Putative Outer Membrane Proteins Research Articles

Calcium Induces the Cleavage of NopA and Regulates the Expression of Nodulation Genes and Secretion of T3SS Effectors in Sinorhizobium fredii NGR234.

The type III secretion system (T3SS) is a key factor for the symbiosis between rhizobia and legumes. In this study, we investigated the effect of calcium on the expression and secretion of T3SS effectors (T3Es) in Sinorhizobium fredii NGR234, a broad host range rhizobial strain. We performed RNA-Seq analysis of NGR234 grown in the presence of apigenin, calcium, and apigenin plus calcium and compared it with NGR234 grown in the absence of calcium and apigenin. Calcium treatment resulted in a differential expression of 65 genes, most of which are involved in the transport or metabolism of amino acids and carbohydrates. Calcium had a pronounced effect on the transcription of a gene (NGR_b22780) that encodes a putative transmembrane protein, exhibiting a 17-fold change when compared to NGR234 cells grown in the absence of calcium. Calcium upregulated the expression of several sugar transporters, permeases, aminotransferases, and oxidoreductases. Interestingly, calcium downregulated the expression of nodABC, genes that are required for the synthesis of nod factors. A gene encoding a putative outer membrane protein (OmpW) implicated in antibiotic resistance and membrane integrity was also repressed by calcium. We also observed that calcium reduced the production of nodulation outer proteins (T3Es), especially NopA, the main subunit of the T3SS pilus. Additionally, calcium mediated the cleavage of NopA into two smaller isoforms, which might affect the secretion of other T3Es and the symbiotic establishment. Our findings suggest that calcium regulates the T3SS at a post-transcriptional level and provides new insights into the role of calcium in rhizobia-legume interactions.

Characterisation of Putative Outer Membrane Proteins from Leptospira borgpetersenii Serovar Hardjo-Bovis Identifies Novel Adhesins and Diversity in Adhesion across Genomospecies Orthologs.

Leptospirosis is a zoonotic bacterial disease affecting mammalian species worldwide. Cattle are a major susceptible host; infection with pathogenic Leptospira spp. represents a public health risk and results in reproductive failure and reduced milk yield, causing economic losses. The characterisation of outer membrane proteins (OMPs) from disease-causing bacteria dissects pathogenesis and underpins vaccine development. As most leptospire pathogenesis research has focused on Leptospira interrogans, this study aimed to characterise novel OMPs from another important genomospecies, Leptospira borgpetersenii, which has global distribution and is relevant to bovine and human diseases. Several putative L. borgpetersenii OMPs were recombinantly expressed, refolded and purified, and evaluated for function and immunogenicity. Two of these unique, putative OMPs (rLBL0972 and rLBL2618) bound to immobilised fibronectin, laminin and fibrinogen, which, together with structural and functional data, supports their classification as leptospiral adhesins. A third putative OMP (rLBL0375), did not exhibit saturable adhesion ability but, together with rLBL0972 and the included control, OmpL1, demonstrated significant cattle milk IgG antibody reactivity from infected cows. To dissect leptospire host-pathogen interactions further, we expressed alleles of OmpL1 and a novel multi-specific adhesin, rLBL2618, from a variety of genomospecies and surveyed their adhesion ability, with both proteins exhibiting divergences in extracellular matrix component binding specificity across synthesised orthologs. We also observed functional redundancy across different L. borgspetersenii OMPs which, together with diversity in function across genomospecies orthologs, delineates multiple levels of plasticity in adhesion that is potentially driven by immune selection and host adaptation. These data identify novel leptospiral proteins which should be further evaluated as vaccine and/or diagnostic candidates. Moreover, functional redundancy across leptospire surface proteins together with identified adhesion divergence across genomospecies further dissect the complex host-pathogen interactions of a genus responsible for substantial global disease burden.

LIC12254 Is a Leptospiral Protein That Interacts with Integrins via the RGD Motif.

