Pneumococcus is an important respiratory pathogen that is associated with high rates of death in newborn children and the elderly. Given the disadvantages of current polysaccharide-based vaccines, the most promising alternative for developing improved vaccines may be to use protein antigens with different roles in pneumococcus virulence. PspA and PhtD, highly immunogenic surface proteins expressed by almost all pneumococcal strains, are capable of eliciting protective immunity against lethal infections.S. pneumoniae infection with PA or PD alone and a combination of PA and PD. The prediction of physicochemical properties, antigenicity, allergenicity, toxicity, and 3D-structure of the constructs, as well as molecular docking with HLA receptor and immune simulation were performed using computational tools. Finally, mice were immunized and the serum levels of antibodies/cytokines and functionality of antibodies in vitro were evaluated after immunization. The mice survival rates and decrease of bacterial loads in the blood/spleen were examined following the challenge.In this study using immunoinformatics approaches, we constructed one fusion construct  by fusing the immunodominant regions of PspA from families 1 & 2 (PA) to the immunodominant regions of PhtD (PD). The objective of this project was to test the immunogenicity of the fusion protein PAD and to compare its protective activity against The computational analyses indicated the proposed constructs could be antigenic, non-allergenic, non-toxic, soluble and able to elicit robust immune responses. The results of actual animal experiments revealed the candidate vaccines could induce the mice to produce high levels of antibodies and cytokines. The complement-mediated bactericidal activity of antibodies was confirmed and the antibodies provided favorable survival in immunized mice after bacterial challenge. In general, the experimental results verified the immunoinformatics studies.For the first time this report presents novel peptide-based vaccine candidates consisting of immunodominant regions of PspA and PhtD antigens. The obtained findings confirmed that the fusion formulation could be relatively more efficient than the individual and combination formulations. The results propose that the fusion protein alone could be used as a serotype-independent pneumococcal vaccine or as an effective partner protein for a conjugate polysaccharide vaccine. Due to the diversity of the PspA molecule in different pneumococcal strains, the amino acid sequences of PspA were retrieved from 123 pneumococcal strains which had unique sequences  (www.cbs.dtu.dk/services/TMHMM-2.0)  database  . MoreoveHMM-2.0) .https://www.ebi.ac.uk/Tools/msa/clustalo/) (https://www.iedb.org/) was used to find the experimentally identified epitopes.The PspA sequences from 123 strains were classified and aligned using the Clustal Omega web server   to identustalo/)  to inves2.1.2https://zhanglab.ccmb.med.umich.edu/I-TASSER/) (http://swissmodel.expasy.org) (https://zhanglab.ccmb.med.umich.edu/ModRefiner/)  (https://saves.mbi.ucla.edu/). BIOVIA Discovery Studio Visualizer was used to display the 3D structure of proteins. Eventually the conformational epitopes were anticipated based on the 3D protein structure using the servers Ellipro (http://tools.iedb.org/ellipro/) (http://www.cbs.dtu.dk/services/DiscoTope/)  server (TASSER/)  or SWISSasy.org) . The proefiner/)  and Gala=REFINE) . The val=REFINE)  and ERRA=REFINE)  programsllipro/)  and DisccoTope/) . The Ell2.1.3http://crdd.osdd.net/raghava/lbtope/protein.php) (http://crdd.osdd.net/raghava/abcpred/) (http://tools.immuneepitope.org/bcell/)  , ABCpredbcpred/) , IEDB Em/bcell/)  and Ellihttp://tools.immuneepitope.org/mhcii) (http://www.cbs.dtu.dk/services/NetMHCIIpan/)  was performed using MHC-II prediction tool in IEDB server (g/mhcii)  and NetMCIIpan/) . To eval2.1.4In the consensus sequence approach, the homologous sequences are analyzed to reach the target sequence. Substitution of amino acids with similar chemical features  often does not significantly affect the structure/function of a protein . The epi2.1.5The peptides predicted by different servers consisting of higher scoring B cell epitopes overlapped with immunodominant T-helper (Th) cell epitopes were analyzed and eventually the selected peptide sequences were linked together using flexible linkers (Gly-Gly-Gly-Ser and Gly-Gly-Ser-Ser-Gly-Gly). In order to efficiently separate and preserve the independent folding and immunological activities, flexible linkers were considered to connect adjacent epitopes . In addi2.1.6in vitro/in vivo half-life, average aliphatic index, and average hydropathicity, were evaluated using ProtParam tool in ExPASy server (http://web.expasy.org/protparam/) (Escherichia coli was predicted by SOLpro tool in SCRATCH server (https://scratch.proteomics.ics.uci.edu/) (http://www.ddg-pharmfac.net/vaxijen/VaxiJen/VaxiJen.html) (http://scratch.proteomics.ics.uci.edu/) (http://www.ddg-pharmfac.net/AllerTOP/)  (https://webs.iiitd.edu.in/raghava/toxinpred/index.html) , total number of positive and negative amino acids, theoretical isoelectric point, instability index, tparam/) . Moreoveci.edu/) . VaxiJenen.html)  and ANTIci.edu/)  were utilerTOP/)  and AlgPlgpred/)  were useex.html) .2.1.7http://robetta.bakerlab.org/)  (https://saves.mbi.ucla.edu/), as well as the ProSA web server (https://prosa.services.came.sbg.ac.at/prosa.php) (3D structure modeling of the protein constructs was performed in the best way by Robetta server (ab.org/)  based on=REFINE)  was used=REFINE)  and ERRA=REFINE)  at the Sosa.php) .2.1.8The structural epitopes which are recognized by antibodies play a major role in the humoral immunity. Therefore, the developed protein constructs should have effective conformational epitopes in their protein domains to induce stronger immunity. The ElliPro server was employed to identify the structural B-cell epitopes in the refined models of the epitope-based vaccine candidates, keeping the default parameters.2.1.9https://cluspro.org/login.php) (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/Generate.html)  . The 3D te.html)  was util2.1.10https://kraken.iac.rm.cnr.it/C-IMMSIM/)  . This se2.1.11http://www.jcat.de/) (E. coli (K12 strain). In addition, this server prevents unwanted sites for restriction enzymes, Rho-independent transcription termination, and prokaryotic ribosome binding. Finally, the optimized gene sequences for the PAD, PA, and PD, with restriction sites NcoI and XhoI respectively added to the 5’ and 3’ ends, were cloned in pET28a vectors using SnapGene tool (https://www.snapgene.com/try-snapgene/).The codon optimization approach was used to enhance the expression of PAD, PA and PD recombinant proteins. The Java Codon Adaptation Tool (JCat) (cat.de/)  online s2.22.2.1E. coli BL21 (DE3) cells, and recombinant clones were identified by colony PCR using T7 universal primers (https://www.addgene.org/mol-bio-reference/sequencing-primers/). Expression of the recombinant proteins was induced by the addition of 1 mM isopropyl-β-thiogalactopyranoside (IPTG) for 18 h, and examined by running on 15% SDS–PAGE and Coomassie Brilliant Blue staining R-250. The expressed recombinant proteins PAD, PA and PD were purified using Ni-NTA chromatography under native conditions, according to the manufacturer’s manual . Finally, the purified proteins were dialyzed overnight and then quantified by NanoDrop spectrophotometer at 280 nm.The pET28a vector harboring the sequence encoding PAD, PA or PD was synthesized by Biomatik Corporation . The recombinant expression vectors  were transferred into 2.2.2Vaccination schedule and timeline of experimental procedures are presented in 2.2.3rd injection were evaluated by indirect enzyme-linked immunosorbent assay (ELISA)  isotypes two weeks after the 3 (ELISA) . In brie2.2.4rd injection were determined using ELISA kits  in accordance with manufacturers’ guidelines.The titers of cytokines interferon-gamma (IFN-γ), interleukin-4 (IL-4), and interleukin-17A (IL-17A) in the mouse sera two weeks after the 32.2.5th injection, the SBA assay was used to evaluate the activity of anti-recombinant protein antibodies in killing of pneumococcal strain ATCC 6303. At first the sera of immunized mice were decomplemented at 56°C for 30 min. Then, 12.5 μl of the bacterial suspension at 105 CFU/ml and 12.5 μl of infant rabbit serum as a source of the complement were added to the 96-well microtiter plates containing serially diluted inactivated serum sample (1:2 to 1:64). After 1 h incubation at 37°C in 5% CO2, 10 μl of each well was cultured on a blood agar plate and incubated for 24 h at 37°C in 5% CO2. The SBA titer was reported as the reciprocal of the serum dilution at which ≥50% of bacteria were lysed in comparison to the negative control without Ab  and PhtD-C antigens are listed in 3.1.5E. coli, more than 20 hours in yeast and more than 