secretome prediction pipeline. The presence of a signal peptide in the N-terminus was predicted using SignalP 4.1Aspergillus species.While combining information from different sources to decide on the presence of signal peptide or GPI anchor or TM domain, a decision is made based on tool predictions using an inclusive rule if UniProt annotation is not available, else decision is made only on UniProt annotation by overriding tool predictions. While combining information from different sources to decide on subcellular localization, the decision is made based on tool predictions using a majority rule if UniProt annotation is not available, else decision is made only on UniProt annotation by overriding tool predictions. Thus, our pipeline gives precedence to published experimental evidence over bioinformatic-based predictions. Supplementary Table et al.132 have evaluated five bioinformatic tools employed here for predictions in fungi, and have found the sensitivity and specificity of SignalP 4.1122 to be 94.9% and 99.7%, respectively, of Phobius123 to be 95.4% and 98.7%, respectively, of WoLF PSORT 0.2129 to be 88% and 99.8%, respectively, of TargetP 1.1130 to be 95.1% and 98.3%, respectively, and of ProtComp 9131 to be 63.3% and 97.2%, respectively. In addition, Pierleoni et al.124 found the sensitivity and specificity of PredGPI124 to be 77.2% and 99.9%, respectively, and of big-PI125 to be 54.5% and 99.7%, respectively. Note that sensitivity measures the fraction of true positive predictions while specificity measures the fraction of true negative predictions.Note that the computational tools used in our secretome prediction pipeline have been widely-used in the scientific literature. Sperschneider Aspergillus species were annotated with information on protein family from Pfam133 database (http://pfam.xfam.org/) and carbohydrate-binding modules from CAZy134 (http://www.cazy.org/)134 and dbCAN v5.0135 (http://csbl.bmb.uga.edu/dbCAN/) databases using hmmpress and hmmscan utilities in HMMER3 (http://hmmer.org/) with profile-specific GA (gathering) thresholds. Furthermore, the predicted secretomes in Aspergillus species were annotated with protein family and domain information from TIGRFAM136 (http://www.jcvi.org/cgi-bin/tigrfams/index.cgi), SFLD137 (http://sfld.rbvi.ucsf.edu/django/), SMART138 (http://smart.embl-heidelberg.de/), CDD139(https://www.ncbi.nlm.nih.gov/cdd), PROSITE128, SUPERFAMILY140 (http://supfam.org/SUPERFAMILY/), PRINTS141 (http://130.88.97.239/PRINTS/index.php), PANTHER142 (http://www.pantherdb.org/), COILS143  and MobiDB-lite144 (http://protein.bio.unipd.it/mobidblite/) accessed through InterPro version 64.0 (https://www.ebi.ac.uk/interpro/) using InterProScan 5146. In addition, the secreted proteins in Aspergillus species were annotated with GO terms using FungiFun2147 (https://elbe.hki-jena.de/fungifun/fungifun.php). Supplementary Table Aspergillus species.The predicted secretomes in Aspergillus species, SSPs were defined as those with sequence length less than or equal to 300 amino acid residues148. The SSPs were then evaluated for effector-like properties based on their EffectorP97 (http://effectorp.csiro.au/) predictions or cysteine-richness. For identifying effector-like SSPs, cysteine-rich sequences are those which contain at least 4 cysteine residues and have greater than 5% of their total amino acid residues as cysteines149. The predicted SSPs in the secretomes of Aspergillus species were also scanned for known fungal or oomycetes effector motifs96  using the FIMO package in MEME program suite150 with E-value cut-off of ≤1e-4 (Supplementary Table In the predicted secretomes of Aspergillus proteins were predicted using Kolaskar-Tongaonkar106 method implemented in EMBOSS package151 with a threshold of 1.0 and BepiPred 2.0107 (http://www.cbs.dtu.dk/services/BepiPred/index.php) with the default threshold of 0.5. Note that only predicted antigenic regions with a length ≥6 amino acids were accounted in the later analysis. For each protein in Aspergillus species, the AAR value was computed following the method proposed by Gomez et al.24. The AAR value of a given protein is computed by dividing the length of its amino acid sequence by the number of predicted antigenic regions. The average AAR value was computed for the set of secreted and cell membrane proteins, respectively, in each of the Aspergillus species considered here . The microarray dataset from infected murine lungs26 gave a reliable signal for 9121 genes in A. fumigatus Af293 which covers more than 90% of the secretome and 88% of the cell membrane proteins in the fungus. Gene expression data26 for A. fumigatus Af293 was next analyzed within the context of the predicted secreted and cell membrane proteins to identify differentially expressed proteins with over 2-fold upregulation in each fraction during pathogenesis for further analysis. Thereafter, secreted and cell membrane proteins in A. fumigatus Af293 with over 2-fold upregulation during pathogenesis and sequence similarity with known drug target proteins from DrugBank25 database were identified using BLASTP (https://blast.ncbi.nlm.nih.gov/Blast.cgi) with E-value cut-off of ≤1e-5 (as specified by DrugBank database).A microarray dataset for A. fumigatus proteins in secretome and cell membrane fraction with over 2-fold upregulation during pathogenesis and with sequence similarity with known drug target proteins from DrugBank25, the structural similarity between filtered proteins and their sequence-based homologs among known drug targets was assessed. Structure of proteins from x-ray diffraction experiments were retrieved from PDB111 (https://www.rcsb.org/pdb/home/home.do). However, experimentally identified protein structures are not available for most of the filtered set of secreted and cell membrane proteins in A. fumigatus Af293, and in such cases, the protein structure models from ModBase112 (https://modbase.compbio.ucsf.edu/modbase-cgi/index.cgi) and SWISS-MODEL repository113 (https://swissmodel.expasy.org/repository) were used to evaluate structural similarity. Note that only protein structure models which have more than 40% sequence identity with the template used to generate the corresponding structure models were selected for subsequent analysis. Further the selected protein structure models were checked for their quality using Verify3D152 (http://servicesn.mbi.ucla.edu/Verify3D/) and PROCHECK153 (http://servicesn.mbi.ucla.edu/PROCHECK/)  and root-mean-square deviation (RMSD) value between locally aligned atoms computed using PyMOL . Filtered proteins in A. fumigatus Af293 with significant structural similarity to known drug target proteins were subsequently tested for binding by the respective drug using molecular protein-ligand docking. Molecular docking is a widely used computational approach in structure-based drug design which predicts with substantial accuracy the plausible binding poses and binding affinity of a ligand to the binding site in the target protein of interest155. Here we employ AutoDock