Pathogenic leptospires can bind to receptors on mammalian cells such as cadherins and integrins. Leptospira effectively adheres to cells, overcomes host barriers and spreads into the bloodstream, reaching internal target organs such as the lungs, liver and kidneys. Several microorganisms produce proteins that act as ligands of integrins through the RGD motif. Here, we characterized a leptospiral RGD-containing protein encoded by the gene lic12254. In silico analysis of pathogenic, intermediate and saprophytic species showed that LIC12254 is highly conserved among pathogenic species, and is unique in presenting the RGD motif. The LIC12254-coding sequence is greatly expressed in the virulent Leptospira interrogans L1-130 strain compared with the culture-attenuated L. interrogans M20 strain. We also showed that the recombinant protein rLIC12254 binds to αVβ8 and α8 human integrins most likely via the RGD motif. These interactions are dose-dependent and saturable, a typical property of receptor-ligand interactions. The binding of the recombinant protein lacking this motif-rLIC12254 ΔRAA-to αVβ8 was almost totally abolished, while that with the α8 human integrin was decreased by 65%. Taken together, these results suggest that this putative outer membrane protein interacts with integrins via the RGD domain and may play a key role in leptospirosis pathogenesis.

The AtoC family response regulator upregulates an operon encoding putative outer membrane proteins sorted by type IX secretion system in Porphyromonas gingivalis

ObjectivesPorphyromonas gingivalis, a keystone periodontopathogen, has multiple two-component systems that are thought to modulate virulence. In this study, we focused on PGN_0775 response regulator (RR), an AtoC homolog, and attempted to identify the target gene that it regulates in P. gingivalis. MethodsComparative proteomic analyses comprising two-dimensional electrophoresis and peptide mass fingerprinting were applied to total protein samples from parent (WT) and atoC gene knockout (KO) strains to screen for affected protein spots. Fluctuations in the expression of corresponding genes were further confirmed using relative quantitative real-time polymerase chain reaction (RQPCR). ResultsFive protein spots with fluctuating expression levels were identified in pgn_0775 KO strains along with their masses and physiological features, which contained two hypothetical proteins with higher expression levels in the WT than in the KO strains. RQPCR analysis confirmed that mRNA levels were consistently decreased in KO and recovered in pgn_0775-complemented KO strains. The two hypothetical proteins appeared to be the products of an operon that comprises four genes encoding three hypothetical but putative type IX secretion system sorting domain-containing proteins and an N-terminal region of the C25 cysteine peptidase. ConclusionsThe AtoC RR homolog in P. gingivalis upregulates the expression of the operon encoding potentially antigenic proteins retained on the cell surface; thus, it could be a promising target for P. gingivalis-specific antivirulence therapy.

Design of a Multi-Epitopes Based Chimeric Vaccine against Enterobacter cloacae Using Pan-Genome and Reverse Vaccinology Approaches.

Enterobacter cloacae (EC) is a significant emerging pathogen that is occasionally associated with lung infection, surgical site infection, urinary infection, sepsis, and outbreaks in neonatal intensive care units. In light of the fact that there is currently no approved vaccine or therapeutic option for the treatment of EC, the current study was developed to concentrate on applications based on modern computational approaches to design a multi-epitope-based E. cloacae peptide vaccine (MEBEPV) expressing the antigenic determinants prioritized from the EC genome. Integrated computational analyses identified two potential protein targets (phosphoporin protein-PhoE and putative outer-membrane porin protein) for further exploration on the basis of pangenome subtractive proteomics and immunoinformatic in-depth examination of the core proteomes. Then, a multi-epitope peptide vaccine was designed, which comprised shortlisted epitopes that were capable of eliciting both innate and adaptive immunity, as well as the cholera toxin’s B-subunit, which was used as an adjuvant in the vaccine formulation. To ensure maximum expression, the vaccine’s 3D structure was developed and the loop was refined, improving the stability by disulfide engineering, and the physicochemical characteristics of the recombinant vaccine sequence were found to be ideal for both in vitro and in vivo experimentation. Blind docking was then used for the prediction of the MEBEPV predominant blinding mode with MHCI, MHCII, and TLR3 innate immune receptors, with lowest global energy of −18.64 kJ/mol, −48.25 kJ/mol, and −5.20 kJ/mol for MHC-I, MHC-II, and TLR-4, respectively, with docked complexes considered for simulation. In MD and MMGBSA investigations, the docked models of MEBEPV-TLR3, MEBEPV-MHCI, and MEBEPV-MHCII were found to be stable during the course of the simulation. MM-GBSA analysis calculated −122.17 total net binding free energies for the TLR3-vaccine complex, −125.4 for the MHC I-vaccine complex, and −187.94 for the MHC II-vaccine complex. Next, MM-PBSA analysis calculated −115.63 binding free energy for the TLR3-vaccine complex, −118.19 for the MHC I-vaccine complex, and −184.61 for the MHC II-vaccine complex. When the vaccine was tested in silico, researchers discovered that it was capable of inducing both types of immune responses (cell mediated and humoral) at the same time. Even though the suggested MEBEPV has the potential to be a powerful contender against E. cloacae-associated illnesses, further testing in the laboratory will be required before it can be declared safe and immunogenic.

