Development of a Recombinant Pichinde Virus-Vectored Vaccine against Turkey Arthritis Reovirus and Its Immunological Response Characterization in Vaccinated Animals

Vaccination may be an effective way to reduce turkey arthritis reovirus (TARV)-induced lameness in turkey flocks. However, there are currently no commercial vaccines available against TARV infection. Here, we describe the use of reverse genetics technology to generate a recombinant Pichinde virus (PICV) that expresses the Sigma C and/or Sigma B proteins of TARV as antigens. Nine recombinant PICV-based TARV vaccines were developed carrying the wild-type S1 (Sigma C) and/or S3 (Sigma B) genes from three different TARV strains. In addition, three recombinant PICV-based TARV vaccines were produced carrying codon-optimized S1 and/or S3 genes of a TARV strain. The S1 and S3 genes and antigens were found to be expressed in virus-infected cells via reverse transcriptase polymerase chain reaction (RT-PCR) and the direct fluorescent antibody (DFA) technique, respectively. Turkey poults inoculated with the recombinant PICV-based TARV vaccine expressing the bivalent TARV S1 and S3 antigens developed high anti-TARV antibody titers, indicating the immunogenicity (and safety) of this vaccine. Future in vivo challenge studies using a turkey reovirus infection model will determine the optimum dose and protective efficacy of this recombinant virus-vectored candidate vaccine.

Transcriptomic and Phenotypic Analyses of the Sigma B-Dependent Characteristics and the Synergism between Sigma B and Sigma L in Listeria monocytogenes EGD-e

Numerous gene expression and stress adaptation responses in L. monocytogenes are regulated through alternative sigma factors σB and σL. Stress response phenotypes and transcriptomes were compared between L. monocytogenes EGD-e and its ΔsigB and ΔsigBL mutants. Targeted growth phenotypic analysis revealed that the ΔsigB and ΔsigBL mutants are impaired during growth under cold and organic-acid stress conditions. Phenotypic microarrays revealed increased sensitivity in both mutants to various antimicrobial compounds. Genes de-regulated in these two mutants were identified by genome-wide transcriptome analysis during exponential growth in BHI. The ΔsigB and ΔsigBL strains repressed 198 and 254 genes, respectively, compared to the parent EGD-e strain at 3 °C, whereas 86 and 139 genes, respectively, were repressed in these mutants during growth at 37 °C. Genes repressed in these mutants are involved in various cellular functions including transcription regulation, energy metabolism and nutrient transport functions, and viral-associated processes. Exposure to cold stress induced a significant increase in σB and σL co-dependent genes of L. monocytogenes EGD-e since most (62%) of the down-regulated genes uncovered at 3 °C were detected in the ΔsigBL double-deletion mutant but not in ΔsigB or ΔsigL single-deletion mutants. Overall, the current study provides an expanded insight into σB and σL phenotypic roles and functional interactions in L. monocytogenes. Besides previously known σB- and σL-dependent genes, the transcriptomes defined in ΔsigB and ΔsigBL mutants reveal several new genes that are positively regulated by σB alone, as well as those co-regulated through σB- and σL-dependent mechanisms during L. monocytogenes growth under optimal and cold-stress temperature conditions.

Redefining the Clostridioides difficile σB Regulon: σB Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide

ABSTRACT In many Gram-positive bacteria, the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile, σB has been implicated in protection against stressors such as reactive oxygen species (ROS) and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allows for the refinement of the previously proposed σB regulon. At least 32% of the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σB was demonstrated through in vitro runoff transcription of specific target genes (cd0350, cd3614, cd3605, and cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to σB-dependent gene activation. IMPORTANCE Sigma B is the alternative sigma factor governing stress response in many Gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together, our data suggest that σB is a key player in dealing with toxic radicals.