Vina to perform rigid docking wherein the target protein of interest is kept rigid while the ligand is flexible. The flexibility of the ligand is determined by torsion tree built while the ligand is prepared for molecular docking using the prepare_ligand4.py script from AutoDockTools156.To further ascertain the similarity between the filtered set of A. fumigatus Af293 were predicted using metaPocket 2.0117 (http://projects.biotec.tu-dresden.de/metapocket/). Secondly, for docking drugs to the predicted protein pockets, both protein and drug molecule were prepared by adding explicit hydrogen atoms and cleaned up using python scripts from AutoDockTools156, prepare_receptor4.py and prepare_ligand4.py respectively, with the default options. Finally, AutoDock Vina118 (http://vina.scripps.edu/) was used for docking the drug against the predicted pockets in proteins with the option for exhaustiveness set at 24 to obtain consistent results157. The binding affinity results obtained by docking drugs to the protein pockets from AutoDock Vina118 were tabulated (Table Firstly, three ligand binding pockets for each filtered protein in All data generated or analyzed during this study is included in this article and its supplementary information.Supplementary TextSupplementary Table S1Supplementary Table S2Supplementary Table S3Supplementary Table S4Supplementary Table S5Supplementary Table S6Supplementary Table S7Supplementary Table S8


Arcobacter species have been associated with human disease, and based on current knowledge, these Gram negative bacteria are considered as potential food and waterborne zoonotic pathogens. At present, only the genome of the species Arcobacter butzleri has been analysed, and still little is known about their physiology and genetics. The species Arcobacter thereius has first been isolated from tissue of aborted piglets, duck and pig faeces, and recently from stool of human patients with enteritis. In the present study, the complete genome and analysis of the A. thereius type strain LMG24486T, as well as the comparative genome analysis with 8 other A. thereius strains are presented. Genome analysis revealed metabolic pathways for the utilization of amino acids, which represent the main source of energy, together with the presence of genes encoding for respiration-associated and chemotaxis proteins. Comparative genome analysis with the A. butzleri type strain RM4018 revealed a large correlation, though also unique features. Furthermore, in silico DDH and ANI based analysis of the nine A. thereius strains disclosed clustering into two closely related genotypes. No discriminatory differences in genome content nor phenotypic behaviour were detected, though recently the species Arcobacter porcinus was proposed to encompass part of the formerly identified Arcobacter thereius strains. The report of the presence of virulence associated genes in A. thereius, the presence of antibiotic resistance genes, verified by in vitro susceptibility testing, as well as other pathogenic related relevant features, support the classification of A. thereius as an emerging pathogen.Four  Arcobacter was created in 1991 as a second genus within the family Campylobacteraceae to include bacteria which differ from the closely related Campylobacter species by their aerotolerance and ability to grow at temperatures below 30° [Arcobacter ebronensis, Arcobacter aquimarinus, Arcobacter faecis, Arcobacter lanthierii and Arcobacter pacificus [Arcobacter butzleri, Arcobacter cryaerophilus and Arcobacter skirrowii are incriminated as food and waterborne pathogens for humans [Arcobacter infection in humans seems to cause enteritis and sometimes bacteraemia, with clinical signs similar to those of campylobacteriosis, but with a higher frequency of persistent watery diarrhoea [The genus acificus –5. Theseacificus . In partr humans . In contiarrhoea –10. Contiarrhoea .Arcobacter thereius was isolated from the stool of two hospitalized patients with symptoms of enteritis [Arcobacter, and were later characterized by Houf et.al. [A. thereius. Though the species has been isolated relatively frequently from the faeces of healthy pigs, still almost nothing is known about its behaviour [in vitro culture has troubled many researchers since. Furthermore, in contrast to the other mammal-associated species, A. thereius isolates are hard to type at strain level, in fact pulsed-field gel electrophoresis (PFGE), amplified fragment-length polymorphism (AFLP) and enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) showed to be not discriminatory for A. thereius [Arcobacter species seem to be lacking in A. thereius [A. thereius is in vitro non-fermenting, nor oxidising carbohydrates, as for the other members of the family Campylobacteraceae, genome analysis represents an ultimate approach to elucidate this species full potential.Recently, the species nteritis . This spnteritis ,12. Somef et.al.  as repreehaviour . Houf etehaviour , and thethereius , and thethereius . Since AA. thereius type strain LMG24486T, as well as the sequences of eight other strains originating from different matrices. The genome of the type strain is analysed with a focus on metabolic pathways, virulence factors, antibiotic resistance genes and adaptation to the environment. The genome is then compared with the only other yet available full genome of a human associated species: A. butzleri type strain RM4018 (= LMG10828T) [A. thereius is assessed in order to comprehend the previously reported strain high heterogeneity [The present study presents the full genome analysis of the G10828T) . Subsequogeneity .A. thereius strains were included   according to the manufacturer’s instructions, followed by evaluation of the DNA integrity by size separation of 10 μL of each DNA sample by agarose gel electrophoresis (1.5%). An Illumina paired-end library was generated from 1 μg of genomic DNA for each of the nine strains. The DNA was fragmented to 800 bp using Covaris S2 sonication  and a sequence library was made for each sample using the NEBNext Ultra DNA Library Prep Kit . During the library preparation, a size selection was performed before and after the enrichment PCR using an Invitrogen 1% E-gel  to select for fragments between 850 and 1200 bp. The libraries were equimolarly pooled and were sequenced on an Illumina MiSeq sequencer , generating 2x300 bp paired-end reads for each sequenced fragment. Base calling and primary quality assessments were performed using Illumina’s Basespace genomics cloud computing environment. Using CLC bio Genomics Workbench version 8.0, the reads were trimmed based on quality scores, library prep adapters were removed, and resulting sequences shorter than 100 nt were discarded. The quality trimmed reads were used to perform a sion 2.0  using alsion 2.0  and the A. thereius LMG24486T was sequenced using PacBio technology [Additionally, the genome of the type strain, chnology , followichnology . The gapA. thereius strains were annotated using a local installation of the Prokka platform