Treponema pallidum genome sequencing from six continents reveals variability in vaccine candidate genes and dominance of Nichols clade strains in Madagascar.

In spite of its immutable susceptibility to penicillin, Treponema pallidum (T. pallidum) subsp. pallidum continues to cause millions of cases of syphilis each year worldwide, resulting in significant morbidity and mortality and underscoring the urgency of developing an effective vaccine to curtail the spread of the infection. Several technical challenges, including absence of an in vitro culture system until very recently, have hampered efforts to catalog the diversity of strains collected worldwide. Here, we provide near-complete genomes from 196 T. pallidum strains–including 191 T. pallidum subsp. pallidum–sequenced directly from patient samples collected from 8 countries and 6 continents. Maximum likelihood phylogeny revealed that samples from most sites were predominantly SS14 clade. However, 99% (84/85) of the samples from Madagascar formed two of the five distinct Nichols subclades. Although recombination was uncommon in the evolution of modern circulating strains, we found multiple putative recombination events between T. pallidum subsp. pallidum and subsp. endemicum, shaping the genomes of several subclades. Temporal analysis dated the most recent common ancestor of Nichols and SS14 clades to 1717 (95% HPD: 1543–1869), in agreement with other recent studies. Rates of SNP accumulation varied significantly among subclades, particularly among different Nichols subclades, and was associated in the Nichols A subclade with a C394F substitution in TP0380, a ERCC3-like DNA repair helicase. Our data highlight the role played by variation in genes encoding putative surface-exposed outer membrane proteins in defining separate lineages, and provide a critical resource for the design of broadly protective syphilis vaccines targeting surface antigens.

Putative Riemerella anatipestifer Outer Membrane Protein H Affects Virulence.

Riemerella anatipestifer causes serious contagious disease in ducks, geese, and other fowl. However, as a harmful pathogen causing significant economic losses in the poultry industry, R. anatipestifer is still poorly understood for its pathogenesis mechanisms. In a previous study, we developed an indirect ELISA method for detecting R. anatipestifer infection using B739_0832 protein, a putative outer membrane protein H (OmpH) that is conserved among different serotypes of R. anatipestifer. Although OmpH in some pathogenic bacteria, such as Pasteurella, has been reported as a virulence factor, it is still not clear whether B739_0832 protein contributes to the virulence of R. anatipestifer. In this study, we confirmed that B739_0832 protein in R. anatipestifer localizes to the outer membrane. We constructed a B739_0832 deletion mutant strain (ΔB739_0832) and assayed various effects from the deletion of B739_0832. ΔB739_0832 strain had a similar growth rate to wild-type R. anatipestifer CH-1. However, the survival rate of ducklings in 10 days after infection from ΔB739_0832 strain was 50%, whereas no ducklings survived from wild-type R. anatipestifer infection. Furthermore, the median lethal dose (LD50) of the ΔB739_0832 strain was approximately 150 times higher than that of the wild-type strain. Pathology examinations on infected ducklings found that, at 36 h after infection, bacterial loads in blood, liver, and brain tissues from ΔB739_0832-infected ducklings were considerably lower than those from wild-type infected ducklings. These results demonstrate that the B739_0832 protein contributes to the virulence of R. anatipestifer CH-1.

Longitudinal TprK profiling of in vivo and in vitro-propagated Treponema pallidum subsp. pallidum reveals accumulation of antigenic variants in absence of immune pressure.

