Nutrient limitation and oxidative stress induce the promoter of acetate operon in Salmonella Typhimurium.

Glyoxylate shunt is an important pathway for microorganisms to survive under multiple stresses. One of its enzymes, malate synthase (encoded by aceB gene), has been widely speculated for its contribution to both the pathogenesis and virulence of various microorganisms. We have previously demonstrated that malate synthase (MS) is required for the growth of Salmonella Typhimurium (S. Typhimurium) under carbon starvation and survival under oxidative stress conditions. The aceB gene is encoded by the acetate operon in S. Typhimurium. We attempted to study the activity of acetate promoter under both the starvation and oxidative stress conditions in a heterologous system. The lac promoter of the pUC19 plasmid was substituted with the putative promoter sequence of the acetate operon of S. Typhimurium upstream to the lacZ gene and transformed the vector construct into E. coli NEBα cells. The transformed cells were subjected to the stress conditions mentioned above. We observed a fourfold increase in the ß-galactosidase activity in these cells resulting from the upregulation of the lacZ gene in the stationary phase of cell growth (nutrient deprived) as compared to the mid-log phase. Following exposure of stationary phase cells to hypochlorite-induced oxidative stress, we further observed a 1.6-fold increase in ß galactosidase activity. These data suggest the induction of promoter activity of the acetate operon under carbon starvation and oxidative stress conditions. Thus, these observations corroborate our previous findings regarding the upregulation of aceB expression under stressful environments.

Sensitivity to antimicrobial peptide A (SapA) contributes to the survival of Salmonella typhimurium against antimicrobial peptides, neutrophils and virulence in mice.

Host-generated antimicrobial peptides (AMPs) play a pivotal role in defense against bacterial pathogens. AMPs kill invading bacteria majorly by disrupting the bacterial cell walls. AMPs are actively synthesized and released into the lumen of the gastrointestinal tract to limit colonization of enteric pathogens like Salmonella typhimurium (S. typhimurium). However, S. typhimurium has evolved several resistance mechanisms to defend AMPs. The multicomponent SapABCDF uptake transporter is one such system that helps in resisting AMPs. In the current study, we analyzed the role of S. typhimurium SapA against stress survival and virulence of this bacterium. ∆sapA mutant strain showed hypersensitivity to AMPs, like melittin and mastoparan. Further, ∆sapA mutant showed more than 22 folds (p = 0.019) hypersensitivity to neutrophils as compared to the WT strain of S. typhimurium. In addition, ∆sapA strain showed defective survival in mice. In conclusion, the results of the current study suggest that the SapA is essential for survival against AMPs and virulence of S. typhimurium.

Deletion of both methionine sulfoxide reductase A and methionine sulfoxide reductase C genes renders Salmonella Typhimurium highly susceptible to hypochlorite stress and poultry macrophages.

Salmonella Typhimurium survives and replicates inside the oxidative environment of phagocytic cells. Proteins, because of their composition and location, are the foremost targets of host inflammatory response. Among others, Met-residues are highly prone to oxidation. Methionine sulfoxide reductase (Msr), with the help of thioredoxin-thioredoxin reductase, can repair oxidized methionine (Met-SO) residues to Met. There are four methionine sulfoxide reductases localized in the cytosol of S. Typhimurium, MsrA, MsrB, MsrC and BisC. MsrA repairs both protein-bound and free 'S' Met-SO, MsrB repairs protein-bound 'R' Met-SO, MsrC repairs free 'R' Met-SO and BisC repairs free 'S' Met-SO. To assess the role(s) of various Msrs in Salmonella, few studies have been conducted by utilizing ΔmsrA, ΔmsrB, ΔmsrC, ΔmsrAΔmsrB, ΔmsrBΔmsrC and ΔbisC mutant strains of S. Typhimurium. Out of the above-mentioned mutants, ΔmsrA and ΔmsrC were found to play important role in the stress survival of this bacterium; however, the combined roles of these two genes have not been determined. In the current study, we have generated msrAmsrC double gene deletion strain (ΔmsrAΔmsrC) of S. Typhimurium and evaluated the effect of gene deletions on the survival of Salmonella against hypochlorite stress and intramacrophage replication. In in vitro growth curve analysis, ΔmsrAΔmsrC mutant strain showed a longer lag phase during the initial stages of the growth; however, it attained similar growth as the wild type strain of S. Typhimurium after 5 h. The ΔmsrAΔmsrC mutant strain has been highly (~ 3000 folds more) sensitive (p < 0.001) to hypochlorite stress. Further, ΔmsrA and ΔmsrAΔmsrC mutant strains showed more than 8 and 26 folds more susceptibility to poultry macrophages, respectively. Our data suggest that the deletion of both msrA and msrC genes severely affect the oxidative stress survival and intramacrophage proliferation of S. Typhimurium.