v1.11 [http://www.vicbioinformatics.com/software.barrnap.shtml), Infernal v1.1.1, and SignalP v4.1 [−6 and 50% of identity) using all available bacterial proteins in the UniProt database 2015_04 [A. thereius strains were also annotated by the Rapid Annotation using Subsystem Technology (RAST) [−30; virulence factors were identified by BLASTn analysis using the human pathogenic bacteria virulence factor database  ,31 platfy (RAST) . Plasmidy (RAST)  and the y (RAST)  plasmid y (RAST) . Antibioy (RAST) ) using dy (RAST) ) by BLASse VFDB;–40) with withA. tA. thereius LMG 24486T, clustered regularly insterspaced short palindromic repeat (CRISPR) regions were identified through CRISPRFinder [For PRFinder  and a gePRFinder . The oriPRFinder . GenomicPRFinder .A. thereius strains that have been sequenced in this study, all other Arcobacter genomes available from NCBI and Campylobacter jejuni NCTC11168 as outgroup. Orthologues were predicted using the OrthoMCL software v1.4 [−6; identity cut-off 50%; reciprocal hit length cut-off: 50%. Further, in order to include only orthologs without evidence of recombination or gene conversion, a stringent filtering step was added. Briefly, the probability of recombination for each orthologous group was calculated using the PhiPack software [A maximum likelihood phylogenetic tree of a concatenated alignment of 239 single-copy orthologs was reconstructed. One-to-one ortholog genes were obtained by comparing a genome database comprising the are v1.4  using thsoftware , and dissoftware , trimmedsoftware  to removA. thereius LMG24486T and A. butzleri RM4018 and within the A. thereius strains were performed using the OrthoMCL software v1.4 [−6; identity cut-off 50%; reciprocal hit length cut-off: 50%. EDGAR [A. thereius species and to construct a synteny plot between A. thereius LMG24486T and A. butzleri RM4018 following default parameters.Comparative analysis between are v1.4  using th%. EDGAR  comparatA. thereius strains. Further, amino acid sequences of Campylobacter and/or Helicobacter species were included in the analysis in the cases where an appropriate amino acid sequence could be retrieved from the NCBI data repository. Each dendrogram was made using the Neighbor-Joining method. Bootstrap values of >50% generated from 100 replicates are shown next to the branches. The evolutionary distance were computed using the p-distance method and are in the units of the number of amino acid differences per site.A phylogenetic analysis using MEGA 6  was perfA. thereius strains using JSpecies Web Server (JSpace WS) with default parameters [A. butzleri RM4018 and A. cibarius LMG21996 were used as an outgroup. In addition, in silico DNA-DNA hybridisation was calculated for all A. thereius strains using the genome-to-genome distance calculator following as alignment method the recommended GGDC 2 BLAST+ [A. butzleri RM4018 and A. cibarius LMG21996 were included as comparison.The Average Nucleotide Identity based on BLAST+ calculation (ANIb) was performed for the nine rameters . The gen2 BLAST+ . GenomesA. thereius strains using the Arcobacter pubMLST database (http://pubmlst.org/Arcobacter/) and according to the protocol of Miller et al. [aspA, atpA, glnA, gltA, pmg and tkt. The gene glyA was however not included as it was absent in A. thereius strains 213, 216, 440, 452 and 11743–4. A dendrogram was constructed with MEGA 6 using the Neighbor-joining method. Bootstrap values of >50% generated from 100 replicates are shown next to the branches. The evolutionary distance were computed using the p-distance method and are in units of the number of bases differences per site.MLST profiles were obtained for the nine r et al. . The nucA. thereius has been generate with ProgressiveMauve [A. thereius strains, using the rps-BLAST program against the NCBI Conserved Domain Database (CDD) and with an e-value cut-off of 1.0 × 10−3. Only the top hits were retained and the genes belonging to each COG categories were counted, and the distribution were compared using a Pearson chi-squared test using the IBM SPSS statistic program v23.Alignment of the nine genomes of iveMauve  using deThe annotated genome sequences were deposited in the DDBJ/EMBL/GenBank database. The sequencing projects and accession numbers are listed in A. thereius strains included in the present study were tested for antimicrobial susceptibility, growth and motility capacity. The latter two parameters were tested under different temperature and atmospheric conditions as shown in Arcobacter broth, 3 g/L Agar Technical n° 3 (Oxoid)).The nine Campylobacter jejuni ATCC33560 and A. butzleri RM4018 were included. Due to the fastidious growth, plates were read after 48 hours. EUCAST breakpoints were used for erythromycin, ciprofloxacin and tetracycline (Campylobacter coli), ampicillin, (non-species-related PK/PD), gentamicin and chloramphenicol (Enterobacteriaceae) and spectinomycin (Neisseria gonorrhoeae) [Based on the genomic findings, antimicrobial susceptibility to eight antibiotics: erythromycin, ciprofloxacin, ampicillin, tetracycline, gentamicin, streptomycin, chloramphenicol and spectinomycin was determined by the gradient strip diffusion method as previously described by Van den Abeele et al. . In brierrhoeae) . For strrrhoeae) .A. thereius LMG24486T, for a total sequence length of 1,909,306 bp with a GC content of 26.93% , 4 , and 27  spacer sequences, respectively , this suggests that the Embdem Meyerhof pathway operates towards gluconeogenesis. This functionality has also been described in the genome of the asaccharolytic Campylobacter jejuni [A. thereius, a complete pathway for the degradation of pyruvate, which can be formed through amino acid degradation, is present (see below). A gene cluster encoding the pyruvate dehydrogenase complex allowing formation of acetyl-CoA is present . The latter can be channelled into the tricarboxylic acid (TCA) cycle or it can be dehydrogenated to acetate by the aldehyde dehydrogenase (AA347_00799). Further, oxaloacetate can be formed from pyruvate by carboxylation by pyruvate carboxylase (AA347_01516). Oxaloacetate can be channelled in the TCA cycle if the quantity of energy is low or it can be converted to glucose if the energy charge is high [r jejuni . All gen is high .A. butzleri RM4018 [Helicobacter pylori [Campylobacter coli RM2228, C. upsaliensis RM3195 and C. lari RM2100 [C. jejuni RM1221 contains all genes encoding the TCA [All genes encoding the enzymes of the TCA were detected, except for the one encoding the succinyl-CoA synthetase. This conformation has also been reported previously in i RM4018  and other pylori , Campyloi RM2100 . In cont the TCA ,65.A. thereius LMG24486T is unable to use carbohydrates as an energy source, alternative metabolic pathways are needed. Amino acids and their degradation products seems to be good candidates since they are present in the habitat of arcobacters, including the animal and human gut. The genome sequence of A. thereius LMG24486T harbours genes encoding different amino acid