Immune evasion by Treponema pallidum subspecies pallidum (T. pallidum) has been attributed to antigenic variation of its putative outer-membrane protein TprK. In TprK, amino acid diversity is confined to seven variable (V) regions, and generation of sequence diversity within the V regions occurs via a non-reciprocal segmental gene conversion mechanism where donor cassettes recombine into the tprK expression site. Although previous studies have shown the significant role of immune selection in driving accumulation of TprK variants, the contribution of baseline gene conversion activity to variant diversity is less clear. Here, combining longitudinal tprK deep sequencing of near clonal Chicago C from immunocompetent and immunosuppressed rabbits along with the newly developed in vitro cultivation system for T. pallidum, we directly characterized TprK alleles in the presence and absence of immune selection. Our data confirm significantly greater sequence diversity over time within the V6 region during syphilis infection in immunocompetent rabbits compared to immunosuppressed rabbits, consistent with previous studies on the role of TprK in evasion of the host immune response. Compared to strains grown in immunocompetent rabbits, strains passaged in vitro displayed low level changes in allele frequencies of TprK variable region sequences similar to that of strains passaged in immunosuppressed rabbits. Notably, we found significantly increased rates of V6 allele generation relative to other variable regions in in vitro cultivated T, pallidum strains, illustrating that the diversity within these hypervariable regions occurs in the complete absence of immune selection. Together, our results demonstrate antigenic variation in T. pallidum can be studied in vitro and occurs even in the complete absence of immune pressure, allowing the T. pallidum population to continuously evade the immune system of the infected host.

Computational identification of significant immunogenic epitopes of the putative outer membrane proteins from Mycobacterium tuberculosis

Novel vaccines are required to effectively combat the epidemic spread of tuberculosis. Using in silico approaches, this study focuses on prediction of potential B cell and T cell binding immunogenic epitopes for 30 putative outer membrane proteins of Mtb. Among these, certain immunodominant epitopes of Rv0172, Rv0295c, Rv1006, Rv2264c, and Rv2525c were found, which are capable of binding B-cell and a maximum number of MHC alleles. The selected immunodominant epitopes were screened for their allergenic and antigenic properties, their percentage identity against the human proteome and their structural properties. Further, the binding efficacy of the immunodominant epitopes of Rv0295c and Rv1006 with HLA-DRB1*04:01 was analyzed using molecular docking and molecular dynamics studies. Hence, the in silico-derived immunogenic peptides (epitopes) could potentially be used for the design of subunit vaccines against tuberculosis.

Dissecting the molecular diversity and commonality of bovine and human treponemes identifies key survival and adhesion mechanisms.

Here, we report the first complete genomes of three cultivable treponeme species from bovine digital dermatitis (DD) skin lesions, two comparative human treponemes, considered indistinguishable from bovine DD species, and a bovine gastrointestinal (GI) treponeme isolate. Key genomic differences between bovine and human treponemes implicate microbial mechanisms that enhance knowledge of how DD, a severe disease of ruminants, has emerged into a prolific, worldwide disease. Bovine DD treponemes have additional oxidative stress genes compared to nearest human-isolated relatives, suggesting better oxidative stress tolerance, and potentially explaining how bovine strains can colonize skin surfaces. Comparison of both bovine DD and GI treponemes as well as bovine pathogenic and human non-pathogenic saprophyte Treponema phagedenis strains indicates genes encoding a five-enzyme biosynthetic pathway for production of 2,3-diacetamido-2,3-dideoxy-d-mannuronic acid, a rare di-N-acetylated mannuronic acid sugar, as important for pathogenesis. Bovine T. phagedenis strains further differed from human strains by having unique genetic clusters including components of a type IV secretion system and a phosphate utilisation system including phoU, a gene associated with osmotic stress survival. Proteomic analyses confirmed bovine derived T. phagedenis exhibits expression of PhoU but not the putative secretion system, whilst the novel mannuronic acid pathway was expressed in near entirety across the DD treponemes. Analysis of osmotic stress response in water identified a difference between bovine and human T. phagedenis with bovine strains exhibiting enhanced survival. This novel mechanism could enable a selective advantage, allowing environmental persistence and transmission of bovine T. phagedenis. Finally, we investigated putative outer membrane protein (OMP) ortholog families across the DD treponemes and identified several families as multi-specific adhesins capable of binding extra cellular matrix (ECM) components. One bovine pathogen specific adhesin ortholog family showed considerable serodiagnostic potential with the Treponema medium representative demonstrating considerable disease specificity (91.6%). This work has shed light on treponeme host adaptation and has identified candidate molecules for future diagnostics, vaccination and therapeutic intervention.