10 hours in mammals. Moreover, the antigenicity, solubility, allergenicity and toxicity of the constructs were investigated as shown in E. coli, the designed recombinant proteins were soluble. AlgPred and AllerTOP servers demonstrated that the protein constructs were non-allergen. The ToxinPred server showed the non-toxicity of the constructs for humans and animals. The output of these computational analyses revealed that the proposed constructs passed the evaluations with satisfactory results.The designed constructs were evaluated and compared with respect to their physicochemical and immunological properties. Various features of the constructs were predicted using the ProtParam tool as represented in 3.1.6The three-dimensional structures of the protein constructs PAD, PA, and PD were predicted by Robetta server and refined by the GalaxyRefine server. The quality evaluation of the models was accomplished using Ramachandran plot, ERRAT value and ProSA Z-score. The predicted 3D structures and validation of the models before and after refinement are represented in 3.1.7Since most of B cell epitopes are discontinuous, the 3D models of the final designed constructs were analyzed for identification of the conformational epitopes by the ElliPro server. It was found that the PAD construct possessed five new epitopes, comprising 20 to 63 residues with the score values of 0.531 to 0.887 (protrusion index). The PA construct had five new epitopes, comprising 4 to 39 residues with the score values of 0.592 to 0.792. For the PD construct, three new epitopes, comprising 19–22 residues were found with a score value of 0. 636 –0.717. The details of the identified structural epitopes of the constructs PAD, PA, and PD are given in 3.1.8The selected immunogenic regions were further validated using molecular docking with MHC Class II (DRB1*01:01) receptor. Since the optimal size of peptides binding to the cleft of MHC-II molecules is approximately 30 residues, the ≤30-mer peptides of the selected epitope-rich regions were considered for the molecular docking. Following the docking process, the complex models ranked according to the energy level were generated by the ClusPro 2.0 server. The best docked complexes were visualized by Chimera software as shown in 3.1.9In silico immune simulations were performed for the evaluation of immunogenic profile of the constructs in real life. Based on the immune-simulation results, the designed constructs are able to generate appropriate primary, secondary and tertiary responses  and show the possibility of high expression of the constructs in E. coli strain K12. The NcoI (CCATGG) and XhoI (CTCGAG) cleavage sites were inserted into the 5’ and 3’ ends of the optimized PAD, PA and PD sequences, respectively  between the restriction enzymes NcoI and XhoI using the SnapGene tool. The developed plasmids were designated as pET28a-PAD, pET28a-PA, and pET28a-PD plasmids . The recombinant plasmids were transferred into E. coli BL21 and the positive clones were detected by colony PCR using T7 universal primers  was seen in the test groups compared to the control group. The bacterial loads in the blood of control mice were about 104The commercial polysaccharide-based pneumococcal vaccines are expensive and serotype-dependent, and their immunogenicity is limited to the serotypes included in the vaccines . TherefoMycobacterium tuberculosis . The pedonovani .The PspA and PhtD proteins, among the pneumococcal virulence proteins, are highly immunogenic which are expressed on all serotypes and have shown very promising results in clinical studies as vaccine candidates . The facStreptococcus pneumoniae. In the present study, the computational approaches were applied for designing potential peptide-based vaccines including the immunodominant regions of PspA (from different families) and PhtD proteins. We assessed the A, B and C regions of the PspA protein to predict the B and T cell epitopes. B regions from clades 1 to 5 were analyzed to provide wider protective coverage and decrease the possibility of PspA variants escaping host immune responses. In addition, the C-terminal of PhtD was analyzed as a promising immunogenic region for predicting B and Th cell epitopes. None of the selected epitope-rich regions of these proteins have been reported before, and this study presents for the first time novel vaccine candidates consisting of immunodominant regions of PspA and PhtD-C . Finally, the immunogenicity and protective effects of the constructs were compared individually, in combination and fusion form in mouse model.According to our knowledge, the proteins PspA and PhtD have not been studied in a fusion form until today, and this study is the first report evaluating the fusion form of these two proteins as a vaccine candidate against On the basis of the immunoinformatics data of the study, the high-scoring and overlapping B and Th cell epitopes shared on multiple servers were taken into account to select the final peptides for PspA  and PhtD-C Table 1.In the next step of this research, the final selected peptides were connected to each other by glycine-rich flexible linkers (GGGS and GGSSGG) capable of improving solubility and enabling the neighboring domains to act freely . A histiE. coli, as well as they are antigenic, non-allergenic and non-toxic.The physicochemical and immunological characteristics of the designed constructs were evaluated using different web servers Table 2.The primary 3D structures of the vaccine candidates were modeled using Robetta Server and then refined by GalaxyRefine software resulting in higher quality 3D models. The quality assessment of the initial and refined models was conducted using Ramachandran diagrams, ProSA Z-scores and ERRAT scores. The obtained results showed that the refined structures possessed better quality than the initial 3D structures Table 3.E. coli K12, the reverse translation and codon optimization of the designed constructs were conducted using the Jcat server. For the optimized sequence of PAD, PA or PD, the GC content and the CAI value were favorable for high protein expression in the bacteria. Finally, for the purpose of in silico cloning, the gene sequences of vaccine candidates were successfully inserted into the pET28a(+) expression vectors. Overall, the in silico data showed that the vaccine candidates may be highly effective against pneumococcal infections, but in order to confirm these in silico results, further experimental studies were performed in vitro and in vivo.In the next step, to ensure high-level expression in E. coli BL21 after induction by IPTG. The computational prediction of the solubility of PAD, PA and PD proteins was confirmed by their in vitro purification under native conditions on Ni-NTA column. In the SDS-PAGE technique, the presence of bands corresponding to PAD, PA and PD recombinant proteins demonstrated acceptable expression and codon optimization. The band near 11, 35, or 48 kDa of the protein ladder corresponds to the PD, PA, or PAD construct, respectively. The predicted MW of PA and PAD are lower than the apparent MW on SDS-PAGE gel, indicating the slow movement of these proteins. In a study by Shirai et al., it was shown that the differences between estimated and apparent MWs could be due to the GRAVY score , while complete clearing of bacteria was observed in the spleen.In this study, the protective effect of epitope-based vaccine candidates against As seen in the present study, in comparison between individual constructs, although the PD construct is a suitable vaccine target, it is not as effective as the PA construct alone as an immunogen and is not sufficient to provide complete protection against pneumococcal infections. The different immunoreactivity results of the two individual formulations PD and PA may be attributed to several factors, e.g. protein molecular weight, chemical/physical properties, composition and degradability . HoweverS. pneumoniae. FlaB–PspA fusion protein stimulated IL-4 and IFN-γ production and significantly enhanced IL-4 production and Th2 responses, being consistent with the IgG subtype responses. Also, our results agree with the results of Lu et al. that showed PsaA–PspA fusion protein could stimulate production of high-titered antibodies against S. pneumoniae strains comparable to each antigen alone  can be found in the article/The animal study was approved by Animal Ethical Committee of Semnan University of Medical Sciences, Semnan, Iran (Approved Number: IR.SEMUMS.REC.1399.128). The study was conducted in accordance with the local legislation and institutional requirements.MS: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing - review & editing, Writing original draft. ZB: Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Writing-original draft. SB: Methodology. EA: Methodology. HM: Validation. SM: Project administration, Supervision. AS: Project administration, Supervision.