transporters, such as symporters specific for glutamate and aspartate/sodium (AA347_00247), an arginine/ornithine antiporter , and a methionine ABC transporter (AA347_00336). This methionine transporter is absent in A. butzleri RM4018 genome, and it could be specific for A. thereius. Furthermore, nine genes were identified encoding several ABC transporter ATP-binding proteins  that could not be further specified based on genome annotation. Nineteen transporters that are present in the genome of A. butzleri RM4018 were absent in all nine A. thereius strains sequenced. These comprised 17 ABC transporter ATP-binding proteins, one sodium:alanine symporter and a sodium:hydrogen antiporter. These additional transporters might allow A. butzleri to use more extracellular substrates compared to A. thereius. For A. thereius LMG24486T, four genes encoding aminopeptidases were retrieved, encompassing a leucine aminopeptidase , a methionine aminopeptidase , one generic aminopeptidase (AA347_00091) and an oligopeptidase A  with a broad specificity.Since Arcobacter thereius LMG24486T has a limited capacity to catabolize amino acids, as only genes involved in aspartate, L-glutamate, and serine catabolism were found in its genome , that allows the production of fumarate and ammonia. Aspartase is a central enzyme in the amino acid catabolism in C. jejuni, and seems to have an important effect on growth in complex media, as a C. jejuni mutant lacking a functional aspartase fails to utilize aspartate, glutamate, glutamine and proline [A gene encoding aspartate aminotransferase was found in the genome of  proline .A. thereius LMG24486T, the gene encoding for CmeR is not present, although an anaerobic regulatory protein (AA347_00524) and a transcriptional regulator (AA347_00794) are present, which function typically in response to environmental change. Therefore, they may be involved in modulation of gene expression by the presence of oxygen. Other reactions involving aspartate allow the production of homoserine  is present together with the aldehyde dehydrogenase complex (AA347_00801), allowing oxidation of NADH by complex I and reduction of NADP+ by an aldehyde dehydrogenase.observed . This ses tested . Remarka1 complex , a cytochrome c oxidase , and a complete cluster of genes encoding a F0/F1 ATPase  are present, enabling production of ATP from ADP or to generate a transmembrane ion gradient. Three genes encoding a cytochrome c peroxidase were found  which may be important in the detoxification of hydrogen peroxide in the periplasm [A cytochrome bcA. thereius LMG 24486T, electrons can be fed to the quinone oxidoreductase by the membrane associated FeNi hydrogenase encoded by hydABCD (AA347_00601—AA347_00604), a malate oxidoreductase (AA347_00064) and a variety of other primary substrate dehydrogenases, for example glutamate dehydrogenase, isocitrate dehydrogenase or pyruvate dehydrogenase. The groups of genes hyaABCD and hupLS encoding for another membrane associate FeNi hydrogenase and for an uptake hydrogenase respectively, result as unique gene for A. butzleri RM4018; although the gene cluster hypABCDEF (AA347_00606 –AA347_00615) is present in both of the species.In nap operon , encoding for proteins involved in nitrate reduction, provides the possibility for anaerobic growth using fumarate and nitrate as electron acceptors instead of oxygen [A. thereius to reduce nitrate has already been reported by Houf et al. [ars . The system is regulated by the repressor protein ArsR, while ArsB is the determinant of the membrane efflux protein that confers resistance by pumping arsenic from the cell, and ArsC is an arsenate reductase. The ars operon has already been described as part of the E. coli chromosome [Staphylococcus aureus [In contrast to the results of the growth tests in anaerobic condition, both in the present study , as wellf oxygen . The abif et al.  Analysisromosome  and on as aureus . The oxys aureus , providiCampylobacter jejuni has been described as only able to express LOS structure and no LPS [A. thereius LMG24486T where a lipooligosaccharide (LOS) biosynthesis gene cluster (AA347_01016 –AA347_01027) was found. The structure of this gene cluster resembles the organization of A. butzleri RM4018 and is present and conserved in all A. thereius strains sequenced in the present study. The genes waaC and waaF  encode a heptosyltransferase I, which is responsible for the linkage between the L-glycero-D-manno-heptose (HEP) and 3-deoxy-D-manno-octulsonic (KDO) and, for a heptosyltransferase II which catalyses the transfer of a second HEP to HEP I [C. jejuni that a mutation in waaF increases the susceptibility to different hydrophobic antibiotics, such as novobiocin, and a mutation in waaC results in an incomplete LOS [A. thereius stains sequenced in this study, resemble the organization of C. jejuni [kps genes involved in the capsular production are not present in A. thereius genomes, as well as, genes encoding for the O-antigen.Lipopolysaccharide (LPS) and lipooligosaccharide (LOS) are important constituents of the outer membrane of bacteria and they can have a role as virulence factor or in antibiotic resistance mechanism . Campylod no LPS . The samto HEP I ,75. It hlete LOS ,76,77; a. jejuni . The kpsArcobacter thereius possesses a polar flagellum [flhA, flhB, flhF, flgL, flgC, flgE, flgB, flgG, flgK, flgH, flgI, fliI, fliR, fliK, fliE, fliN, fliG, fliF, fliS, fliD, fliQ, fliM, flaB) were found in A. thereius LMG24486T, which were organized into three gene clusters . The same gene organization is also found in the other A. thereius sequenced, suggesting that this gene region is conserved within this species.lagellum  and all Arcobacter thereius LMG24486T contains a complete pseudaminic acid biosynthesis pathway , which has been reported to constitute a major virulence factor in C. jejuni [C. jejuni pseH (AA347_00584), which may be involved in flagellar glycosylation. This pathway was only partially present in A. butzleri RM4018, as both the pseG  and psebutzleri .A. thereius LMG22486T harbours 18 methyl-accepting chemotaxis proteins, nine two-component response regulators, and eight sensor histidine kinases. During the comparison, in the genome of A. butzleri RM4018 36 sensor histidine kinase, 41 response regulators have been found as unique features of A. butzleri RM4018 [A. thereius might be less reactive to the surrounding changes, although a full set of genes encoding for chemotaxis proteins were present, including CheARDB (AA347_01490 –AA347_01494), CheV (AA347_01730), CheY (AA347_01490). These proteins play a role in signal transmission from the receptor to the flagellum to regulate or change the bacterial movements.Interaction between bacteria and the environment is a fundamental aspect for the survival and adaptation of the microorganism. In fact, bacteria have a lot of different mechanisms which enable them to respond to environmental changes. i RM4018 . This inin silico search for genes putatively involved in antibiotic resistance revealed the presence