Peptidoglycan-Binding Anchor Is a Pseudomonas aeruginosa OmpA Family Lipoprotein With Importance for Outer Membrane Vesicles, Biofilms, and the Periplasmic Shape.

The outer membrane protein A (OmpA) family contains an evolutionary conserved domain that links the outer membrane in Gram-negative bacteria to the semi-rigid peptidoglycan (PG) layer. The clinically significant pathogen Pseudomonas aeruginosa carries several OmpA family proteins (OprF, OprL, PA0833, and PA1048) that share the PG-binding domain. These proteins are important for cell morphology, membrane stability, and biofilm and outer membrane vesicle (OMV) formation. In addition to other OmpAs, in silico analysis revealed that the putative outer membrane protein (OMP) with gene locus PA1041 is a lipoprotein with an OmpA domain and, hence, is a potential virulence factor. This study aimed to evaluate PA1041 as a PG-binding protein and describe its effect on the phenotype. Clinical strains were confirmed to contain the lipoprotein resulting from PA1041 expression with Western blot, and PG binding was verified in enzyme-linked immunosorbent assay (ELISA). By using a Sepharose bead-based ELISA, we found that the lipoprotein binds to meso-diaminopimelic acid (mDAP), an amino acid in the pentapeptide portion of PGs. The reference strain PAO1 and the corresponding transposon mutant PW2884 devoid of the lipoprotein were examined for phenotypic changes. Transmission electron microscopy revealed enlarged periplasm spaces near the cellular poles in the mutant. In addition, we observed an increased release of OMV, which could be confirmed by nanoparticle tracking analysis. Importantly, mutants without the lipoprotein produced a thick, but loose and unorganized, biofilm in flow cells. In conclusion, the lipoprotein from gene locus PA1041 tethers the outer membrane to the PG layer, and mutants are viable, but display severe phenotypic changes including disordered biofilm formation. Based upon the phenotype of the P. aeruginosa PW2884 mutant and the function of the protein, we designate the lipoprotein with locus tag PA1041 as “peptidoglycan-binding anchor” (Pba).

Transcriptional and immunological analysis of the putative outer membrane protein and vaccine candidate TprL of Treponema pallidum.

BackgroundAn effective syphilis vaccine should elicit antibodies to Treponema pallidum subsp. pallidum (T. p. pallidum) surface antigens to induce pathogen clearance through opsonophagocytosis. Although the combination of bioinformatics, structural, and functional analyses of T. p. pallidum genes to identify putative outer membrane proteins (OMPs) resulted in a list of potential vaccine candidates, still very little is known about whether and how transcription of these genes is regulated during infection. This knowledge gap is a limitation to vaccine design, as immunity generated to an antigen that can be down-regulated or even silenced at the transcriptional level without affecting virulence would not induce clearance of the pathogen, hence allowing disease progression.Principal findingsWe report here that tp1031, the T. p. pallidum gene encoding the putative OMP and vaccine candidate TprL is differentially expressed in several T. p. pallidum strains, suggesting transcriptional regulation. Experimental identification of the tprL transcriptional start site revealed that a homopolymeric G sequence of varying length resides within the tprL promoter and that its length affects promoter activity compatible with phase variation. Conversely, in the closely related pathogen T. p. subsp. pertenue, the agent of yaws, where a naturally-occurring deletion has eliminated the tprL promoter region, elements necessary for protein synthesis, and part of the gene ORF, tprL transcription level are negligible compared to T. p. pallidum strains. Accordingly, the humoral response to TprL is absent in yaws-infected laboratory animals and patients compared to syphilis-infected subjects.ConclusionThe ability of T. p. pallidum to stochastically vary tprL expression should be considered in any vaccine development effort that includes this antigen. The role of phase variation in contributing to T. p. pallidum antigenic diversity should be further studied.