A 36-year-old pregnant woman presented with 1 week of burning pain on the upper labial mucosa and new onset headache. Six days prior, the patient’s 3-year-old child tested positive for streptococcal pharyngitis after exposure at daycare and was treated with antibiotics. Physical examination was remarkable for pink thin plaques with white, polycyclic borders on the upper labial mucosa  that couQuestion 1: What is the most likely diagnosis?A.Lichen planusB.CandidiasisC.Aphthous stomatitisD.Geographic lipE.Herpetic gingivostomatitisAnswers:A.Lichen planus – Incorrect. Oral lichen planus may involve labial mucosa and can be associated with sensitivity to acidic or spicy food similar to geographic tongue. However, this condition presents more commonly with bilateral white reticulations, which are frequently associated with erythema and erosions and yellow fibrinous exudate.B.Candidiasis – Incorrect. Although oral candidiasis may present with burning pain and can involve any mucosal surface, this condition classically presents as curd-like white papules and plaques that can be wiped off.C.Aphthous stomatitis – Incorrect. This common condition affects approximately 20% to 50% of the population and favors labial mucosa but presents more commonly with ovoid ulcers with a white-to-yellow base and an erythematous rim.D.Geographic lip – Correct. Geographic tongue is a benign, inflammatory disorder of unclear etiology that predominantly involves the dorsal and lateral tongue. Ectopic geographic tongue  may involve any oral mucosa and classically presents as flattened or atrophic lesions varying from a few millimeters to several centimeters with a serpiginous white border.E.Herpetic gingivostomatitis – Incorrect. Herpetic gingivostomatitis may involve the labial mucosa but frequently presents with vesicles at the vermillion border that break to form erosions and ulcers that subsequently crust.Question 2: Geographic tongue shares strong similarities with what common dermatologic condition?A.PsoriasisB.Erythema multiformeC.Pityriasis roseaD.Discoid lupus erythematosusE.RosaceaAnswers:A.,P = .03).Psoriasis – Correct. Current literature suggests clinical, histologic, and immunohistochemical similarities between geographic tongue and psoriasis.B.Erythema multiforme – Incorrect. There is no clear relationship between erythema multiforme and geographic tongue.C.Pityriasis rosea – Incorrect. There is no clear relationship between pityriasis rosea and geographic tongue.D.Discoid lupus erythematosus – Incorrect. There is no clear relationship between discoid lupus erythematosus and geographic tongue.E.Rosacea – Incorrect. There is no clear relationship between rosacea and geographic tongue.Question 3: Which of the following statements is FALSE?A.Migratory stomatitis is another, appropriate name for geographic lip.B.Ectopic geographic tongue was first described in 1955.C.Spicy or acidic foods may exacerbate symptoms of geographic tongue, both lingual and ectopic.D.Although the diagnosis of ectopic geographic tongue is clinical only, biopsies may demonstrate elongation of rete edges, parakeratosis, Munro abscesses, and suprapapillary thinning.E.Geographic tongue affects approximately 1 in 5 individuals.Answers:A.Migratory stomatitis is another, appropriate name for geographic lip – Incorrect. This is true. There are many names that have been used for ectopic geographic tongue, such as migratory stomatitis, stomatitis areata migrans, erythema circinata migrans, annulus migrans, and geographic stomatitis.B.Ectopic geographic tongue was first described in 1955 – Incorrect. This is true. This phenomena was first described in a 30-year-old Maltese man in 1955.C.Spicy or acidic foods may exacerbate symptoms of geographic tongue, both lingual and ectopic – Incorrect. This is true. Although some patients may be asymptomatic, burning pain is often related to eating spicy or acidic foods.D.Although the diagnosis of ectopic geographic tongue is clinical only, biopsies may demonstrate elongation of rete edges, parakeratosis, Munro abscesses, and suprapapillary thinning – Incorrect. This is true. Similar to psoriasis, biopsies of these lesions may demonstrate elongation of rete edges, parakeratosis, Munro abscesses, and suprapapillary thinning.E.Geographic tongue affects approximately 1 in 5 individuals – Correct. This statement is false. The prevalence of geographic tongue is 1% to 4.8%, and ectopic geographic tongue is rarely described.The authors declare no conflicts of interest with respect to the research, authorship, and/or publication of this article. Nicole R. LeBoeuf is a consultant and has received honoraria from Bayer, Seattle Genetics, Sanofi, Silverback, Fortress Biotech, and Synox Therapeutics outside the scope of the submitted work.


Candida bloodstream isolates in a hospital setting. The resistance mechanisms towards the two most commonly administered antifungals, fluconazole and anidulafungin, were determined. Blood culture isolates between 1 January 2018 and 30 June 2021 positive for Candida spp. were included. Susceptibility testing was performed using Etest. Whole-genome-sequencing was performed using Illumina NovaSeq with bioinformatics analysis performed. A total of 203 isolates were sequenced: 56 C. glabrata, 53 C. tropicalis, 44 C. albicans, 36 C. parapsilosis complex , six C. krusei, five C. dubliniensis, and three C. auris. A single cluster of azole-resistant C. tropicalis, and four clusters of C. parapsilosis isolates were observed, suggesting possible transmission occurring over several years. We found 11.3%, and 52.7 % of C. tropicalis and C. parapsilosis, respectively, clustered with other isolates, suggesting exogenous sources may play a significant role of transmission, particularly for C. parapsilosis. The clusters spanned over several years suggesting the possibility of environmental reservoirs contributing to the spread. Limited clonality was seen for C. albicans. Several sequence types appeared to be dominant for C. glabrata, however the SNP differences varied widely, indicating absence of sustained transmission.Invasive candida infections are significant infections that may occur in vulnerable patients with high rates of mortality or morbidity. Drug-resistance rates also appear to be on the rise which further complicate treatment options and outcomes. The aims of this study were to describe the prevalence, molecular epidemiology, and genetic features of  All genome data for this study have been deposited in GenBank. The genome raw reads have been deposited in the Short Read Archive as SRR19696069 with the BioProject No. PRJNA848263. The authors confirm all supporting data and protocols have been provided within the article or through supplementary data files.Candida spp. are traditionally thought to be endogenous in nature (arising from one’s own flora). Our data demonstrate differences in diversity between Candida spp., with clustering of non-albicans Candida spp. over several years. Whilst limited clustering is seen for some species, >50 % of C. parapsilosis clustered with other isolates. This suggests the possibility of exogenous sources of infection and occult transmission may occur in healthcare settings. The sporadic occurrence of clustered isolates over varying locations and at different times suggest the possibility of periodic breaks in infection control, and possible environmental reservoirs of infection.Infections with Candida spp. [Invasive candidiasis are significant infections that may occur in patients, particularly those who have critical illness or are immunocompromised. Typical risk factors for candidaemia include ICU admission, total parenteral nutrition use, malignancy, and neutropaenia. High mortality rates are reported even with antifungal therapy . Currentida spp. . In suchida spp. .Candida spp. are Candida albicans, Candida tropicalis, Candida parapsilosis, and Candida glabrata. In addition, Candida auris has joined the fray causing a global outbreak in ICUs, with the capacity to acquire resistance against azoles, echinocandins, as well as polyenes [Candida spp. are not limited to C. auris and has been reported in C. parapsilosis and C. tropicalis [Candida spp. are usually caused by an individual’s own normal flora.Historically, the four most commonly isolated pathogenic polyenes . This haopicalis . This alCandida bloodstream isolates in a hospital setting. We investigated possible transmission patterns of not just drug-resistant but also drug-susceptible Candida spp., providing insight into how candidaemia infections may have been acquired.The aims of this study were to describe the prevalence, molecular epidemiology, and genetic features of The study was performed at a 1200-bed tertiary referral hospital. The range of services offered here are broad including adult, paediatric, obstetrics and gynaecology services, medicine and surgery across a wide range of specialties including solid organ and haematology oncology, solid organ transplant , and stem cell transplant, as well as intensive care services . Inpatients services are provided across two separate blocks with wards. The wards were broadly categorized in this study into intensive care units (ICU), high dependency units (HDU), general ward, and emergency department. Individual wards are usually assigned particular patient types and these are broadly categorized as ward category  and associated primary treatment teams  ..Candida spp. were included in this study. Blood cultures were incubated using the BacT/Alert Virtuo  system, upon positive signal, an aliquot of blood was subcultured onto routine culture media  and Sabouraud dextrose agar when fungi were seen on Gram-stain. Identification of isolates was performed using MALDI-TOF .Blood culture isolates between 1 January 2018 and 30 June 2021 positive for C. krusei due to the presence of intrinsic resistance mechanisms), and a susceptible breakpoint of ≤1 mg l−1 was applied for amphotericin B as this was the common breakpoint used for Candida spp. Only non-duplicate isolates (first isolate from a patient) were included in describing the susceptibility profiles.Susceptibility testing for fluconazole, anidulafungin, and amphotericin B was performed using Etest . The minimum-inhibitory-concentration (MIC) results were interpreted based on EUCAST breakpoints. Where breakpoints were not available, non-species-specific breakpoints were applied for fluconazole (with the exception of https://github.com/tseemann/shovill) coupled with SPAdes v. 3.14.0 [https://github.com/tseemann/snp-dists). FastTree [https://github.com/tseemann/snippy) was used for SNP variant calling of drug-resistance associated loci. Snippy uses BWA MEM [ERG3, ERG11, FKS genes, efflux-transporters and transcriptional factors that mediate drug resistance  were used as a reference for Snippy to determine the presence of non-synonymous mutations which may confer drug resistance phenotype. Copy number variation in RTA3, ERG3 and ERG11 genes of C. parapsilosis genomes was determined using pipeCoverage (https://github.com/MrOlm/pipeCoverage).Illumina NovaSeq was used to generate 150 bp paired-end reads. The raw reads were then assembled using Shovill v1.0.4  schemes have been published for C. albicans, C. dubliniensis, C. tropicalis, C. glabrata, and C. krusei [https://pubmlst.org/organisms/). C. parapsilosis does not currently have an MLST scheme. For C. albicans clade assignment, the MLST alleles were concatenated for cluster analysis and the clade defined as previously described [C. glabrata), T_ , A_  and P_ (for C. parapsilosis).For pathogenic . krusei . The MLSescribed . The genCandida spp. isolated from blood cultures. Seven different Candida spp. were isolated and C. glabrata was the most common cause of bloodstream infection (28.2 %), followed by C. tropicalis (25.3 %) and C. albicans (21 %) (C. glabrata complex (renamed as Nakaseomyces glabrata complex and includes Nakaseomyces nivariensis and Nakaseomyces bracarensis) [C. tropicalis, 44 C. albicans, 36 C. parapsilosis complex , six C. krusei (renamed as Pichia kudriavzevii) [C. dubliniensis, and three C. auris [Between 1 January 2018 and 30 June 2021, there were 203 non-duplicate s (21 %) . The detarensis) , 53 C. tavzevii) , five C.C. auris .C. parapsilosis, 31 isolates were C. parapsilosis sensu stricto, four were C. orthopsilosis and one isolate was C. metapsilosis. A similar prevalence and distribution of the three species was also observed in other hospitals where C. parapsilosis sensu stricto was consistently the most dominant species and C. metapsilosis the least common [Of the 36 t common .C. nivariensis and C. bracarensis, rare pathogens which are closely related to C. glabrata sensu stricto and also known to cause invasive disease, were not detected in our cohort. This was similar to global findings that both C. nivariensis and C. bracarensis were extremely uncommon  [solates) .C. albicans was most frequently isolated (41.3%) while C. tropicalis was the leading non-albicans species [The species distribution parallels other observational studies. A 12 month, laboratory-based surveillance of candidaemia at 25 hospitals from China, Hong Kong, India, Singapore, Taiwan and Thailand showed that  species . DiffereCandida spp. in Tables S3-S6. These are further stratified based on whether isolates formed clusters versus non-clustered isolates for C. tropicalis and C. parapsilosis, and sequence type for C. glabrata. The average age was 61 years with more male (n=124) than female (n=79) patients with no clear predilection seen for any particular pathogen. Overall, the treating teams with the most number of candidaemia cases were the haematology team , followed by medical oncology (9.9 %), and nephrology (7.4 %). This is consistent with known risk factors for candidaemia.The demographics and primary treatment teams for included patients are summarized based on C. parapsilosis complex and C. tropicalis isolates. C. albicans was found to be completely susceptible to both azole and echinocandins .Overall, echinocandin resistance rates was low. In esistant . Previouopicalis . One casC. tropicalis isolates carried Y132F and S154F double mutation in ERG11p which mediates high-level azole resistance [ERG11. Overexpression of MDR transporters and CDR efflux pumps (Table S1) could increase resistance to azole antifungal drugs [C. tropicalis.Five out of nine azole resistant sistance   were correlated with azole resistance. Gain-of-function (GOF) mutations were not observed in transcriptional factors UPC2, NDT80, MRR1 and TAC1. This suggests that there may be a novel mechanism of azole resistance perhaps yet undetected in the clinical isolates.Y132F substitution in ERG11p is correlated with azole resistance in psilosis . HoweverRTA3, encoding a lipid translocase, has been implicated in antifungal resistance, when found in multiple copies [C. parapsilosis genomes for RTA3, ERG3 and ERG11 did not reveal any duplications.Gene copy number gains have been implicated in fluconazole resistance. The gene e copies . AnalysiC. glabrata has an intrinsic resistance to fluconazole, with no obvious ERG11 polymorphism correlated to resistance [CDR1, CDR2 and SNQ2 [C. glabrata PDR1 has a high allelic diversity. This was demonstrated in a study looking at 122 clinical C. glabrata isolates. At least 12 different alleles recovered only from azole-susceptible isolates containing combinations of eight different mutations and 58 different alleles specific for azole-resistant isolates with 58 distinct mutations [CDR1 thus contributing to azole resistance of clinical isolates [−1). A previously observed missense mutation L935M was seen in an isolate with azole MIC of 16 mg l−1 (Table S7). This L935M mutation is located on the central regulatory domain [sistance   in Fks2p. The F659 position has been established to be important for decreased echinocandin susceptibility [Both echinocandin resistant tibility   and could potentially represent a novel drug mutation.Of the three anidulafungin resistant had none . S654P iC. parapsilosis has an intrinsically reduced susceptibility to echinocandins resulting from a naturally occurring P660A substitution in Fks1p. This mutation leads to a characteristic 2-log decrease in echinocandin sensitivity [−1 did not display any mutations in the FKS genes , copy-number variation (CNV) [C. albicans. We did not look at those global gene arrangements here as that analysis would be better suited with long-read sequencing.We focussed on investigating well-established mechanisms of resistance including drug target alteration of on (CNV)  includinC. glabrata sensu stricto, the most prevalent sequence types (STs) for C. glabrata were: ST7, ST55, and ST59  isolates from the C. tropicalis cluster was isolated from haematology patients who were admitted to haematology-oncology wards  were unique and previously unassigned , with <31 SNPs intra-cluster  of cases were patients whose primary treating team was Nephrology, although all three patients were in different locations at the time of culture, with all three cultures from different years . The other clusters do not appear to have a clear association location and patient population. Clustered C. parapsilosis isolates spanned all the years of collection (2018–2021)  . Persist studies . Guinea fections .C. parapsilosis [Our data indicates clustering of both azole-resistant and azole-susceptible isolates . Currentpsilosis .C. krusei isolates had different MLST allelic profiles .None of the four C. auris are recognised: South Asian (clade I), East Asian (clade II), South African (clade III), and South American (clade IV). C. auris was first detected in Singapore in 2012. Studies on the local genomic epidemiology of isolates have been limited but thus far the majority of the isolates belong to the clade I [et al. [C. auris isolates had >30 SNPs between them and in comparison to isolates isolated in earlier years from another tertiary hospital, the SNP differences were large (>100 SNPs).Four major geographic clades of  clade I . This wa [et al. . Here, tC. auris which has repeatedly caused global outbreaks [Candida spp. can also occur. Although the overall population structure of C. glabrata, C. tropicalis, C. parapsilosis, C. albicans, and C. krusei was made up of diverse clones and not dominated by a single clone, several clusters of isolates were noted  isolates. These were separated between four clusters with varying azole-resistance  clustered in a single azole-resistant C. tropicalis cluster predominantly involving haematology-oncology patients in haematology-oncology wards (21–68 SNPs) . There were no overlaps in the primary treating team for patients and often cases were located in different wards and were months to years apart. This suggests that patient-to-patient spread may play a less significant role however transmission between patients without clinical infection cannot be excluded. The sporadic nature (spread out over years and location) may also indicate intermittent breaks in infection control procedures and act as a signal to remind healthcare workers to maintain vigilance in daily care of patients. The interspersed nature of these isolates also suggests the possibility of these isolates being part of a lineage that has successfully adapted in hospital/healthcare environments. Clonal outbreaks of other Candida spp. has been reported, particularly isolates with acquired azole-resistance [Clustering of isolates occurred most commonly with sistance . This wa68 SNPs) . Two clusistance . This maCandida spp. have been demonstrated to survive on inanimate surfaces, particularly if there is moisture [C. albicans appears to be least viable on inanimate surfaces, which correlates with our findings that they are most heterozygous with no evidence of C. albicans transmission between different patients. Conversely, C. parapsilosis survives well on inanimate surfaces, which supports its potential as a persistent nosocomial pathogen [moisture . C. albipathogen .C. tropicalis, and multiple clusters of C. parapsilosis. Azole-susceptibilities of C. parapsilosis varied between and within the clustered isolates. Clustering amongst other Candida spp. (with the exception of C. auris) was not evident. This may be due to endogenous infections being a more common source of some Candida spp. while exogenous  being more common for C. tropicalis and C. parapsilosis. Environmental testing was not performed for this study but would be of interest for future research directions to identify reservoirs that may contribute to nosocomial candidaemia infections. Other factors that were not investigated include antifungal usage and patient-specific risk factors.In summary, we report the genomic epidemiology of candidaemia isolates, demonstrating a single cluster of isolates in azole-resistant Future investigations should investigate these factors in parallel in order to delineate relative risk that each factor contributes. This may identify potential interventions that can reduce overall candidaemia rates in hospital and particularly transmission of drug-resistant isolates.Click here for additional data file.