Identification of oxidant susceptible proteins in Salmonella Typhimurium.

The human gut pathogen, Salmonella Typhimurium (S. Typhimurium) not only survives but also replicates inside the phagocytic cells. Bacterial proteins are the primary targets of phagocyte generated oxidants. Because of the different amino acid composition, some proteins are more prone to oxidation than others. Many oxidant induced modifications to amino acids have been described. Introduction of carbonyl group is one of such modifications, which takes place quite early following exposure of proteins to oxidants and is quite stable. Therefore, carbonyl groups can be exploited to identify oxidant susceptible proteins. Hypochlorous acid (HOCl) is one of the most potent oxidants produced by phagocytes. Incubation of S. Typhimurium with 3 mM HOCl resulted in more than 150 folds loss of bacterial viability. Proteins extracted from HOCl exposed S. Typhimurium cells showed about 60 folds (p < 0.001) more carbonyl levels as compared to unexposed cells. Similarly, 2, 4-Dinitrophenylhydrazine (2, 4-DNPH) derivatized proteins of HOCl treated S. Typhimurium cultures reacted strongly with anti-DNP antibodies as compared to buffer treated counterpart. Next, we have derivatized carbonyl groups on the proteins with biotin hydrazide. The derivatized proteins were then isolated by avidin affinity chromatography. Mass spectrometry based analysis revealed the presence of 204 proteins.

Isolation and Identification of Protein L-Isoaspartate-O-Methyltransferase (PIMT) Interacting Proteins in Salmonella Typhimurium.

Protein L-isoaspartate-O-methyltransferase (PIMT) plays an important role in restoration of covalently damaged Asn/Asp residues. It repairs the racemized forms of these amino acids in protein by forming a labile isoAsp methyl ester which readily converts back to the succinimide intermediate. Spontaneous hydrolysis of the intermediate further restores a minor portion to the normal Asp residues. While significant numbers of PIMT targets have been identified in eukaryotes, very few are documented from prokaryotes. Temperature (42 °C) induced elevation in PIMT expression level has been recently shown in a poultry isolate of Salmonella Typhimurium (ST). The enzyme was also found to be crucial for survival, virulence and colonization of ST in poultry. In the present study, co-immunoprecipitation (Co-IP) approach was used (for isolation) followed by LC-MS analysis to identify the PIMT interacting proteins of ST. Four different proteins were identified among which cytochrome C biogenesis protein A (CcmA) was further expressed in recombinant form and analysed for interaction with recombinant PIMT (rPIMT) by microtiter plate assay. Additionally, the findings were supported by alterations in secondary structure of the proteins upon co-incubation.

Methionine sulfoxide reductase A of Salmonella Typhimurium interacts with several proteins and abets in its colonization in the chicken.