of several genes within the A. thereius LMG24486T genome that could contribute to antibiotic resistance. For instance, a gene cluster encoding a CmeABC efflux pump was found (AA347_00510 –AA347_00512), which is associated with efflux of various antibiotics in C. jejuni [A. thereius strains sequenced , described to be involved in penicillin tolerance in Staphyloccocus aureus [Arcobacter cibarius towards ciprofloxacin [C. coli, A. butzleri and A. cryaerophilus [Arcobacter thereius LMG24486T possesses no specific mechanisms towards macrolide resistance, such as the presence of a major outer membrane porin (MOMP) or macrolide resistance-associated mutations. The latter include specific point mutations at positions 2074 and 2075 in the 23S rRNA gene, which are involved in erythromycin resistance in Campylobacter species [A. thereius 213, A. thereius 216, A. thereius 440, A. thereius 11743–4, and A. thereius DU22 , which was also observed in the A. thereius strains 440 and DU22.Further, evidence for chloramphenicol resistance was found, as a chloramphenicol O-acetyltransferase was present  was found in the genomes of A. thereius strains 213 (AAW29_01543), 216 (AAW30_00055), DU19 (AAX30_01180), and 11743–4 (AAX28_00760), which is involved in resistance towards streptomycin/spectinomycin in Pasteurella multocida [A. thereius 11743–4. This efflux pump, usually present in Gram-negative bacteria, confers resistance to tetracycline by exporting it out of the cell thereby reducing the intracellular concentration [Campylobacteriaceae.Several other antibiotic resistant related genes were found in the other ultocida . Furtherntration . This isA. thereius strains are shown in A. thereius 11743–4 carried the TetA protein. Interesting, the ciprofloxacin susceptibility results showed that only A. thereius 11743–4, carrying the DNA GyraseA point mutation Thr85Ile, is resistant against ciprofloxacin while, A. thereius DU22, 440 and 452, that carry the Thr85Ser point mutation remain sensitive towards ciprofloxacin. Streptomycin and chloramphenicol show MICs around breakpoint value, while 80–100% of the strains are resistant to spectinomycin. A. thereius 11743–4 presented with a divergent resistance pattern in comparison to the other strains, pointing towards acquired multiresistance, which has to be further investigated.The susceptibility results obtained by gradient strip diffusion for the nine A. thereius LMG24486T, among which the fibronectin binding protein Cj1349 (AA347_00304), the invasion protein CiaB (AA347_00973), the virulence factor MviN (AA347_01129), the phospholipase PldA (AA347_01541), the hemolysin TlyA (AA347_01277), and the enterobactin receptor IrgA (AA347_01908 and AA347_01909) [A. butzleri RM4018, which encodes a putative siderophore esterase IroE adjacent to a single copy of the IrgA gene [A. butzleri, A. cryaerophilus and A. skirrowii such as HecAB [A. thereius LMG24486T. The virulence factors present in the other eight A. thereius strains sequenced in this study were identical to the type strain . The putative virulence genes cj1349, ciaB, mviN, pldA, tlyA and irgA were conserved within the species A. thereius  ,88. The rgA gene . Next toas HecAB ,88, weree strain , except thereius . Althoug species ,88, this strains , which mA. thereius strains sequenced in the present study, displays a similar genomic architecture, although a few structural rearrangements were found  functional categories across genomes  and a UDP-N-acetyl-D-glucosamine 6-dehydrogenase (DU22: AAX29_01729 and DU19: AAX30_01828). The seven strains isolated from pigs contained two genes which were absent in all strains originating from duck cloaca, both encoding hypothetical proteins which are adjacent to each other. The genome of A.thereius LMG24486T harboured 394 accessory genes  were detected in the type strain. The occurrence of two unique additional hydrogenase might enable this strain to obtain energy in microaerobic environments. Because they are not found in related, pathogenic strains, these genes, unique to A. thereius LMG24486T, might confer an advantage in some aspects of A. thereius’ behaviour, but it is unlikely that they play a role in pathogenicity.Comparison of the genomes from all re found , S1 Fig.structed . The int genomes . This rery genes , i.e., gT and A. thereius LMG24487 exhibited a mean DNA-DNA relatedness value of 79% [in silico DDH as well as Average Nucleotide Identity (ANI) analysis suggest a separation into two species, each inclosing one of the strains. The in silico DDH values and the ANI data for the nine A. thereius strains are shown in A. thereius strains examined. However, strains of both clusters have been included in the previous characterization of the species [isDDH data analysis on the strains included in the present study proposed the classification into a new species, Arcobacter porcinus, including the majority of strains previously identified as A. thereius [Arcobacter genus.Though in the initial characterization of the species, DNA-DNA hybridization of strains LMG24486e of 79% , in sili species , with ovthereius . FurtherA. thereius genomes revealed the consistent presence of the loci aspA, atpA, glnA, gltA, pmg and tkt. In contrast glyA locus was lacking in A. thereius 213, 216, 440, 452 and 11743–4, while two copies were present in the genomes of A. thereius DU19, DU22, LMG24487 and LMG24486T  and eight draft genome sequences, the A. thereius core genome showed to have a stable size with the largest proportion of the genes part of the core-genome, while the pan-genome is predicted to be open.A. thereius genomes sequenced, A. thereius 440 recovered from pig faeces and LMG24487 isolated from piglet aborted foetus, carried the gene encoding for the zonula occludens toxin (ZOT) . The ZOT is known to work on the intracellular tight junction and allows pathogenic bacteria, like Vibrio cholera or Neisseria meningitidis, to increase tissue permeability. Recently, this gene has been detected also in the genome of Campylobacter concisus 13826 [jejuni Campylobacter found in samples of patients with gastrointestinal disorders and now suggested as a potential pathogen [C. concisus, allowing the bacteria to attach and invade host cells through a paracellular mechanism. It was not detect in other Arcobacter species, and further research is needed, taking into account current genome knowledge, to elucidate which genes participate in cell adhesion and invasion.Of the eight us 13826 , a non-jpathogen . Accordipathogen , the previrB4, virB6, virB9, virB10, virB11; AAW29_01609; AAW29_01614; AAW29_01615; AAW29_01616; AAW29_001618) was present in the genome of A. thereius 213 as a singleton. The type IV secretion contributes in a lot of transport like, exchange of genetic material between bacteria, movement of plasmid and injection of virulence factor in the host cell. The subgroup VirB is specific for the T-DNA transfer and is built with 11 different proteins (VirB1-VirB11), but a conserved core of five proteins is always present  [A. butzleri