Antigenic outer membrane proteins prediction of Pasteurella multocida serotype B:2

Outer membrane proteins (OMPs) are one of the prominent virulence factor or immunogenic element of Pasteurella multocida which are responsible for eliciting immune responses in multiple infected hosts. Identification of these proteins allows researchers to target OMPs to be manipulated as a vaccine against bacterial infection. Precise and rapid bioinformatics tools allow researchers to perform in silico analysis to extract putative OMPs from the genome information. In this study, we have successfully identified 105 putative OMPs of P. multocida subsp. multocida strain PMTB2.1 through computational prediction tools including a subcellular localisation predictor, PSORTb v3.0 followed by a lipoprotein predictor, LipoP 1.0 and a β-barrel transmembrane protein predictor, BOMP for sub-classification of the OMPs into 53 integral and 52 peripheral OMPs of this strain. The manipulation of antigenic epitope predictors and the antigenicity score filtering identified nine putative antigenic OMPs. These putative predicted antigenic OMPs of this pathogen will provide crucial initial guidance for the experimental identification and selection of antigenic protein(s) for the development of future haemorrhagic septicaemia (HS) vaccine.

Proteomic analysis of Rickettsia akari proposes a 44\u2009kDa-OMP as a potential biomarker for Rickettsialpox diagnosis

BackgroundRickettsialpox is a febrile illness caused by the mite-borne pathogen Rickettsia akari. Several cases of this disease are reported worldwide annually. Nevertheless, the relationship between the immunogenicity of R. akari and disease development is still poorly understood. Thus, misdiagnosis is frequent. Our study is aiming to identify immunogenic proteins that may improve disease recognition and enhance subsequent treatment. To achieve this goal, two proteomics methodologies were applied, followed by immunoblot confirmation.ResultsThree hundred and sixteen unique proteins were identified in the whole-cell extract of R. akari. The most represented protein groups were found to be those involved in translation, post-translational modifications, energy production, and cell wall development. A significant number of proteins belonged to amino acid transport and intracellular trafficking. Also, some proteins affecting the virulence were detected. In silico analysis of membrane enriched proteins revealed 25 putative outer membrane proteins containing beta-barrel structure and 11 proteins having a secretion signal peptide sequence. Using rabbit and human sera, various immunoreactive proteins were identified from which the 44 kDa uncharacterized protein (A8GP63) has demonstrated a unique detection capability. It positively distinguished the sera of patients with Rickettsialpox from other rickettsiae positive human sera.ConclusionOur proteomic analysis certainly contributed to the lack of knowledge of R. akari pathogenesis. The result obtained may also serve as a guideline for a more accurate diagnosis of rickettsial diseases. The identified 44 kDa uncharacterized protein can be certainly used as a unique marker of rickettsialpox or as a target molecule for the development of more effective treatment.

Putative β-Barrel Outer Membrane Proteins of the Bovine Digital Dermatitis-Associated Treponemes: Identification, Functional Characterization, and Immunogenicity.

Bovine digital dermatitis (BDD), an infectious disease of the bovine foot with a predominant treponemal etiology, is a leading cause of lameness in dairy and beef herds worldwide. BDD is poorly responsive to antimicrobial therapy and exhibits a relapsing clinical course; an effective vaccine is therefore urgently sought. Using a reverse vaccinology approach, the present study surveyed the genomes of the three BDD-associated Treponema phylogroups for putative β-barrel outer membrane proteins and considered their potential as vaccine candidates. Selection criteria included the presence of a signal peptidase I cleavage site, a predicted β-barrel fold, and cross-phylogroup homology. Four candidate genes were overexpressed in Escherichia coli BL21(DE3), refolded, and purified. Consistent with their classification as β-barrel OMPs, circular-dichroism spectroscopy revealed the adoption of a predominantly β-sheet secondary structure. These recombinant proteins, when screened for their ability to adhere to immobilized extracellular matrix (ECM) components, exhibited a diverse range of ligand specificities. All four proteins specifically and dose dependently adhered to bovine fibrinogen. One recombinant protein was identified as a candidate diagnostic antigen (disease specificity, 75%). Finally, when adjuvanted with aluminum hydroxide and administered to BDD-naive calves using a prime-boost vaccination protocol, these proteins were immunogenic, eliciting specific IgG antibodies. In summary, we present the description of four putative treponemal β-barrel OMPs that exhibit the characteristics of multispecific adhesins. The observed interactions with fibrinogen may be critical to host colonization and it is hypothesized that vaccination-induced antibody blockade of these interactions will impede treponemal virulence and thus be of therapeutic value.