Defending phagocyte generated oxidants is the key for survival of Salmonella Typhimurium (S. Typhimurium) inside the host. Met residues are highly prone to oxidation and convert into methionine sulfoxide (Met-SO). Methionine sulfoxide reductase (Msr) can repair Met-SO to Met thus restoring the function(s) of Met-SO containing proteins. Using pull down method we have identified several MsrA interacting proteins in the S. Typhimurium, one of them was malate synthase (MS). MS is an enzyme of glyoxylate cycle. This cycle is essential for survival of S. Typhimurium inside the host under nutrient limiting conditions. By employing in vitro cross-linking and blot overlay techniques we showed that purified MsrA interacted with pure MS. Treatment of pure malate synthase with H2O2 resulted in reduction of MS activity. However, MsrA along with thioredoxin-thioredoxin reductase system partially restored the activity of oxidized MS. Our mass spectrometry data demonstrated H2O2 mediated oxidation and MsrA mediated repair of Met residues in MS. Further in comparison to S. Typhimurium, the msrA gene deletion (∆msrA) strain showed reduced (60%) malate synthase specific activity. Oral inoculation with wild type, ∆msrA and ∆ms strains of S. Typhimurium resulted in colonization of 100, 0 and 40% of the poultry respectively.

Salmonella Typhimurium methionine sulfoxide reductase A (MsrA) prefers TrxA in repairing methionine sulfoxide.

Intraphagocytic survival of Salmonella Typhimurium (ST) depends (at least in part) upon its ability to repair oxidant-damaged macromolecules. Met residues either free or in protein bound form are highly susceptible to phagocyte-generated oxidants. Oxidation of Mets leads to Met-SO formation, consequently loss of protein functions that results in cell death. Methionine sulfoxide reductase (Msr) reductively repairs Met-SO to Met in the presence of thioredoxin (trx) and thioredoxin reductase (trxR). Earlier we reported that methionine sulfoxide reductase A (msrA) gene deletion strain of ST suffered oxidative stress.[1] Thioredoxin system of ST comprises of two thioredoxins (trxA and trxC) and one thioredoxin reductase (trxB). Preferred trx utilized in MsrA-mediated repair of Met-SO is not known. In current study, we cloned, expressed, and purified ST TrxA, TrxB, TrxC, and MsrA in recombinant forms. The migration of TrxA, TrxB, TrxC, and MsrA proteins was approximately 10, 36, 16, and 26 kDa on SDS-gels. The nicotinamide adenine dinucleotide phosphate hydrogen (NADPH)-linked reductase assays interpreted that MsrA utilized two times more NADPH for the reduction of S-methyl p-tolyl sulfoxide when TrxA was included in the assays as compared to TrxC.

Contribution of protein isoaspartate methyl transferase (PIMT) in the survival of Salmonella Typhimurium under oxidative stress and virulence.

The enteric pathogen Salmonella Typhimurium (ST) survives inside the oxidative environment of phagocytic cells. Phagocyte generated oxidants primarily target proteins and modify amino acids in them. These modifications render the targeted proteins functionally inactive. Conversion of Asp to iso-Asp is one of the several known oxidant mediated amino acids modifications. By repairing iso-Asp to Asp, protein-isoaspartyl methyltransferase (PIMT) maintains the activities of proteins and thus helps in cellular survival under oxidative stress. To elucidate the role of PIMT in ST survival under oxidative stress, we have constructed a pimt gene deletion strain (Δpimt strain) of ST. The Δpimt strain grows normally in various culture media in vitro. However, in comparison to wild type ST, the Δpimt strain is found significantly (p<0.001) more susceptible to H2O2 and hypochlorite (HOCl). Further, the Δpimt mutant strain shows hypersusceptibility (p<0.001) to INF-γ stimulated macrophages. This susceptibility is reversed by pharmacological inhibition of reactive oxygen species (ROS) but not reactive nitrogen species (RNS) production. Further, plasmid based complementation enhances the survival of Δpimt mutant strain against oxidants in vitro and also inside the macrophages. In mice model, the LD50 for wild type ST and mutant Δpimt has been 1.73×10(4) and 1.38×10(5), respectively. Further, the mutant strain shows reduced dissemination to spleen and liver in mice. Following infection with a mixture of wild type ST and the Δpimt mutant (co-infection experiment), we recover significantly (p<0.001) less numbers of mutant bacteria from the spleen and liver of mice.

Role of Helicobacter pylori methionine sulfoxide reductase in urease maturation.

