In Silico Screening of Putative Corynebacterium pseudotuberculosis Antigens and Serological Diagnosis for Caseous Lymphadenitis in Sheep by Enzyme-Linked Immunosorbent Assay

Corynebacterium pseudotuberculosis is the etiologic agent of Caseous Lymphadenitis (CLA), a disease leading to severe damage in sheep and goats farming due to the lack of serological diagnosis, treatment, and effective prophylaxis. In this context, several strategies in an attempt to discover new antigens to compose diagnosis assays or vaccines are fundamental. Therefore, this study aimed to use bioinformatics software to evaluate the critical chemical characteristics of unknown proteins of C. pseudotuberculosis by selecting them for heterologous expression in Escherichia coli. For this purpose, six protein sequences of ascorbate transporter subunit, UPF protein, MMPL family transporter, Ribonuclease, Iron ABC transporter domain-containing permease, and fimbrial subunit were obtained. In silico analyses were performed using amino acid sequences to access immunodominant epitopes and their antigenic and allergenic potential and physicochemical characterization. The expressed proteins were used as an antigen for serological diagnosis by ELISA. All proteins showed distinct immunodominant epitopes and potential antigenic characteristics. The only proteins expressed were PTS and Ribonuclease. In parallel, we expressed CP40 and all were used with ELISA antigen in 49 CLA positive sera and 26 CLA negative sera. The proteins alone showed 100% sensitivity and 96.2%, 92.3%, and 88.5% specificity for rPTS, rRibonuclease, and rCP40, respectively. When proteins were combined, they showed 100% sensitivity and 84.6%, 92.3%, 88.5%, and 92.3% specificity for rPTS/rCp40, rRibonuclease/rCP40, rPTS/rRibonuclease, and rPTS/rRibonuclease/rCP40, respectively. The results of this study show an excellent correlation of sensitivity and specificity with all proteins. None of the specificity values preclude the potential of rPTS, rRibonuclease, or rCP40 for use in ELISA diagnostic assays since the results of this work are superior to those of other studies on CLA diagnosis described in the literature.

Deletion of FaeG alleviated Enterotoxigenic Escherichia coli F4ac-induced apoptosis in the intestine

Enterotoxigenic Escherichia coli (ETEC) F4ac is a major constraint to the development of the pig industry, which is causing newborn and post-weaning piglets diarrhea. Previous studies proved that FaeG is the major fimbrial subunit of F4ac E. coli and efficient for bacterial adherence and receptor recognition. Here we show that the faeG deletion attenuates both the clinical symptoms of F4ac infection and the F4ac-induced intestinal mucosal damage in piglets. Antibody microarray analysis and the detection of mRNA expression using porcine neonatal jejunal IPEC-J2 cells also determined that the absence of FaeG subunit alleviated the F4ac promoted apoptosis in the intestinal epithelial cells. Thus, targeted depletion of FaeG is still beneficial for the prevention or treatment of F4ac infection.

Peptide-Based Inhibitors of Fimbrial Biogenesis inPorphyromonas gingivalis

ABSTRACTPeriodontitis is a progressive inflammatory disease that affects roughly half of American adults. Colonization of the oral cavity by the Gram-negative bacterial pathogenPorphyromonas gingivalisis a key event in the initiation and development of periodontal disease. Adhesive surface structures termed fimbriae (pili) mediate interactions ofP. gingivaliswith other bacteria and with host cells throughout the course of disease. TheP. gingivalisfimbriae are assembled via a novel mechanism that involves proteolytic processing of lipidated precursor subunits and their subsequent polymerization on the bacterial surface. Given their extracellular assembly mechanism and central roles in pathogenesis, theP. gingivalisfimbriae are attractive targets for anti-infective therapeutics to prevent or treat periodontal disease. Here we confirm that conserved sequences in the N and C termini of the Mfa1 fimbrial subunit protein perform critical roles in subunit polymerization. We show that treatment ofP. gingivaliswith peptides corresponding to the conserved C-terminal region inhibits the extracellular assembly of Mfa fimbriae on the bacterial surface. We also show that peptide treatment interferes with the function of Mfa fimbriae by reducingP. gingivalisadhesion toStreptococcus gordoniiin a dual-species biofilm model. Finally, we show that treatment of bacteria with similar peptides inhibits extracellular polymerization of the Fim fimbriae, which are also expressed byP. gingivalis. These results support a donor strand-based assembly mechanism for theP. gingivalisfimbriae and demonstrate the feasibility of using extracellular peptides to disrupt the biogenesis and function of these critical periodontal disease virulence factors.