and it could play an important role in gene transfer within Arcobacter [A gene cluster, coding for type IV secretion system ( VirB11) . This seArcobacter butzleri is the most representative human related Arcobacter and, after the announcement of its genome, its role as potential human pathogen has been confirmed in several reported cases. The genomes of A. thereius LMG24486T and A. butzleri RM4018 show little synteny  genes, A. thereius LMG24486T contained 393 singletons , and A. butzleri RM4018 contained 688 singletons . Besides the differences and similarities already mentioned above, among the singletons of A. butzleri RM4018 a complete cluster of genes encoding for sulphur uptake and assimilation was present [sox gene cluster, necessary for the sulphur oxidation, were also present in A. thereius LMG24486T , this strain could be able to oxidise sulphite to sulphate. synteny , which sbutzleri . Indeed, present . HoweverthiDEHGFS, AA347_00322 –AA347_00327), enabling thiamine autotrophy in A. thereius LMG24486T. Production of thiamine is an important mechanism because it is an essential cofactor of different metabolic enzymes and it was not present in other Arcobacter [A. thereius. However, it remains to be determined if this is going to be maintained when genomes of other Arcobacter species will be available.Further, a thiamine biosynthetic gene cluster was found .Another interesting difference present as a singleton in cleavage . Type I cleavage . For theA. butzleri RM4018, the genome sequence of LMG24486T contains the genes of the ectoine biosynthesis pathway . Ectoine is a compatible solute, important for its function as osmoprotector; it helps microorganism to survive extreme osmotic stress and temperature stress. As A. thereius LMG 24486T contains also an aspartate kinase (AA347_01497), and an aspartate semialdehyde dehydrogenase (AA347_01037), ectoine may be produced out of aspartate. Further, all six genes involved in urea degradation (ureABCDEFD) in A. butzleri RM4018 [A. thereius strains.In contrast to i RM4018  were notA. thereius strains, including the type strain, confirmed that they do not ferment nor oxidize carbohydrates, and energy provision depends rather on a limited group of amino acids. The species is regarded as difficult to grow under laboratory conditions, and growth temperature and atmosphere requirements are not exactly similar to those previous described for A. butzleri. However, we could not identify the possible genetic determinants for these differences in the genomes of A. thereius.Whole genome analysis of nine Arcobacter thereius is predominantly present in the pig intestinal tract, but, due to the apparent lack of virulence factors previously based on specific PCR detection, has not been considered an important species from the food safety perspective so far. However, the species was recently isolated from the stool of human enteritis patients, and the present study reveals the presence of six out of eight virulence associated genes previously reported in A. butzleri. Further research will elucidate the role of these genes in the potential pathogenicity of A. thereius.A. thereius strains revealed the delineation of two closely related subgroups, for which, besides the group including the A. thereius reference strain, a new species, A. porcinus has recently be proposed [A. butzleri, reveals a large correlation, though also unique features are present. The occurrence of virulence associated genes, genes for antibiotic resistance as well as other pathogenic related relevant features, justifies a further exploration of the reservoir, transmission routes, consolidated in a risk assessment in both human and veterinary medicine.Comparative genome and phylogenetic analysis of the nine proposed . HoweverS1 Fig(TIF)Click here for additional data file.S2 FigA. thereius strains sequenced. No significant differences in the distribution of genes belonging to a functional category among the different strains has been found (see The number of genes in each categories was compared among the nine ound see . (C) = E(PDF)Click here for additional data file.S3 FigArcobacter and Campylobacter species are included as outgroup. For Campylobacter jejuni ATCC43439 the gene uncA (atpA) has been used.A neighbour-joining phylogenetic tree representing the six genes involved in the MLST method. (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.


Neisseria gonorrhoeae is capable of causing gonorrhoea and more complex diseases in the human host. Neisseria meningitidis is a closely related pathogen that shares many of the same genomic features and virulence factors, but causes the life threatening diseases meningococcal meningitis and septicaemia. The importance of non-coding RNAs in gene regulation has become increasingly evident having been demonstrated to be involved in regulons responsible for iron acquisition, antigenic variation, and virulence. Neisseria spp. contain an IS-like element, the Correia Repeat Enclosed Element, which has been predicted to be mobile within the genomes or to have been in the past. This repeat, present in over 100 copies in the genome, has the ability to alter gene expression and regulation in several ways. We reveal here that Correia Repeat Enclosed Elements tend to be near non-coding RNAs in the Neisseria spp., especially N. gonorrhoeae. These results suggest that Correia Repeat Enclosed Elements may have disrupted ancestral regulatory networks not just through their influence on regulatory proteins but also for non-coding RNAs. Gonorrhoea poses a global threat that may become virtually untreatable due to antibiotic resistance, including increasing prevalence of azithromycin-resistant isolates ,2,3,4 reNeisseria spp. [Non-coding RNAs (ncRNAs) can be important regulators of gene expression with roles in both physiology and disease ,12. Smalria spp. ,18,19,20aniS was annotated as a coding sequence (NMB1205 in the meningococcus and NGO0796 in the gonococcus), leading to its inclusion in the design of microarrays that have shown its differential expression [nrrF has been shown to be part of the fur regulon [pilE was identified and shown to be required for pilin antigenic variation [Neisseria meningitidis in the rat model [Neisseria lactamica. RNA-seq and transposon insertion site sequence mapping identified 253 ncRNAs in Neisseria gonorrhoeae [Neisseria gonorrhoeae MS11 strain.Small ncRNA pression ,24,25,26 regulon ,16. RNA- regulon ,20,27. Aariation ,19. Deleat model . Eleven orrhoeae ; 59 are Neisseria spp. genomes are the Correia Repeat Enclosed Elements (CREE) [N. gonorrhoeae genome contains just over 100 copies [N. meningitidis has roughly twice as many CREE within its genome [Other non-coding features of the s (CREE) . A N. go0 copies  of the C0 copies . N. menis genome ,31. Corrs genome ,33. CREEs genome .N. gonorrhoeae, N. meningitidis, and N. lactamica. RNA-seq was used to support ncRNA predictions.This study investigated whether CREE are located near predicted small ncRNAs in Neisseria spp. investigated