Paracoccidioides lutzii causes systemic mycosis Paracoccidioidomycosis (PCM), which presents a broad distribution in Latin America. Upon infection, the fungus undergoes a morphological transition to yeast cells and provokes an inflammatory granulomatous reaction with a high number of neutrophils in the lungs. In this work, we employed proteomic analysis to investigate the in vitro response of the fungus to the interaction with human neutrophils. Proteomic profiling of P. lutzii yeast cells harvested at 2 and 4 h post interaction with human polymorphonuclear cells allowed the identification of 505 proteins differentially accumulated. The data indicated that P. lutzii yeast cells underwent a shift in metabolism from glycolysis to Beta oxidation, increasing enzymes of the glyoxylate cycle and upregulating enzymes related to the detoxification of oxidative and heat shock stress. To our knowledge, this is the first study employing proteomic analysis in the investigation of the response of a member of the Paracoccidioides genus to the interaction with neutrophils.The fungal pathogen  Paracoccidioides. At present, PCM is spread in Central and South America, with a higher prevalence in Brazil 1-Fibrinopeptide B human ([M + 2H]2+ = 785.8426) at 200 fmol/µL  was used to ensure accuracy during the analysis. Mas spectrometric experiments were performed on a Synapt G1 MSTM  equipped with a NanoElectronSpray source and mass analyzers hybrid quadrupole/time-of-flight (TOF) operating in V-mode. The mass spectrometric full-scan data were acquired in the positive ion mode nano-ESI(+) in the m/z range of 50–2000 with a scan time of 0.4 s. The spectrometer was automatically programmed to switch between low collision energy MS (6 V) and elevated collision energies (40 V). The peptide fractions were analyzed in triplicates.Chromatography was performed on a precolumn nanoEaseP. lutzii database . The parameters were as follows: strict trypsin specificity, allowing one missed cleavage; minimum peptide length was five amino acids; the detection of at least two fragment ions per peptide; carbamidomethylation of cysteine was a fixed modification; maximum protein mass (600 kDa); and phosphorylation STY of proteins and oxidation of methionine were set as variable modifications. A minimum of two peptides were required for protein identification, and peptide spectral matches were filtered using a target–decoy approach at a false discovery rate (FDR) of 4%. Proteins identified with low accuracy were excluded, and proteins with two or more peptides were admitted in at least two out of three technical replicate injections. The identified proteins were organized by the expression algorithm into a statistically significant list corresponding to induced and reduced regulation ratios between internalized and control. The mathematic model used to calculate the ratios is part of the expression algorithm inside the PLGS from Waters Corporation [www.PeptideAtlas.org, accessed on 15 February 2023) with the data set identifier: PASS03809 . Regulated proteins were functionally categorized using FungiDB , Uniprot  and KEGG  databases. The annotation of non-characterized proteins was performed via homology from the proteins present in the NCBI . For the experiment dynamic range, the peptide parts per million error (ppm) and peptide detection type using software such as MassPivot v1.0.1. and Spotfire v8.0. Microsoft Excel (Microsoft®), which was also used for table manipulations. The data obtained via mass spectrometry of the internalization assay was also analyzed against the Human neutrophil database .Raw files were analyzed together using the ProteinLynx Global Server software version 3.0.2 (PLGS) . The protein identifications and quantitative packaging were generated using specific algorithms ,26, and poration . The sofp-values < 0.05 were considered statistically significant. Bar charts and heat maps were generated through the use of GraphPad Prism 8.0.1 software .Excel software (Microsoft Office 365) was used to run the statistical analysis, and data with P. lutzii during the infective process in human neutrophils in 2 and 4 h. Microscopical analysis depicts P. lutzii internalized by human neutrophils, as demonstrated via staining the preparation with Giemsa , enoyl CoA hydratase (PAAG_06309) and 3 ketoacyl CoA thiolase (PAAG_02664), were also increased after neutrophil internalization, suggesting a potential utilization of fatty acids as fuel within neutrophils.Enzymes of beta oxidation were increased in yeast cells internalized by the neutrophils, as depicted in The interaction with neutrophils also increased the expression of proteins related to amino acid catabolism. One subunit from 2-oxoisovalerate dehydrogenase (PAAG_01310), which generates acetyl-CoA through the degradation of valine, leucine and isoleucine, was increased after 4 h of neutrophil internalization. The glutaryl CoA dehydrogenase (PAAG_05984) transforms glutaryl-CoA into 2-trans-enoyl-CoA, which enters the beta oxidation or into crotonoyl-CoA in the lysine and tryptophan oxidation pathways, whose final product is acetyl-CoA. The enzymes from the pyruvate dehydrogenase complex, dihydrolipoyl dehydrogenase (PAAG_03330), pyruvate dehydrogenase E1 component  and pyruvate dehydrogenase protein X component (PAAG_00050), were increased upon 4 h of neutrophil internalization. This complex is responsible for the oxidation and decarboxylation of pyruvate to produce acetyl-CoA. Dihydrolipoyl dehydrogenase can also participate in the degradation of valine, leucine and isoleucine, producing acetyl-CoA, together with 2-oxoisovalerate dehydrogenase and pyruvate dehydrogenase X component (PAAG_02769), which was exclusively found at both neutrophil interaction times. Most of the cited enzymes had their expression increased over time, suggesting a need for acetyl-CoA production during neutrophil internalization.3 in the presence of NAD+, thus providing the compound to the TCA cycle. The enzyme alanine glyoxylate aminotransferase (PAAG_03138), which releases pyruvate, also increased. The enzymes 4 aminobutyrate aminotransferase (PAAG_00468) and succinate semialdehyde dehydrogenase (PAAG_08718) belong to the GABA shunt, where the first converts GABA and α-ketoglutarate into succinate semialdehyde, which is then converted into succinate by the second enzyme. The argininosuccinate lyase (PAAG_06407) releases arginine and fumarate, a TCA intermediate, also increased. Another increased enzyme was phosphoglycerate kinase (PAAG_02869), which produces 3-phosphoglycerate, an isocitrate dehydrogenase regulator, which leads to an increase in TCA [In the present assay, enzymes related to the TCA pathway increased upon neutrophil interaction, as depicted in e in TCA . The incP. lutzii yeast cells interacting with neutrophils. As observed, the figure depicts the integration of metabolic pathways presumably affected by neutrophils in P. lutzii. The fungal cells internalized by neutrophils may undergo a metabolic shift where two regulatory enzymes are decreased. The first is 6-phosphofructo-2-kinase (PFK) from glycolysis, and the other is isocitrate dehydrogenase (IDH) from TCA. The enzyme pyruvate dehydrogenase (PDH) exhibited an increase, potentially aimed at supporting the TCA cycle, which might be affected due to the reduced expression of IDH. In addition, malate synthase (MLS) is induced, taking part in anaplerotic reactions that enable cells to use substrates that enter central carbon metabolism; the amino acid synthesis is decreased. Finally, enzymes related to reactive oxygen species protection increased , suggesting that P. lutzii cells may be exposed to elevated levels of reactive species within neutrophils.P. lutzii. P. lutzii. Among those proteins, we can see Azurocidin (CAP7_HUMAN), a serine protease with antimicrobial effects. Additionally, defensin 1 (DEF1_HUMAN), a major family of antimicrobial peptides expressed predominantly in neutrophils and epithelial cells, was detected. Additionally, proteins that compose NETs, such as elastase (ELNE_HUMAN), myeloperoxidase (PERM_HUMAN), lactotransferrin (TRFL_HUMAN) and histones, were detected in neutrophils during interaction with P. lutzii.We also focused on neutrophil proteins when interacting with P. lutzii upon interaction with neutrophils. The environment within neutrophils induces a series of metabolic changes in P. lutzii, including proteins related to the response to the formation of oxidative species that leads to metabolic stress. This was also confirmed in C. albicans through the use of different approaches [C. albicans internalized by neutrophils overexpresses enzymes that detoxify oxidative species, such as SOD [Cryptococcus neoformans in a macrophage infection assay [Histoplasma capsulatum yeast cells from host-derived oxidative stress. Mutant strains lacking the SOD gene reduced the ability of fungi to initiate respiratory infections due to the rapid clearance performed by the immune system [P. brasiliensis with human PMNs led to an increase in SODs, including SOD1, as seen in the present work. The silencing of SOD1 provoked fungal susceptibility to PMNs, decreased fungal burden in the lung, eliminated fungal burden in the liver and impaired the ability to eliminate intracellular H2O2 [P. lutzii during