Methionine Sulfoxide Reductases Are Essential for Virulence of Salmonella Typhimurium

Production of reactive oxygen species represents a fundamental innate defense against microbes in a diversity of host organisms. Oxidative stress, amongst others, converts peptidyl and free methionine to a mixture of methionine-S- (Met-S-SO) and methionine-R-sulfoxides (Met-R-SO). To cope with such oxidative damage, methionine sulfoxide reductases MsrA and MsrB are known to reduce MetSOs, the former being specific for the S-form and the latter being specific for the R-form. However, at present the role of methionine sulfoxide reductases in the pathogenesis of intracellular bacterial pathogens has not been fully detailed. Here we show that deletion of msrA in the facultative intracellular pathogen Salmonella (S.) enterica serovar Typhimurium increased susceptibility to exogenous H2O2, and reduced bacterial replication inside activated macrophages, and in mice. In contrast, a ΔmsrB mutant showed the wild type phenotype. Recombinant MsrA was active against free and peptidyl Met-S-SO, whereas recombinant MsrB was only weakly active and specific for peptidyl Met-R-SO. This raised the question of whether an additional Met-R-SO reductase could play a role in the oxidative stress response of S. Typhimurium. MsrC is a methionine sulfoxide reductase previously shown to be specific for free Met-R-SO in Escherichia (E.) coli. We tested a ΔmsrC single mutant and a ΔmsrBΔmsrC double mutant under various stress conditions, and found that MsrC is essential for survival of S. Typhimurium following exposure to H2O2, as well as for growth in macrophages, and in mice. Hence, this study demonstrates that all three methionine sulfoxide reductases, MsrA, MsrB and MsrC, facilitate growth of a canonical intracellular pathogen during infection. Interestingly MsrC is specific for the repair of free methionine sulfoxide, pointing to an important role of this pathway in the oxidative stress response of Salmonella Typhimurium

Methionine Sulfoxide Reductase A (MsrA) Deficient Mycoplasma genitalium Shows Decreased Interactions with Host Cells

Mycoplasma genitalium is an important sexually transmitted pathogen that affects both men and women. In genital-mucosal tissues, it initiates colonization of epithelial cells by attaching itself to host cells via several identified bacterial ligands and host cell surface receptors. We have previously shown that a mutant form of M. genitalium lacking methionine sulfoxide reductase A (MsrA), an antioxidant enzyme which converts oxidized methionine (Met(O)) into methionine (Met), shows decreased viability in infected animals. To gain more insights into the mechanisms by which MsrA controls M. genitalium virulence, we compared the wild-type M. genitalium strain (G37) with an msrA mutant (MS5) strain for their ability to interact with target cervical epithelial cell lines (HeLa and C33A) and THP-1 monocytic cells. Infection of epithelial cell lines with both strains revealed that MS5 was less cytotoxic to HeLa and C33A cell lines than the G37 strain. Also, the MS5 strain was more susceptible to phagocytosis by THP-1 cells than wild type strain (G37). Further, MS5 was less able to induce aggregation and differentiation in THP-1 cells than the wild type strain, as determined by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the cells, followed by counting of cells attached to the culture dish using image analysis. Finally, MS5 was observed to induce less proinflammatory cytokine TNF-α by THP-1 cells than wild type G37 strain. These results indicate that MsrA affects the virulence properties of M. genitalium by modulating its interaction with host cells

Expression during Host Infection and Localization of Yersinia pestis Autotransporter Proteins