here were downloaded on 24 October 2012, accession numbers: N. gonorrhoeae strain NCCP11945 (NC_011035); N. gonorrhoeae strain FA1090 (NC_002946); N. meningitidis strain MC58 (NC_003112); N. meningitidis strain Z2491 (NC_003116); N. meningitidis strain FAM18 (NC_008767); N. meningitidis strain alpha14 (NC_013016); N. meningitidis strain 53442 (NC_010120); N. lactamica strain 020-06 (NC_014752). SIPHT settings used: MaxE 5.00E-3; minimum length 30; maximum length 550; minimum TT confidence 86; and maximum RNAMotif score −6. These are the default settings when a new analysis is launched on the SIPHT web interface (http://newbio.cs.wisc.edu/sRNA/apps/sRNA/submitjob_pegasus_web.php).Candidate small ncRNAs were identified using SIPHT . GenBankN. gonorrhoeae strains FA1090 and NCCP11945 were reported previously [N. meningitidis strains MC58, Z2491, FAM18, alpha14, and 053442, and N. lactamica strain 020-06, CREE were identified using the Fuzznuc pattern finder [xBASE [The locations of the CREE within eviously . Within n finder  within xr [xBASE , as had r [xBASE . Search r [xBASE ,32,33.N. gonorrhoeae strain NCCP11945. Statistical analyses used the Kolmogorov-Smirnov test and non-parametric Spearman’s Rho correlation analysis in IBM SPSS Statistics version 21 to assess the distribution of CREE in the genome and between the CREE and ncRNAs.CREE and ncRNA location data was organised in Excel matching CREE with the closest ncRNA for N. gonorrhoeae strain NCCP11945 was grown on GC agar (Oxoid) at 37°C, 5% CO2 overnight. Growth was immediately removed to RNALater (LifeTechnologies) and RNA extracted using the RNasy kit (Qiagen). RNA quality was determined on the 2100 Bioanalyzer (Agilent) to have a RIN of 9 or above. RNA-seq used the Ion Personal Genome Machine, Ion Total RNA-Seq Kit with ERCC RNA Spike-In Control Mix, Ion Express Template kit, Ion Sequencing kit (Life Technologies), and 1 μg of rRNA-depleted RNA. RNA-seq data (GEO accession numbers GSE58650 and GSE73032) was aligned against the reference genome NCCP11945 using NextGENe v2.3.4.2.N. gonorrhoeae strains NCCP11945 and FA1090, N. meningitidis strains MC58, Z2491, FAM18, alpha14, and 053442, and N. lactamica strain 020-06 . SIPHT uses only intergenic regions for its predictions, therefore the aniS annotation as a CDS excludes it from detection by SIPHT. Annotations as CDSs may account for otherwise conserved ncRNAs being absent from some SIPHT predictions.Small ncRNAs were predicted using SIPHT  for N. gn 020-06 . Between genomes . These res  (The output of SIPHT was combined with the CREE location information for each genome and those within 1000 bp were identified . Startinca (64%) .N. meningitidis and N. lactamica, where on average 37% and 39%, respectively, of all CREE overlap with predicted ncRNAs, compared to a 58% average in N. gonorrhoeae (Many of the CREE overlap the sequences of predicted ncRNAs . In gonoorrhoeae .p < 0.001). Using the same test, CREE distribution around the genome was shown to be neither random  nor uniform . Spearman’s Rho correlation demonstrated that CREE locations were very strongly correlated with ncRNA locations . The ncRNA locations were all either located within or very close to CREE regions  than in N. meningitidis (average 37%) and N. lactamica (39%). This is particularly of note because there are only half as many CREE in N. gonorrhoeae, yet those that are present are far more likely to be located close to ncRNAs and to overlap the ncRNA sequences. In N. lactamica, the number of CREE in the genome is closer to that seen in the gonococcus and yet the overlaps between CREE and ncRNAs are on par with N. meningitidis (CREE tend to be located within 300 bp of predicted ncRNAs, with many overlapping . The frengitidis . Given tN. gonorrhoeae strain NCCP11945. Those with the highest detected transcription are upstream of virulence determinants (RNA-seq data supports the SIPHT predictions , demonstrminants , furtherNeisseria spp., CREE are frequently found near or overlapping ncRNAs in the genome. CREE may influence the expression of ncRNAs through the presence of their promoters and/or insertional activation, similar to the role of CREE in gene regulation. It may be possible to exploit differences between the species with regards to ncRNAs and their interaction with CREE in the design of RNA-based therapies to restrict the meningococcus to the mucosal surface, where it is as harmless as N. lactamica, and to prevent gonococcal antigenic variation, enabling rapid clearance and immunity.In the


Apolygus lucorum, Lygus pratensis as well as three Adelphocoris spp., including Adelphocoris lineolatus, A. suturalis, and A. fasciaticollis are major pests of multiple agricultural crops in China, which have distinct geographical distribution and occurrence ranges. Like many insect species, these bugs heavily rely on olfactory cues to search preferred host plants, thereby investigation on functional co-evolution and divergence of olfactory genes seems to be necessary and is of great interest. In the odorant detection pathway, olfactory receptor co-receptor (Orco) plays critical role in the perception of odors. In this study, we identified the full-length cDNA sequences encoding three putative Orcos  in bug species of A. suturalis, A. fasciaticollis, and L. pratensis based on homology cloning method. Next, sequence alignment, membrane topology and gene structure analysis showed that these three Orco orthologs together with previously reported AlinOrco and AlucOrco shared high amino acid identities and similar topology structure, but had different gene structure especially at the length and insertion sites of introns. Furthermore, the evolutional estimation on the ratios of non-synonymous to synonymous (Ka/Ks) revealed that Orco genes were under strong purifying selection, but the degrees of variation were significant different between genera. The results of quantitative real-time PCR experiments showed that these five Orco genes had a similar antennae-biased tissue expression pattern. Taking these data together, it is thought that Orco genes in the mirid species could share conserved olfaction roles but had different evolution rates. These findings would lay a foundation to further investigate the molecular mechanisms of evolutionary interactions between mirid bugs and their host plants, which might in turn contribute to the development of pest management strategy for mirid bugs.The phytophagous mirid bugs of  Bacillus thuringiensis) cotton and the associated reduction in broad-spectrum insecticide used for controlling Helicoverpa spp. , chemosensory proteins (CSPs), odorant receptors (ORs), sensory neuron membrane proteins (SNMPs) and odorant degradation enzymes (ODEs) play important roles in odorant signal transduction pathway Leal, . ORs locDrosophila melanogaster, OR2 in Bombyx mori, and OR7 in mosquito species  or Kuerle  experimental station of the Chinese Academy of Agricultural Sciences. The laboratory colony was kept in 20 × 10 × 6 cm rearing containers and