Redefining the Clostridioides difficile σB regulon: σB activates genes involved in detoxifying radicals that can result from the exposure to antimicrobials and hydrogen peroxide

In many gram-positive bacteria the general stress response is regulated at the transcriptional level by the alternative sigma factor sigma B (σB). In C. difficile σB has been implicated in protection against stressors such as reactive oxygen species and antimicrobial compounds. Here, we used an anti-σB antibody to demonstrate time-limited overproduction of σB in C. difficile despite its toxicity at higher cellular concentrations. This toxicity eventually led to the loss of the plasmid used for anhydrotetracycline-induced σB gene expression. Inducible σB overproduction uncouples σB expression from its native regulatory network and allowed for the refinement of the previously proposed σB regulon. At least 32% the regulon was found to consist of genes involved in the response to reactive radicals. Direct gene activation by C. difficile σB was demonstrated through in vitro run-off transcription of specific target genes (cd0350, cd3614, cd3605, cd2963). Finally, we demonstrated that different antimicrobials and hydrogen peroxide induce these genes in a manner dependent on this sigma factor, using a plate-based luciferase reporter assay. Together, our work suggests that lethal exposure to antimicrobials may result in the formation of toxic radicals that lead to σB-dependent gene activation.ImportanceSigma B is the alternative sigma factor governing stress response in many gram-positive bacteria. In C. difficile, a sigB mutant shows pleiotropic transcriptional effects. Here, we determine genes that are likely direct targets of σB by evaluating the transcriptional effects of σB overproduction, provide biochemical evidence of direct transcriptional activation by σB, and show that σB-dependent genes can be activated by antimicrobials. Together our data suggest that σB is a key player in dealing with toxic radicals.

Transketolase of Staphylococcus aureus in the Control of Master Regulators of Stress Response During Infection

Staphylococcus aureus is a leading cause of both acute and chronic infections in humans. The importance of the pentose phosphate pathway (PPP) during S. aureus infection is currently largely unexplored. In the current study, we focused on one key PPP enzyme, transketolase (TKT). We showed that inactivation of the unique gene encoding TKT activity in S. aureus USA300 (∆tkt) led to drastic metabolomic changes. Using time-lapse video imaging and mice infection, we observed a major defect of the ∆tkt strain compared with wild-type strain in early intracellular proliferation and in the ability to colonize kidneys. Transcriptional activity of the 2 master regulators sigma B and RpiRc was drastically reduced in the ∆tkt mutant during host cells invasion. The concomitant increased RNAIII transcription suggests that TKT—or a functional PPP—strongly influences the ability of S. aureus to proliferate within host cells by modulating key transcriptional regulators.

Transketolase is involved in the control of Sigma B during chronic infection by Staphylococcus aureus

Staphylococcus aureus is a leading cause of both acute and chronic infections in humans. Its ability to persist within host cells is thought to play an important role in chronicity and treatment failures. The importance of the pentose phosphate pathway (PPP) during S. aureus chronic infection is currently largely unexplored. Here, we focused on one key PPP enzyme, transketolase. We showed that inactivation of the unique gene encoding transketolase activity in S. aureus USA300 (Δtkt) led to an impaired growth in broth. Using time-lapse video imaging, we correlated this phenotype with a defect in early intracellular proliferation compared to wild-type strain. As determined by metabolomic analysis, tkt inactivation also had an important impact on S. aureus metabolism. We then monitored long-term intracellular persistence over 10 days by counting of viable bacteria. Unexpectedly for such a slow-growing strain, the Δtkt mutant was almost completely eliminated by endothelial cells after ten days, as opposed to a prototypical slow-growing ΔhemDBL mutant for which we recovered 1,000 fold more viable bacteria. We found that in infected cells, the transcriptional activity of the two master regulators Sigma B and RpiRc was drastically reduced in the Δtkt mutant compared to wild-type strain. Concomitantly, RNAIII transcription was strongly increased. This transcriptional profile is likely to explain the inability of this slow-growing mutant to sustain long-term intracellular survival, suggesting that TKT -or a functional PPP-is required for intracellular bacteria to enable a transcriptional program geared towards persistence.ImportanceStaphylococcus aureus is a leading cause of severe bacterial infections. This bacterium is readily internalized by non-professional phagocytes and infected cells have been proposed to play an important role in chronic infections and treatment failures.Here, we show the importance of the unique transketolase TKT of S. aureus USA300 in bacterial adaptation during chronic intracellular infection. We show that TKT is mandatory for the metabolomic homeostasis of S. aureus during intracellular persistence. This work unravels the critical role of TKT in the transcriptional regulation of the master regulators Sigma B, RpiRc and RNAIII linking the pentose phosphate pathway to the control of chronic S. aureus infections.