oxidative stress provoked by H2O2 and macrophage infection [P. brasiliensis during macrophage [C. albicans [Paracoccidioides spp., and inhibitors reduce fungal load in infected mice [In this work, we focused on the analysis of proteins regulated in proaches ,31. To ah as SOD . Anotheron assay  showed te system . The intlar H2O2 . Similarnfection  and in Pcrophage  and muricrophage  infectioalbicans . In addited mice .P. lutzii by neutrophils increased proteins of β-oxidation, as expected. Previous studies have shown that pathogens infecting macrophages can catabolize host lipids [P. brasiliensis internalized by macrophages [P. lutzii internalized by neutrophils. Enzymes from the pyruvate dehydrogenase complex increased in P. lutzii after neutrophil internalization, similar to the findings in macrophage infection [P. brasiliensis and P. restrepiensis exoantigen [P. lutzii secreted proteins [The internalization of t lipids . Similarrophages , as wellrophages , both shnfection . From thoantigen ,42. Consproteins . Additioproteins . These dC. albicans showed ineffective growth on arginine as a sole carbon and nitrogen source. The enzyme activity favors NADPH production as a way to fulfill the energy requirements of cells under limitations in terms nutrients, such as inside host cells [C. albicans [P. brasiliensis [Our proteomic results depicted an increase in glutamate dehydrogenase (GDH), an enzyme that releases α-ketoglutarate. The latter is critical for maintaining the α-ketoglutarate pool in the cytosol and feeding TCA as an alternative for ATP production in starving pathogenic cells. A high-throughput assay testing the effect of glutamate dehydrogenase deletion in st cells . Additioalbicans . Of the iliensis . This suP. lutzii internalized by neutrophils; they are phosphoglycerate kinase (PGK) and glucose-6-phosphate 1-epimerase. PGK can be modulated by stress and under limited nutritional conditions. Higher levels of this enzyme were found in C. albicans in conditions of intense need for energy [C. neoformans [C. albicans [P. lutzii, a secreted PGK was identified as a plasminogen-binding protein [P. brasiliensis after macrophage infection [Two glycolytic enzymes were increased in r energy . In addir energy  and fungr energy  and is poformans  and C. aalbicans  cell wal protein , suggestnfection ; this enP. lutzii when internalized by neutrophil. It plays an important role in C. neoformans infection, and its absence significantly reduces cell fitness and fungal burden in host cells and makes the pathogen hypersusceptible to fluconazole [C. neoformans [A. fumigatus deprived of 3-isopropylmalate dehydratase has its virulence attenuated [C. neoformans under the same conditions does not grow in a leucine-limited medium, showing decreased pathogenicity [In this way, the enzyme ATP-citrate lyase (ACL), which sustains the replenishment of the TCA cycle, is increased in conazole . Anotheroformans . A similtenuated , and C. genicity .Paracoccidioides spp. assays under different conditions, such as during infection of alveolar macrophages [P. lutzii upon neutrophil internalization. The same was observed in P. brasiliensis internalized by macrophages [C. albicans during neutrophil internalization [S. cerevisiae and C. albicans [C. neoformans, ICL increased in fungus isolated from cerebrospinal fluid [C. albicans [P. brasiliensis, ICL increased during mouse lung infection [P. lutzii potentially shifts to a non-glycolytic substrate metabolism to circumvent the lack of carbohydrates within the neutrophils.Another TCA enzyme, succinyl-CoA ligase (SCL), plays a key role in cellular metabolism, and its increase in this assay suggests that the fungal cells internalized by neutrophils possibly undergo severe metabolic changes. This enzyme is overexpressed in several rophages , oxidatirophages  and durirophages ,58. The rophages  and in Clization . The rolalbicans ,60. In Cal fluid  and afteal fluid ; howeveralbicans . In P. bnfection . The incP. lutzii after neutrophil internalization. The enzymeshorismitee synthase (CS), asparagine synthetase (AS) and glutamine synthase (GS) decreased in the present assay. Fungal cells subjected to stress conditions tend to inhibit the synthesis of amino acids since the process is energetically expensive. Stress conditions mimic the microenvironment faced by the pathogen within host cells during infection. AS decreased in a proteomic approach that assessed metabolic alterations of P. lutzii under carbon starvation [P. brasiliensis internalized by macrophages [The amino acid biosynthesis decreased In arvation , and GS arvation . Both corophages .P. lutzii. One of the identified proteins is Azurocidin, a protein with antimicrobial effects that is rapidly produced by migrating PMN. Additionally, Azurocidin may have a crucial role as a mediator of the initiation of the immune response by alerting the immune system through different mechanisms, such as the activation of monocytes and macrophages [P. lutzii. Analyses of the role of the protein components of NETs during the internalization of C. albicans identified that the proteins elastase, myeloperoxidase, lactotransferrin and histones—also identified in the present work—were involved in the interaction with the fungal cell surface [P. brasiliensis, presenting a fungistatic effect [Another relevant finding is the detection of some proteins in neutrophils after the internalization of rophages . Additio surface . It has c effect . ROS genc effect ,67. The P. lutzii to in vitro interaction with human neutrophils. Quantitative and qualitative changes in the set of proteins related to beta oxidation, stress response, glyoxylate cycle, among others, demonstrated a potential shift in metabolism, as well as the fungus response to the hostile environment of neutrophils and provided new molecules to focus on in new studies involving fungal responses to a host.In synthesis, we employed mass spectrometry coupled with high-performance liquid chromatography to analyze the response of the dimorphic fungus


Porphyromonas gingivalis. We previously reported an antagonistic relationship between Streptococcus cristatus and P. gingivalis and the role of S. cristatus in inhibition of the biofilm formation, invasion, and gingipain enzymatic activity of P. gingivalis. Given the importance of P. gingivalis as a keystone pathogen of polymicrobial communities, the determinants of P. gingivalis levels, its interaction with the core microbiota, and association with the pathogenic potential of the microbial communities need to be addressed.Periodontitis has been recently defined as a dysbiotic disease resulting from imbalanced oral microbiota. The transition of microbial communities from commensal to periodontitis-associated ones likely requires colonization by specific pathogens, including S. cristatus in altering interactions of P. gingivalis with other oral bacteria in a complex context. We collected dental plaque samples from periodontitis patients and assigned them into two groups based on their ratios of S. cristatus and P. gingivalis. We then characterized microbial profiles of the dental plaque samples using shotgun metagenomic sequencing and subsequently compared oral microbial composition and functional capabilities between groups with high or low S. cristatus-P. gingivalis ratios. Taxonomic annotation showed significant differences in microbial compositions at both genus and species levels between the two groups. Notably, a higher microbial composition diversity was observed in the samples with low S. cristatus-P. gingivalis ratios. The antibiotic resistance gene profiles of the two groups are also distinct, with significantly increased diversity and abundance of antibiotic resistance genes in the dental plaque samples with low S. cristatus-P. gingivalis ratios, which likely lead to elevated virulence potential.This present study intends to determine the role of S. cristatus-P. gingivalis ratios in influencing the virulence of the oral microbiome. Approaches to enhance S. cristatus-P. gingivalis ratios in oral microbial communities will be attractive for revising the dysbiotic oral microbiome.Overall, our work highlights the importance of  Periodontitis is one of the most common diseases in humans, affecting ~ 42% of adults aged 30 years and older in the US. The prevalence of periodontitis is influenced by multiple factors including age, gender, socioeconomic status, and race/ethnicity . The oraPorphyromonas gingivalis is known to play a vital role in the development of dysbiotic microbial communities and can disrupt host-microbial homeostasis and induce inflammatory responses by acting with other oral bacteria. Therefore, it is considered a keystone-pathogen of periodontitis  that can effectively inhibit biofilm formation, invasion, and gingipain enzymatic activity of P. gingivalis  periodontitis patients, compared to those from African Americans (AAs) and Hispanic Americans (HAs) patients . Candidates were screened during routine dental visits in clinic at the School of Dentistry, University of Texas Health Science Center at Houston between 2017 and 2022. Individuals aged 21 –75 with self-reported ethnicity/race of AAs, CAs, or HAs were enrolled after the initial periodontal examination that included determination of plaque index (PI) and bleeding on probing (BOP) . RadiogrDental plaque samples were collected by board certified periodontists using sterile paper points at baseline prior to any dental treatment and labelled with numbers according to sampling sequences. The paper points were placed in ≥ 5mm pockets in different quadrants for 1 minute and then immersed immediately in an Eppendorf tube with 0.5 ml of Tris-EDTA (TE) buffer (pH 7.5) . Oral baThe samples were sent to Novogene Co.  for metagenomic sequencing. Briefly, DNA extracted from human dental plaques was randomly sheared into short fragments. The obtained fragments were end repaired, A-tailed and further ligated with Illumina adapters. The fragments with adapters were then PCR amplified, size selected, and purified. Quality control of the library was conducted with Qubit  and real-time PCR for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on an Illumina NovaSeq high throughput sequencer by Novogene Corporation, Inc., with paired-end sequencing length of 150bp and an output of ~ 6GB raw data per sample.The amount of raw data generated per sample is 6.276GB on average. To ensure accuracy and reliability of the subsequent data analysis, we trimmed all low-quality bases. We also discarded the reads which contain uncalled N nucleotide stretching over 10bp and reads which overlap with adapter for more than 15 bp. To minimalize host DNA contamination, we also discarded the raw reads mapping to the human reference genome using Bowtie2 . After qSoftware MetaGeneMark (version 2.10) was used to predict Open Reading Frames (ORFs) from Scaftigs (≥ = 500bp) , 21. ORFt-tests were performed to compare the samples with high S. cristatus and P. gingivalis ratios to those with low ratios. The t-tests were two-sided, and an assumption of identical variances on the sample distributions was used. The threshold of statistical significance was set to p-value ≤ 0.05.Statistical analyses were performed using scipy.stats in SciPy (version 1.4.1), an open-source Python library for scientific computing. Independent two sample P. gingivalis and its antagonistic species S. cristatus in the composition of the oral microbiota, we selected 14 samples with relatively low S. cristatus-P. gingivalis ratios (< 1) and 16 samples with the higher ratios (> 100) for metagenome shotgun sequencing. The general characteristics of the participants included in the present study is shown in We previously investigated the oral microbial profiles of dental plaques derived from periodontitis patients with different racial/ethnic backgrounds and using qPCR measured the distribution and levels of several well-studied oral bacteria in their dental plaque samples. These included keystone pathogens, accessory pathogens, and pathobionts . To examS. cristatus-P. gingivalis ratio (< 1). While the mean and median of gene counts is 294,355 and 316,927, respectively, in samples with higher S. cristatus-P. gingivalis ratio (> 100). Although a statistically significant difference in numbers of the non-redundant genes was not reached between the groups with different S. cristatus-P. gingivalis ratios, significant abundance dissimilarities were found at bacterial genus and species levels between these groups using ANOSIM analysis, a non-parametric test based on ranked dissimilarity measure (p = 0.016) and the species level . Moreover, non-redundant genes were observed in both the groups as shown in Venn diagrams , while 113,466 (11.75%) were observed in the group with the high ratios (G2). Further, taxonomic annotations identified a total of 1484 microbial species in the cohort. Microbial taxa were found more diverse in the samples with low S. cristatus-P. gingivalis ratios. A total of 1201 species were identified in the group with low S. cristatus-P. gingivalis ratios, while only 630 species in the group with high S. cristatus-P. gingivalis ratios. Among the 1201 microbial taxa annotated in the group with low S. cristatus-P. gingivalis ratios, 854 group-specific species (58.6%) were found compared to 283 unique species (19.1 %) in the group with the high ratios. A total of 347 species annotated in both groups. These results indicate a more complex profile of microbial species in the group with low S. cristatus-P. gingivalis ratios , with samples within each group gathering closer together. The PCA plot in We also conducted Principal Coordinates Analysis (PCoA), Principal Component Analysis (PCA), and Non-metric MultiDimensional Scaling (NMDS) analysis, an indirect gradient analysis approach that produces an ordination based on a distance matrix. S. cristatus-P. gingivalis ratios. As shown in P. gingivalis, Treponema denticola, and Desulfomicrobium orale, out of the 12 most abundant taxa, were detected 56.08, 5.83, and 5.62-fold more abundant, respectively, in samples with low S. cristatus-P. gingivalis ratios, compared to the samples with high ratios. The other nine common bacterial taxa  were 1.80-fold to 6.77-fold more abundant in samples with the high ratios than those with the low ratios. In addition, the top 35 microbial taxa with differential abundance between groups are shown in an abundance heatmap   , Carbohye (CARD) , Kyoto Ee (CARD) , and Gene (CARD) . As shows ratios . These rS. cristatus-P. gingivalis. The results revealed that genes involved in human diseases-associated antimicrobial drug resistance were enriched in the samples with the low ratios (p = 0.0498) (p = 0.0324), which was in agreement with the analyses on the CAZy database , energy (p = 0.0463), amino acid metabolism (p = 0.0403) and nucleotide (p = 0.0296) as well as glycan biosynthesis and metabolism (p = 0.0495) were significantly abundant in the group with high ratios  . While gdatabase . The funh ratios . HoweverS. cristatus on violence potential of P. gingivalis and on formation and composition of oral microbial biofilms using qPCR   . Our disS. cristatus-P. gingivalis ratios, significant differences in abundance and diversity of bacterial species as well as functional pathways between the two groups exist. We postulate that S. cristatus-P. gingivalis ratios associated increase in abundance and diversity of periodontitis- associated bacteria and antibiotic resistance genes accelerate progression of periodontitis and reduce response to periodontitis treatment. This notion is supported by our previous observations that the ratio of S. cristatus-P. gingivalis was significantly higher in Caucasians Americans (CAs) than in Africa Americans (AAs) and that better gains in clinical attachment levels were observed in CA periodontitis patients compared to those found in AAs after nonsurgical periodontal treatment (As reported in several studies of 16S rRNA sequencing , 44, abureatment , 45.S. cristatus-P. gingivalis ratios in virulence potentials of the oral microbiome. Microbial communities with low S. cristatus-P. gingivalis ratios had an elevated level of several well-studied periodontitis-associated bacteria, decreased levels of streptococcus and actinomyces species, and much diversified microbial profiles and antibiotic resistance genes. Differential diversity and abundance of the oral microbiome are likely regulated by core bacteria in microbiota, including the ratios of S. cristatus-P. gingivalis, which may lead to functional changes of the oral microbiome. Therefore, approaches to enhance S. cristatus-P. gingivalis ratio will be attractive for rebuilding and maintaining healthy oral microbiome.In conclusion, our work highlights the role of


Songling virus (SGLV), a newly discovered tick-borne orthonairovirus, was recently identified in human spleen tissue. It exhibits cytopathic effects in human hepatoma cells and is associated with clinical symptoms including headache, fever, depression, fatigue, and dizziness, but no treatments or vaccines exist for this pathogenic virus. In the current study, immunoinformatics techniques were employed to identify potential vaccine targets within SGLV by comprehensively analyzing SGLV proteins. Four proteins were chosen based on specific thresholds to identify B-cell and T-cell epitopes, validated through IFN-γ epitopes. Six overlap MHC-I, MHC-II, and B cell epitopes were chosen to design a comprehensive vaccine candidate, ensuring 100% global coverage. These structures were paired with different adjuvants for broader protection against international strains. Vaccine constructions’ 3D models were high-quality and validated by structural analysis. After molecular docking, SGLV-V4 was selected for further research due to its lowest binding energy  and its suitable immunological and physiochemical properties. The vaccine gene is expressed significantly in E. coli bacteria through in silico cloning. Immunological research and MD simulations supported its molecular stability and robust immune response within the host cell. These findings can potentially be used in designing safer and more effective experimental SGLV-V4 vaccines.  