Yersinia pestis CO92 has 12 open reading frames encoding putative conventional autotransporters (yaps), nine of which appear to produce functional proteins. Here, we demonstrate the ability of the Yap proteins to localize to the cell surface of both Escherichia coli and Yersinia pestis and show that a subset of these proteins undergoes processing by bacterial surface omptins to be released into the supernatant. Numerous autotransporters have been implicated in pathogenesis, suggesting a role for the Yaps as virulence factors in Y. pestis. Using the C57BL/6 mouse models of bubonic and pneumonic plague, we determined that all of these genes are transcribed in the lymph nodes during bubonic infection and in the lungs during pneumonic infection, suggesting a role for the Yaps during mammalian infection. In vitro transcription studies did not identify a particular environmental stimulus responsible for transcriptional induction. The primary sequences of the Yaps reveal little similarity to any characterized autotransporters; however, two of the genes are present in operons, suggesting that the proteins encoded in these operons may function together. Further work aims to elucidate the specific functions of the Yaps and clarify the contributions of these proteins to Y. pestis pathogenesis

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Not AvailableIn this study, the investigation on the prevalence of Coxiella burnetii antibodies in cattle dairy farms (n = 44) associated with a history of abortions /and reproductive disorders located in various geographical areas in different states of India was carried out. A total of 323 serum samples (n = 323) collected from apparently healthy cattle associated with a history of abortions, infertility, repeat breeding, anoestrus, endometritis, etc., were screened for anti-C. burnetii antibodies by employing PrioCHECK™ Ruminant Q Fever AB Plate ELISA Kit. The overall seropositivity of 44.0% (142/323, 95% confident interval: 39–49%) was observed in cattle with the presence of antibodies associated with abortions and other reproductive disorders (χ2 = 18.75, p < 0.01). Further, the seropositivity of the dairy farms [84.1% (37/44)] implies at least one or two animals in each farm was found to be positive for C. burnetii antibodies. Moreover, on screening of the serum samples from seronegative and seropositive dairy farms, by Trans PCR, 84 out of 107 samples were found positive for C. burnetii genomic DNA, which indicated the ongoing active infection in all the studied farms. This study revealed that C. burnetii infection was prevalent in dairy cattle associated with abortions, infertility, and other reproductive problems and it implies the cattle have a role as a reservoir and may act as a potential zoonotic source to farm associated risk-groups personnel namely farmers, animal handlers, veterinarians, etc., that necessitates the adoption of appropriate mitigating strategies to reduce the incidence of Q fever in cattle dairy farms.Not Availabl

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Not AvailableAIM: In this study, the prevalence and the distribution status of Leptospira serogroup-specific antibodies among cattle and buffaloes in enzootic districts of Andhra Pradesh, a South Indian state was carried out. MATERIALS AND METHODS: A total of 426 serum samples were randomly sampled from various villages from Prakasam, Kurnool, Guntur, Chittoor, Srikakulam, Visakhapatnam, and Godavari districts of Andhra Pradesh between 2016 and 2017. Serum samples from cattle (n=106) and buffaloes (n=320) having a history of pyrexia, and reproductive problems such as agalactia, infertility, abortions, and stillbirth. The serum samples were screened for Leptospira-specific antibodies by microscopic agglutination test using a reference panel of 18 live cultures of pathogenic Leptospira serovars. RESULTS: The overall seropositivity of 68.08% (290/426) was observed with 70.8% (75/106) in cattle and 67.18% (215/320) in buffaloes. The frequency distribution of predominant serogroup-specific Leptospira antibodies in the sampled areas was determined against the employed serovars as follows: Icterohaemorrhagiae - 21.38%, Hebdomadis - 18.97%, Australis - 18.62%, Pomona - 17.24%, Sejroe - 15.86%, Tarassovi - 15.86%, Autumnalis - 15.52%, Panama - 14.83%, Shermani - 12.07%, Javanica - 11.38%, Hurstbridge - 11.03%, and Pyrogenes - 10.69%. CONCLUSION: It was evident that bovines had a role in maintaining several predominant Leptospira serovars with the change in the trend over a period. The results from this study would also help in strategizing and mitigating the disease burden in cattle and buffaloes of the enzootic area.Not Availabl