A Novel Bvg-Repressed Promoter Causesvrg-Like Transcription offim3but Does Not Result in the Production of Serotype 3 Fimbriae in Bvg−ModeBordetella pertussis

ABSTRACTInBordetella pertussis, two serologically distinct fimbriae, FIM2 and FIM3, undergo on/off phase variation independently of each other via variation in the lengths of C stretches in the promoters for their major subunit genes,fim2andfim3. These two promoters are also part of the BvgAS virulence regulon and therefore, if in an on configuration, are activated by phosporylated BvgA (BvgA~P) under normal growth conditions (Bvg+mode) but not in the Bvg−mode, inducible by growth in medium containing MgSO4or other compounds, termed modulators. In theB. pertussisTohama I strain (FIM2+FIM3−), thefim3promoter is in the off state. However, a high level of transcription of thefim3gene is observed in the Bvg−mode. In this study, we provide an explanation for this anomalous behavior by defining a Bvg-repressed promoter (BRP), located approximately 400 bp upstream of the Pfim3transcriptional start. Although transcription of thefim3gene in the Bvg−mode resulted in Fim3 translation, as measured by LacZ translational fusions, no accumulation of Fim3 protein was detectable. We propose that Fim3 protein resulting from translation of mRNA driven by BRP in the Bvg−mode is unstable due to a lack of the fimbrial assembly apparatus encoded by thefimBCgenes, located within thefhaoperon, and therefore is not expressed in the Bvg−mode.IMPORTANCEInBordetella pertussis, the promoter Pfim3-15C for the major fimbrial subunit genefim3is activated by the two-component system BvgAS in the Bvg+mode but not in the Bvg−mode. However, many transcriptional profiling studies have shown thatfim3is transcribed in the Bvg−mode even when Pfim3is in a nonpermissive state (Pfim3-13C), suggesting the presence of a reciprocally regulated element upstream of Pfim3. Here, we provide evidence that BRP is the cause of this anomalous behavior offim3. Although BRP effectsvrg-like transcription offim3in the Bvg−mode, it does not lead to stable production of FIM3 fimbriae, because expression of the chaperone and usher proteins FimB and FimC occurs only in the Bvg+mode.

BPSL1626: Reverse and Structural Vaccinology Reveal a Novel Candidate for Vaccine Design Against Burkholderia pseudomallei

Due to significant advances in computational biology, protein prediction, together with antigen and epitope design, have rapidly moved from conventional methods, based on experimental approaches, to in silico-based bioinformatics methods. In this context, we report a reverse vaccinology study that identified a panel of 104 candidate antigens from the Gram-negative bacterial pathogen Burkholderia pseudomallei, which is responsible for the disease melioidosis. B. pseudomallei can cause fatal sepsis in endemic populations in the tropical regions of the world and treatment with antibiotics is mostly ineffective. With the aim of identifying potential vaccine candidates, we report the experimental validation of predicted antigen and type I fimbrial subunit, BPSL1626, which we show is able to recognize and bind human antibodies from the sera of Burkholderia infected patients and to stimulate T-lymphocytes in vitro. The prerequisite for a melioidosis vaccine, in fact, is that both antibody- and cell-mediated immune responses must be triggered. In order to reveal potential antigenic regions of the protein that may aid immunogen re-design, we also report the crystal structure of BPSL1626 at 1.9 Å resolution on which structure-based epitope predictions were based. Overall, our data suggest that BPSL1626 and three epitope regions here-identified can represent viable candidates as potential antigenic molecules.