This muin silico method has gained significant appeal  (The systematic methodology employed in this study to design a multi-epitope vaccine construct targeting the s (SGLV) Figure 12.1Songling virus proteome data was downloaded from the National Centre for Biotechnology Information (NCBI) database under reference taxonomy ID: 2795181, and sequences were verified from Virus Pathogen Resource (ViPR) (e (ViPR) . BLASTp e (ViPR) . For idee (ViPR) . The ante (ViPR) . For thee (ViPR) .2.2MHC-I epitopes were predicted using the IEDB MHC-I stickiness predictions program  accessedhttp://crdd.osdd.net/raghava/abcpred/) tool was used for this analysis  was utilized to check the designed vaccine had successfully covered the entire world population   .2.5http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/) was used to determine the secondary structure of the new vaccine construct, which was then validated through the PSIPRED server (http://bioinf.cs.ucl.ac.uk/psipred/) (https://swissmodel.expasy.org/) was used to model proteins, and then ProSAweb Server (https://prosa.services.came.sbg.ac.at/prosa.php) was used to analyze protein structure and validate the model (https://servicesn.mbi.ucla.edu/ERRAT/) and RAMPAGE server (http://mordred.bioc.cam.ac.uk/rapper/rampage.php) (PDBsum (sipred/) –24. SWIShe model , 26. Godage.php) .2.6http://hdock.phys.hust.edu.cn/) and HawkDock (http://cadd.zju.edu.cn/hawkdock/), in order to evaluate the chemical reactions between immune receptors  and vaccine constructs   . Another and V4) .2.7https://imods.iqfr.csic.es/)  research simulation. MD simulations, energy efficiency, and protein flexibility were all calculated using the iMODS web server (sic.es/) . iMODS isic.es/) .2.8http://www.cbs.dtu.dk/services/C-ImmSim-10.1/) was used to model computational immunological simulation of our prioritized vaccine design. This platform employs a potent combination of predictive modeling techniques, including Position-Specific Scoring Matrices (PSSM) and a variety of cutting-edge machine-learning algorithms, to assess and predict the cellular and humoral responses elicited by our antigenic vaccine candidate (The C-ImmSim webserver (andidate . The appandidate . Our focandidate .2.9http://www.jcat.de) was used to optimize the coding and execute a reverse translation of the sequence of amino acid sequences for the suggested immunization (The JAVA Codon optimization Tool (Jcat)  was employed to acquire the mRNA sequence of the vaccine. To predict the secondary structure of the vaccine mRNA, two web-based servers, Mfold v2.3 (http://www.unafold.org/mfold/applications/rna-folding-form-v2.php) (http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi) (The Transcription and Translation web based Tool (-v2.php)  and RNAfold.cgi) , were ut33.1The complete proteome of the SGLV strain, containing 40 proteins from different strains across the world, was extracted from NCBI in FASTA format  epitopes were determined for the selected proteins using the IEDB server, with an ICf twelve  overlappf twelve Table 1.The epitopes chosen exhibited 100% coverage across the global population Figure 33.3To generate a multi-epitope vaccine EAAAK, CTL, HTL, and GGGS/HEYGAEALERAG linkers and adjuvants were used. When administered intramuscularly, vaccine containing linkers offer superior protection against each epitope . Increas3.4Based on their immunological features, none of the immunization strategies were found to be harmful or allergic. Each one of the multi-epitope vaccine formulations has a substantial antigenic property, as demonstrated by antigenicity scores > 0.8 as estimated by the VaxiJen 2.0 server. Using cross-validation on the peptide sequence based on established datasets, VaxiJen 2.0 determines the antigenicity of viral sequences and identifies their protective properties. Each structure VaxiJen 2.0 score fell between 0.4333 and 0.5197, which is the same as the default threshold for viruses . Using t3.5Computational approaches that anticipated secondary structure components were used to analyze the structural characteristics of the vaccine constructs. The PDBsum server displays proteins in their residue conservation and 2D structure. It shows which parts of the protein are not the same (colored blue) and which parts are very similar (colored red). 3.6Molecular docking is used for predicting the suitable binding between multi-epitope vaccine (MEV) and receptor molecules. Human surface TLR3, TLR4, and TLR8 immune system receptors were used to dock MEV with the help of Hdock Server (a blind docking technique) and Hawkdock Server. In the result, only one structure is prioritized for each dock based on its high score and lowest binding energy Table 4.3.7The TLR4 receptor was chosen due to its lowest binding energy with the SGLV-V4 construct. To comprehensively evaluate the stability of proteins and the enthalpy efficiency within SGLV-V4-TLR4 complexes, molecular dynamics (MD) simulations were employed. In parallel, the iMODS platform facilitated an in-depth analysis of atomic and molecular movements within the vaccine’s biological context, elucidating macromolecular mobility via the normal mode analysis (NMA) methodology. For a more detailed understanding, Furthermore, NMA-derived B-factor analysis was instrumental in portraying the relative amplitude of atomic displacements within the molecular complex. 3.8The focused MEV significantly boosted secondary responses, as predicted by immune modeling. In principle, this sequence can help the immune system quickly respond to threats. High levels of IgM were the prime simulated response. The simulated secondary and tertiary responses revealed considerable increases in B-cell populations as well as high concentrations of IgG1 + IgG2, IgM, and IgM + IgG antibodies. However, there was a decrease in the antigen levels , which makes the E. coli K12 strain a great host organism. The estimated GC content of the improved sequence was 52.7%, which is significantly higher than the value of 50.73 found in E. coli. The modified sequence had a CAI (codon adaptation index) of 1.0. The multi-epitope vaccine (MEV) vector’s codon usage curve is shown in Designing a vaccine with an appropriate expression system is the initial stage in evaluating a vaccination candidate, which requires a serological study. Prior to 3.10in vivo.The RNAfold server predicts the vaccine mRNA’s secondary structure with a minimum free energy of -268.90 kcal/mol (4In response to the growing concern over the flow of Songling virus (SGLV) cases worldwide and the absence of available vaccines, this study investigated the challenge of preventing future Songling virus epidemics. Employing cutting-edge immunoinformatics techniques, we embarked on designing innovative multi-epitope Songling virus vaccine constructs by examining the proteome of the Songling virus to pinpoint targets for a potential vaccine. Using strict standards, they forecasted epitopes for B-cells, MHC-I, and MHC-II. These epitopes play a crucial role by sparking a protective immune response that blocks viruses and establishes long-term defense .The overlapped epitopes are prioritized from MHCI & II, and B-cell which are highly antigenic, non-allergen, and produce humoral response, that combats infections by eliminating infected cells or releasing antiviral substances to establish lasting immunity . To enhaMolecular docking analysis was then employed to explore the interaction between the vaccine constructs and the crucial immune cell receptors, i.e., TLRs. TLRs are known for their pivotal role in immune cell activation and the recognition of viral peptide structures . The resin vitro immunological assays to be conducted to confirm and validate the immunogenicity of the designed vaccine. Subsequently, a challenge-protection preclinical trial will be initiated, presenting a crucial opportunity to rigorously evaluate and substantiate the efficacy and safety of the SGLV-V4-TLR4 vaccine construct. These endeavors aim to provide a comprehensive framework to combat Songling virus infections effectively, potentially mitigating their impact and safeguarding public health against this evolving threat. However, to determine the vaccine’s safety and efficacy, further experimental validation is required, which may involve the production of vaccine proteins with thorough in vivo and in vitro tests. However, the current research relies entirely on the results of computational approaches for technical equipment.As we progress toward the next critical steps, we anticipate 5In our study on Songling virus (SGLV), a tick-borne pathogen lacking treatment or vaccines, we employed immunoinformatics to identify four potential vaccine target proteins. Designing a comprehensive vaccine candidate with broad global coverage, we combined B-cell and T-cell epitopes and validated them through IFN-γ epitopes. SGLV-V4, selected for its strong performance in molecular docking and favorable properties, was efficiently expressed in E. coli bacteria. Immunological research and simulations confirmed its stability and robust immune response, offering a promising avenue for safer and more effective SGLV-V4 vaccine development.The original contributions presented in the study are included in the article/SA: Writing – original draft, Writing – review & editing, Conceptualization, Data curation, Formal Analysis, Investigation, Methodology. AAli: Writing – original draft, Writing – review & editing, Investigation, Software, Visualization. AAla: Funding acquisition, Validation, Writing – review & editing. AB: Validation, Writing – review & editing. MD: Writing – review & editing. AAb: Writing – review & editing.