was reared on green beans (Phaseolus vulgaris L.) and a 10% sucrose solution  following the manufacturer's instructions. The RNA quantity and integrity were checked using 1.2% agarose gel electrophoresis and a NanoDrop 2000 spectrophotometer . Total RNA was treated with RQ1 RNase-Free DNase  at 37°C for 30 min to remove residual DNA. The cDNAs were synthesized using the Superscript III Reverse Transcriptase system .AsutOrco, AfasOrco, and LpraOrco genes were cloned using degenerate primers (Table rs Table . Each rers Table . Touchdors Table .http://topcons.cbr.su.se/) The full length Orco sequences were aligned by ClustalX 2.1 and edited by GeneDoc 2.7.0 software. TOPCONS  followed the manufacturer's instruction. Introns of Orco genes were amplified using specific primers Tables –S4.The nGQ477013, KU230353, KF921006, KU188517, and MG397129, separately) were used as the endogenous control to normalize the target gene expression and correct for any sample-to-sample variation. The primers .The reference genes β-actin  using degenerate primers  of AsutOrco, AfasOrco, and LpraOrco were 1416, 1416, and 1422 bp, respectively, which resembled the full length of previously reported Orco genes  in comparison to rather conserved amino acids (data not shown) Figure . Orco wi Figures . NotablyA. lucorum displays the most extensive distribution in China, whereas L. pratensis mainly occurred in Xinjiang Uygur Autonomous Region  . Phylogenetic relationship was largely consistent with the species tree constructed from the alignment of species-specific cytochrome oxidase subunit I , which was significantly different to that of AlucOrco (0.365–0.496) from Apolygus genus, or LpraOrco, LlinOrco and LhesOrco (0.361–0.497) from Lygus genus. These findings indicted there might be a strong constraint on functional variation within Orco from same genus, as illustrated above. In addition, these results were faultlessly correlated to the phylogenetic analyses  revealed that their levels of the purifying selection pressure significantly varied in the genera and species. Generally, the values of Ka/Ks were similar among species within same genus, but were different from species across genera. As shown in Table ucOrco 0.65–0.496  Figures , we propL. hesperus (Hull et al., A. lucorum of the olfactory co-receptor Orco gene by RNA interference could induce EAG response declining to two putative semiochemicals (Zhou et al., B. dorsalis could fulfill a role involved in the perception of Rhodojaponin-III, a non-volatile compound (Yi et al., In general, target gene with different tissue expressions would play different physiological function. To figure out the potential roles of Orco in mirid bugs species, qPCR measurement was conducted to assess their tissue-specific expressions Figure . The resY-JZ and LS conceived and designed the experimental plan. QiW, Y-LZ, and H-HC performed the experiments. QiW, QianW, SS, YX, KD, and AK analyzed the data. QiW and QianW drafted the manuscript. LS and Y-JZ refined and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Treponema pallidum ssp. pallidum , the causative agent of the sexually transmitted disease syphilis, is an uncultivatable human pathogen. The geographical differences in T. pallidum genomes leading to differences in pathogenicity are not yet understood. Presently, twelve T. pallidum genomes are available to the public, all of which are American in origin and often co-infect patients with human immunodeficiency virus (HIV). In this study, we examined the T. pallidum subsp. pallidum strain Amoy, a syphilis pathogen found in Xiamen, China. We sequenced its genome using Illumina next-generation sequencing technology and obtained a nearly (98.83%) complete genome of approximately 1.12 Mbps. The new genome shows good synteny with its five T. pallidum sibling strains , among which SS14 is the strain closest to the Amoy strain. Compared with strain SS14, the Amoy strain possesses four uncharacterized strain-specific genes and is likely missing six genes, including a gene encoding the TPR domain protein, which may partially account for the comparatively low virulence and toxicity of the Amoy strain in animal infection. Notably, we did not detect the 23S rRNA A2058G/A2059G mutation in the Amoy strain, which likely explains the sensitivity of Amoy strain to macrolides. The results of this study will lead to a better understanding of the pathogenesis of syphilis and the geographical distribution of T. pallidum genotypes. Treponema pallidum ssp. pallidum  is the cause of the sexually transmitted disease syphilis. However, the exact pathogenesis of this disease is poorly understood. Sub Subde noT. pallidum strains,[T. pallidum strains, adopting Treponema pallidum subsp. pertenue str. Gauthier as an outlier, using the online tool REALPHY 1.10. The REALPHY program uses the maximum likelihood method PhyML [We annotated the genome using the NCBI PGAAP pipeline,  tagging  strains, includinod PhyML  to inferod PhyML  Pan-genood PhyML  and Orthod PhyML  algorithod PhyML  EventualT. pallidum SS14, the average sequencing depth was approximately 300X, which is sufficient for high-quality genome assembly. After employing a computational pipeline for assembly, re-scaffolding, gap closing, and scaffold ordering, we eventually obtained a draft circular genome of 1,139,223 base pairs, which consisted of 15 scaffolds (98.83%) and approximately 1.17% Ns (Genome sequencing of the Amoy strain on the Illumina HiSeq 2000 platform yielded about 2.82G base pair raw paired-end reads. Approximately 367 Mb of clean reads passed the QC and contamination checks. Compared with the genome size (1.14 Mb) of the sibling strain 1.17% Ns .T. pallidum strains.[ strains. In totalT. pallidum genomes  according to their sequence similarities, using a close strain, Treponema pallidum subsp. pertenue str. Gauthier, as an outlier. Genome clustering assigned the six T. pallidum genomes to two groups  belonged to the SS14-like group. The reasnicillin. However,nicillin. A previonicillin. and in Snicillin. In the pT. pallidum Amoy strain, providing the first genome sequence of a clinical syphilis isolate from China. According to analysis of genomic similarity, the Amoy strain is mostly closely related to the SS14-like group. At the same time, we also illustrated the genomic differences of the T. pallidum Amoy strain compared with other strains from various host populations and different geographic regions, identifying two uncharacterized proteins specific to the Amoy strain. Unlike current prevalent isolates, we did not detect a 23S rRNA A2058G/A2059G mutation in the Amoy strain, which partially explains the absence of macrolide resistance in the Amoy strain. Overall, the sequenced genome of the T. pallidum Amoy strain will lead to a better understanding of different types of pathogenesis of T. pallidum strains and will contribute to the goal of achieving syphilis eradication.In this study, we sequenced the genome of the S1 Fig(TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.