The persistence of the gastric pathogen Helicobacter pylori is due in part to urease and Msr (methionine sulfoxide reductase). Upon exposure to relatively mild (21% partial pressure of O2) oxidative stress, a Δmsr mutant showed both decreased urease specific activity in cell-free extracts and decreased nickel associated with the partially purified urease fraction as compared with the parent strain, yet urease apoprotein levels were the same for the Δmsr and wild-type extracts. Urease activity of the Δmsr mutant was not significantly different from the wild-type upon non-stress microaerobic incubation of strains. Urease maturation occurs through nickel mobilization via a suite of known accessory proteins, one being the GTPase UreG. Treatment of UreG with H2O2 resulted in oxidation of MS-identified methionine residues and loss of up to 70% of its GTPase activity. Incubation of pure H2O2-treated UreG with Msr led to reductive repair of nine methionine residues and recovery of up to full enzyme activity. Binding of Msr to both oxidized and non-oxidized UreG was observed by cross-linking. Therefore we conclude Msr aids the survival of H. pylori in part by ensuring continual UreG-mediated urease maturation under stress conditions.

Comparative analysis reveals the trivial role of MsrP in defending oxidative stress and virulence of Salmonella Typhimurium in mice.

Sulphur containing amino acids, methionine and cysteine are highly prone to oxidation. Reduction of oxidized methionine (Met-SO) residues to methionine (Met) by methionine sulfoxide reductases (Msrs) enhances the survival of bacterial pathogens under oxidative stress conditions. S. Typhimurium encodes two types (cytoplasmic and periplasmic) of Msrs. Periplasmic proteins, due to their location are highly vulnerable to host-generated oxidants. Therefore, the periplasmic Msr (MsrP) mediated repair (as compared to the cytoplasmic counterpart) might play a more imperative role in defending host-generated oxidants. Contrary to this, we show that in comparison to the ΔmsrP strain, the mutant strains in the cytoplasmic Msrs (ΔmsrA and ΔmsrAC strains) showed many folds more susceptibility to chloramine-T and neutrophils. Further ΔmsrA and ΔmsrAC strains accumulated higher levels of ROS and showed compromised fitness in mice spleen and liver. Our data suggest the pivotal role of cytoplasmic Msrs in oxidative stress survival of S. Typhimurium.

Alkyl hydroperoxide reductase repair by Helicobacter pylori methionine sulfoxide reductase.

Protein exposure to oxidants such as HOCl leads to formation of methionine sulfoxide (MetSO) residues, which can be repaired by methionine sulfoxide reductase (Msr). A Helicobacter pylori msr strain was more sensitive to HOCl-mediated killing than the parent. Because of its abundance in H. pylori and its high methionine content, alkyl hydroperoxide reductase C (AhpC) was hypothesized to be prone to methionine oxidation. AhpC was expressed as a recombinant protein in Escherichia coli. AhpC activity was abolished by HOCl, while all six methionine residues of the enzyme were fully to partially oxidized. Upon incubation with a Msr repair mixture, AhpC activity was restored to nonoxidized levels and the MetSO residues were repaired to methionine, albeit to different degrees. The two most highly oxidized and then Msr-repaired methionine residues in AhpC, Met101 and Met133, were replaced with isoleucine residues by site-directed mutagenesis, either individually or together. E. coli cells expressing variant versions were more sensitive to t-butyl hydroperoxide than cells expressing native protein, and purified AhpC variant proteins had 5% to 39% of the native enzyme activity. Variant proteins were still able to oligomerize like the native version, and circular dichroism (CD) spectra of variant proteins revealed no significant change in AhpC conformation, indicating that the loss of activity in these variants was not related to major structural alterations. Our results suggest that both Met101 and Met133 residues are important for AhpC catalytic activity and that their integrity relies on the presence of a functional Msr.

Identification of a novel Francisella tularensis factor required for intramacrophage survival and subversion of innate immune response.