Vaccinomics to design a novel single chimeric subunit vaccine for broad-spectrum immunological applications targeting nosocomial Enterobacteriaceae pathogens

Healthcare associated infections (HAIs) are major cause of elevated mortality, morbidity, and high healthcare costs. Development of a vaccine targeting these pathogens could benefit in reducing HAIs count and excessive use of antibiotics. This work aimed to design a multi-epitope based prophylactic/ therapeutic vaccine directing against carbapenem resistant Enterobacter cloacae and other leading nosocomial members of Enterobacteriaceae group. Based on subtractive proteomics and immunoinformatics in-depth investigation of E. cloacae reference proteome, we prioritize four targets: outer membrane usher protein-lpfC, putative outer membrane protein A-OmpA, putative outer membrane protein-FimD, and arginine transporter fulfilling criteria of vaccine candidacy. A multi-epitope peptide vaccine construct is then formulated comprising predicted epitopes with potential to evoke both innate and adaptive immunity and B-subunit of cholera toxin as an adjuvant. The construct is modelled, loop refined, improved for stability via disulfide engineering and optimized for codon usage as per Escherichia coli expression system to ensure its maximum expression. Cross-conservation analysis carried out to evaluate broad-spectrum applicability by providing cross protection against nosocomial pathogens. A blind docking method is applied further to predict predominant binding mode of the construct with TLR4 innate immune receptor, followed by molecular dynamics simulation protocol to probe complex dynamics and exposed topology of the construct epitopes for recognition and immune processing by the host. Towards the end, binding free energies of the vaccine construct-TLR4 receptor were estimated to test docking predictions and affirm complex stability. We believe these findings to be highly useful for vaccinologists in making a highly effective vaccine for E. cloacae specifically, and other notorious Enterobacteriaceae nosocomial pathogens in general.

How BamA recruits OMP substrates via poly-POTRAs domain.

Almost all the outer membrane proteins (OMPs) fold into an invariant β-barrel fold via the polypeptide-transport-associated (POTRA) motif and β-barrel assembly machinery (BAM). However, whether and how poly-POTRAs interact with OMPs remain largely unknown. Here, we have characterized the structures of Haemophilus influenzae poly-POTRAs via X-ray crystallography, small angle X-ray scattering, and molecular dynamics simulation. Unexpectedly, crystal packing reveals a putative OMP travel pathway spiraled by the conserved α2-β2 edges in poly-POTRAs. Supportively, the structure-based mutations targeting the OMP binding sites significantly disrupt OMP biogenesis, resulting in severe cell growth defects. Another notable feature in H. influenzae POTRA structures is flexibility. As characterized by ELISA assays, poly-POTRAs could recruit OMP substrates in a step-wise manner. More importantly, the restriction of POTRA-POTRA linkage and flexibility significantly impairs the BamA function and causes cell growth defect. Altogether, these results suggest that the β-strand augmentations and intrinsic flexibility are important factors for BamA-OMP recruitment.-Ma, X., Wang, Q., Li, Y., Tan, P., Wu, H., Wang, P., Dong, X., Hong, L., Meng, G. How BamA recruits OMP substrates via poly-POTRAs domain.

Directly Sequenced Genomes of Contemporary Strains of Syphilis Reveal Recombination-Driven Diversity in Genes Encoding Predicted Surface-Exposed Antigens.

