Gastrointestinal signals in supplemented media reveal a role in adherence for the Shigella flexneri sap autotransporter gene.

Shigella flexneri causes severe diarrheal disease worldwide. While many aspects of pathogenesis have been elucidated, significant knowledge gaps remain regarding the role of putative chromosomally-encoded virulence genes. The uncharacterized sap gene encoded on the chromosome has significant nucleotide sequence identity to the fluffy (flu) antigen 43 autotransporter gene in pathogenic Escherichia coli. Here, we constructed a Δsap mutant in S. flexneri strain 2457T and examined the effects of this mutation on bacterial cell aggregation, biofilm formation, and adherence to colonic epithelial cells. Analyses included the use of growth media supplemented with glucose and bile salts to replicate small intestinal signals encountered by S. flexneri. Deletion of the sap gene in 2457T affected epithelial cell adherence, resulted in quicker bacterial cell aggregation, but did not affect biofilm formation. This work highlights a functional role for the sap gene in S. flexneri pathogenesis and further demonstrates the importance of using relevant and appropriate gastrointestinal signals to characterize virulence genes of enteropathogenic bacteria.

Bacteriophages against enteropathogens: rediscovery and refinement of novel antimicrobial therapeutics.

PURPOSE OF REVIEW: Alarming rates of antibiotic resistance in bacteria and gastrointestinal dysbiosis associated with traditional antimicrobial therapy have led to renewed interests in developing bacteriophages as novel therapeutics. In this review, we highlight some of the recent advances in bacteriophage therapeutic development targeting important enteropathogens of the gastrointestinal tract. RECENT FINDINGS: Bacteriophages are viruses that infect bacteria, either to utilize the bacterial machinery to produce new progeny or stably integrate into the bacterial chromosome to ensure maintenance of the viral genome. With recent advances in synthetic biology and the discovery of CRISPR-Cas systems used by bacteria to protect against bacteriophages, novel molecular applications are taking us beyond the discovery of bacteriophages and toward innovative applications, including the targeting of bacterial virulence factors, the use of temperate bacteriophages, and the production of bacteriophage proteins as antimicrobial agents. These technologies offer promise to target enteropathogens without disrupting the healthy microbiota of the gastrointestinal tract. Moreover, the use of nanoparticle technology and other modifications are helping researchers circumvent the harsh gastrointestinal conditions that could limit the efficacy of bacteriophages against enteric pathogens. SUMMARY: This era of discovery and development offers significant potential to modify bacteriophages and overcome the global impact of enteropathogens.

Intranasal Immunization of Mice with Multiepitope Chimeric Vaccine Candidate Based on Conserved Autotransporters SigA, Pic and Sap, Confers Protection against Shigella flexneri.

Shigellosis is a diarrheal disease and the World Health Organization prompts the development of a vaccine against Shigella flexneri. The autotransporters SigA, Pic and Sap are conserved among Shigella spp. We previously designed an in silico vaccine with immunodominat epitopes from those autotransporters, and the GroEL protein of S. typhi as an adjuvant. Here, we evaluated the immunogenicity and protective efficacy of the chimeric multiepitope protein, named rMESF, in mice against lethal infection with S. flexneri. rMESF was administered to mice alone through the intranasal (i.n.) route or accompanied with Complete Freund's adjuvant (CFA) intradermically (i.d.), subcutaneously (s.c.), and intramuscular (i.m.), as well as with Imject alum (i.m.). All immunized mice increased IgG, IgG1, IgG2a, IgA and fecal IgA titers compared to PBS+CFA and PBS+alum control groups. Furthermore, i.n. immunization of mice with rMESF alone presented the highest titers of serum and fecal IgA. Cytokine levels (IFN-γ, TNF-α, IL-4, and IL-17) and lymphocyte proliferation increased in all experimental groups, with the highest lymphoproliferative response in i.n. mice immunized with rMESF alone, which presented 100% protection against S. flexneri. In summary, this vaccine vests protective immunity and highlights the importance of mucosal immunity activation for the elimination of S. flexneri.

A Zinc-Dependent Metalloproteinase of Brucella abortus Is Required in the Intracellular Adaptation of Macrophages.

Brucella abortus is a pathogen that survives in macrophages. Several virulence factors participate in this process, including the open reading frame (ORF) BAB1_0270 codifying for a zinc-dependent metalloproteinase (ZnMP). Here, its contribution in the intracellular adaptation of B. abortus was analyzed by infecting RAW264.7 macrophages with the mutant B. abortus Δ270 strain. Results showed that this ZnMP did not participated in either the adherence or the initial intracellular traffic of B. abortus in macrophages. Nevertheless, its deletion significantly increased the co-localization of B. abortus Δ270 with phagolysosomal cathepsin D and reduced its co-localization with calnexin present in endoplasmic reticulum (RE)-derived vesicles. Although B. abortus Δ270 showed an upregulated expression of genes involved in virulence (vjbR, hutC, bvrR, virB1), it was insufficient to reach a successful intracellular replication within macrophages. Furthermore, its attenuation favored in macrophages infected the production of high levels of cytokines (TNF-α and IL-6) and co-stimulatory proteins (CD80 and CD86), signals required in T cell activation. Finally, its deletion significantly reduced the ability of B. abortus Δ270 to adapt, grow and express several virulence factors under acidic conditions. Based on these results, and considering that this ZnMP has homology with ImmA/IrrE proteases, we discuss its role in the virulence of this pathogen, concluding that ZnMP is required in the intracellular adaptation of B. abortus 2308 during the infection of macrophages.

In silico design of a vaccine candidate based on autotransporters and HSP against the causal agent of shigellosis, Shigella flexneri.

Shigellosis is a diarrheal disease that causes high mortality every year, especially in children, elderly and immunocompromised patients. Recently, resistance strains to antibiotic therapy are in the rise and the World Health Organization prioritizes the development of a safe vaccine against the most common causal agent of shigellosis, Shigella flexneri. This pathogen uses autotransporter proteins such as SigA, Pic and Sap to increase virulence and some of them have been described as highly immunogenic proteins. In this study, we used immune-informatics analysis to identify the most antigenic epitope as a vaccine candidate on three passenger domains of auto-transporter proteins encoded on the pathogenic island SHI-1, to induce immunity against S. flexneri. Epitope identification was done using various servers such as Bepipred, Bcepred, nHLAPRED, NetMHCII, Rankpep and IEDB and the final selection was done based on its antigenicity using the VaxiJen server. Moreover, to enhance immunity, the GroEL adjuvant was added to the final construct as a Toll-like receptor 2 (TLR2) agonist. On the other hand, to predict the tertiary structure, the I-TASSER server was used, and the best model was structurally validated using the ProSA-web software and the Ramachandran plot. Subsequently, the model was refined and used for docking and molecular dynamics analyses with TLR2, which demonstrated an appropriate and stable interaction. In summary, a potential subunit vaccine candidate, that contains B and T cell epitopes with proper physicochemical properties was designed. This multiepitope vaccine is expected to elicit robust humoral and cellular immune responses and vest protective immunity against S. flexneri.