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Not AvailableBackground and aim For understanding the epidemiology of leptospirosis, the confined abundance of several species of pathogenic leptospires and knowledge on the serovar(s) prevalent in the reservoir and carrier hosts may be a useful indicator of transmission to incidental or accidental hosts in a geographical niche. The present study was carried out to ascertain the frequency distribution of Leptospira serovars and the prevalence of anti leptospiral antibodies in small ruminants (sheep and goats) in the epidemiological units (villages) in the coastal districts of enzootic regions in South Peninsular India. Materials and methods A total of 1167 serum samples (sheep 299 and goats 868) from apparently healthy animals, randomly collected from various epidemiological units were tested in microscopic agglutination test (MAT) using 18 reference Leptospira serovars antigens. Results The overall seroprevalence of 40% (at 95% confidence intervals 36.82 to 42.43) in small ruminants (44% at 95% confidence interval 40.49 to 52.26) in sheep and 38% (95% confidence interval 34.96 to 41.41) in goats was observed with the predominance of Icterohaemorrhagiae, Javanica, Australis, Hurstbridge, and Pyrogenes serogroup anti leptospiral antibodies in the studied region. The Chi squared test revealed that the presence of anti leptospiral antibodies is significantly not independent (associated) across the administrative division (Chisquare 105.80 p less than 0.05) as well as for sheep (Chisquare 34.67 p less than 0.01) and goats (Chi square 68.78 p less than 0.01). Among seropositive samples (462 reactors), the MAT was positive for more than one serovar in 73% of sheep (95 out of 131) and 53% of goats (177 out of 331), representing an overall 59% cross reactive prevalence in small ruminants. The determined frequency distribution (varied among small ruminants) of the employed serovars representing major reactive serogroup was Icterohaemorrhagiae (29.87), Javanica (20.78), Australis (20.35), Hurstbridge (16.23), Pyrogenes (15.8), Djasmin (15.58), Bataviae (15.37), Autumnalis (14.5), Canicola (14.5), Hebdomadis (14.07), Shermani (13.64), Panama (13.42), Sejroe (12.77), etc. Conclusion: This study indicates alarmingly high seroprevalence of leptospirosis in small ruminants with existing endemicity in the studied region in South Peninsular India. Further, these prevalent serovars in the administrative division may be of use in the reference panels of antigens in the MAT in both humans and animal disease diagnostic laboratories for effective and timely diagnosis of leptospirosis and to combat the challenges in public health.Not Availabl

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Not AvailableThe expressed recombinant leptospiral surface adhesion lipoprotein (Lsa27) of pathogenic Leptospira in E. coli was evaluated for the detection of Leptospira antibodies in cattle sera by latex agglutination test (LAT). The Lsa27 lacking signal peptide coding gene sequences from L. interrogans serovar Pomona was amplified (~ 660 bp) by PCR and the amplicon was cloned into pETiteN-HisKan vector. The expressed recombinant Lsa27 histidine-tagged fusion protein (rLsa27) was Ni-NTA affinity purified under denaturation followed by renaturation methods. The purified rLsa27 was characterized by SDS-PAGE and immunoblot, which confirmed the leptospiral protein with a MW of ~ 25 kDa. Further, the prepared sensitized latex beads coated with rLsa27 were evaluated as a diagnostic antigen for detection of pathogenic Leptospira antibodies by using known microscopic agglutination test (MAT) positive (n = 74) and negative (n = 62) sera for Leptospira antibodies in LAT, which revealed the relative diagnostic sensitivity of 91.89% and specificity of 87.10% against the gold standard serological test, MAT. Furthermore, on evaluation of developed rLsa27 LAT using serum samples from cattle associated with the history of abortions and reproductive disorder (n = 309), the relative sensitivity of 96.15%, and specificity of 89.11% were observed. Therefore, this rapid field test using the rLsa27 is first of its kind and it could be used as a screening test for the detection of Leptospira antibodies or it can be complemented by other diagnostics for the diagnosis /surveillance of bovine leptospirosis.Not Availabl