Syphilis, caused by Treponema pallidum subsp. pallidum (TPA), remains an important public health problem with an increasing worldwide prevalence. Despite recent advances in in vitro cultivation, genetic variability of this pathogen during infection is poorly understood. Here, we present contemporary and geographically diverse complete treponemal genome sequences isolated directly from patients using a methyl-directed enrichment prior to sequencing. This approach reveals that approximately 50% of the genetic diversity found in TPA is driven by inter- and/or intra-strain recombination events, particularly in strains belonging to one of the defined genetic groups of syphilis treponemes: Nichols-like strains. Recombinant loci were found to encode putative outer-membrane proteins and the recombination variability was almost exclusively found in regions predicted to be at the host-pathogen interface. Genetic recombination has been considered to be a rare event in treponemes, yet our study unexpectedly showed that it occurs at a significant level and may have important impacts in the biology of this pathogen, especially as these events occur primarily in the outer membrane proteins. This study reveals the existence of strains with different repertoires of surface-exposed antigens circulating in the current human population, which should be taken into account during syphilis vaccine development.

Roles of YcfR in Biofilm Formation in Salmonella Typhimurium ATCC 14028

An increasing number of foodborne diseases are currently attributable to farm produce contaminated with enteric pathogens such as Salmonella enterica. Recent studies have shown that a variety of enteric pathogens are able to colonize plant surfaces by forming biofilms and thereby persist for long periods, which can subsequently lead to human infections. Therefore, biofilm formation by enteric pathogens on plants poses a risk to human health. Here, we deciphered the roles of YcfR in biofilm formation by Salmonella enterica. YcfR is a putative outer membrane protein associated with bacterial stress responses. The lack of YcfR facilitated the formation of multicellular aggregates on cabbage leaves as well as glass surfaces while reducing bacterial motility. ycfR deletion caused extensive structural alterations in the outer membrane, primarily in lipopolysaccharides, outer membrane proteins, cellulose, and curli fimbria, thereby increasing cell surface hydrophobicity. However, the absence of YcfR rendered Salmonella susceptible to stressful treatments, despite the increased multicellular aggregation. These results suggest that YcfR is an essential constituent of Salmonella outer membrane architecture and its absence may cause multifaceted structural changes, thereby compromising bacterial envelope integrity. In this context, YcfR may be further exploited as a potential target for controlling Salmonella persistence on plants.

Non-pathogenic Borrelia burgdorferi expressing Treponema pallidum TprK and Tp0435 antigens as a novel approach to evaluate syphilis vaccine candidates

BackgroundSyphilis is resurgent in many developed countries and still prevalent in developing nations. Current and future control campaigns would benefit from the development of a vaccine, but although promising vaccine candidates were identified among the putative surface-exposed integral outer membrane proteins of the syphilis spirochete, immunization experiments in the rabbit model using recombinant antigens have failed to fully protect animals upon infectious challenge. We speculated that such recombinant immunogens, purified under denaturing conditions from Escherichia coli prior to immunization might not necessarily harbor their original structure, and hypothesized that enhanced protection would result from performing similar immunization/challenge experiments with native antigens. MethodsTo test our hypothesis, we engineered non-infectious Borrelia burgdorferi strains to express the tp0897 (tprK) and tp0435 genes of Treponema pallidum subsp. pallidum and immunized two groups of rabbits by injecting recombinant strains intramuscularly with no adjuvant. TprK is a putative integral outer membrane protein of the syphilis agent, while tp0435 encodes the highly immunogenic T. pallidum 17-kDa lipoprotein, a periplasmic antigen that was also shown on the pathogen surface. Following development of a specific host immune response to these antigens as the result of immunization, animals were challenged by intradermal inoculation of T. pallidum. Cutaneous lesion development was monitored and treponemal burden within lesions were assessed by dark-field microscopy and RT-qPCR, in comparison to control rabbits. ResultsPartial protection was observed in rabbits immunized with B. burgdorferi expressing TprK while immunity to Tp0435 was not protective. Analysis of the humoral response to TprK antigen suggested reactivity to conformational epitopes. ConclusionsImmunization with native antigens might not be sufficient to obtain complete protection to infection. Nonetheless we showed that non-infectious B. burgdorferi can be an effective carrier to deliver and elicit a specific host response to T. pallidum antigens to assess the efficacy of syphilis vaccine candidates.