Francisella tularensis, the causative agent of tularemia, is one of the deadliest agents of biological warfare and bioterrorism. Extremely high virulence of this bacterium is associated with its ability to dampen or subvert host innate immune response. The objectives of this study were to identify factors and understand the mechanisms of host innate immune evasion by F. tularensis. We identified and explored the pathogenic role of a mutant interrupted at gene locus FTL_0325, which encodes an OmpA-like protein. Our results establish a pathogenic role of FTL_0325 and its ortholog FTT0831c in the virulent F. tularensis SchuS4 strain in intramacrophage survival and suppression of proinflammatory cytokine responses. This study provides mechanistic evidence that the suppressive effects on innate immune responses are due specifically to these proteins and that FTL_0325 and FTT0831c mediate immune subversion by interfering with NF-κB signaling. Furthermore, FTT0831c inhibits NF-κB activity primarily by preventing the nuclear translocation of p65 subunit. Collectively, this study reports a novel F. tularensis factor that is required for innate immune subversion caused by this deadly bacterium.

Periplasmic methionine sulfoxide reductase (MsrP)-a secondary factor in stress survival and virulence of Salmonella Typhimurium.

Among others, methionine residues are highly susceptible to host-generated oxidants. Repair of oxidized methionine (Met-SO) residues to methionine (Met) by methionine sulfoxide reductases (Msrs) play a chief role in stress survival of bacterial pathogens, including Salmonella Typhimurium. Periplasmic proteins, involved in many important cellular functions, are highly susceptible to host-generated oxidants. According to location in cell, two types of Msrs, cytoplasmic and periplasmic are present in S. Typhimurium. Owing to its localization, periplasmic Msr (MsrP) might play a crucial role in defending the host-generated oxidants. Here, we have assessed the role of MsrP in combating oxidative stress and colonization of S. Typhimurium. ΔmsrP (mutant strain) grew normally in in-vitro media. In comparison to S. Typhimurium (wild type), mutant strain showed mild hypersensitivity to HOCl and chloramine-T (ChT). Following exposure to HOCl, mutant strain showed almost similar protein carbonyl levels (a marker of protein oxidation) as compared to S. Typhimurium strain. Additionally, ΔmsrP strain showed higher susceptibility to neutrophils than the parent strain. Further, the mutant strain showed very mild defects in survival in mice spleen and liver as compared to wild-type strain. In a nutshell, our results indicate that MsrP plays only a secondary role in combating oxidative stress and colonization of S. Typhimurium.

Pan msr gene deleted strain of Salmonella Typhimurium suffers oxidative stress, depicts macromolecular damage and attenuated virulence.

Salmonella encounters but survives host inflammatory response. To defend host-generated oxidants, Salmonella encodes primary antioxidants and protein repair enzymes. Methionine (Met) residues are highly prone to oxidation and convert into methionine sulfoxide (Met-SO) which compromises protein functions and subsequently cellular survival. However, by reducing Met-SO to Met, methionine sulfoxide reductases (Msrs) enhance cellular survival under stress conditions. Salmonella encodes five Msrs which are specific for particular Met-SO (free/protein bound), and 'R'/'S' types. Earlier studies assessed the effect of deletions of one or two msrs on the stress physiology of S. Typhimurium. We generated a pan msr gene deletion (Δ5msr) strain in S. Typhimurium. The Δ5msr mutant strain shows an initial lag in in vitro growth. However, the Δ5msr mutant strain depicts very high sensitivity (p < 0.0001) to hypochlorous acid (HOCl), chloramine T (ChT) and superoxide-generating oxidant paraquat. Further, the Δ5msr mutant strain shows high levels of malondialdehyde (MDA), protein carbonyls, and protein aggregation. On the other side, the Δ5msr mutant strain exhibits lower levels of free amines. Further, the Δ5msr mutant strain is highly susceptible to neutrophils and shows defective fitness in the spleen and liver of mice. The results of the current study suggest that the deletions of all msrs render S. Typhimurium highly prone to oxidative stress and attenuate its virulence.

Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase.

Hypochlorous acid (HOCl) produced via the enzyme myeloperoxidase is a major antibacterial oxidant produced by neutrophils, and Met residues are considered primary amino acid targets of HOCl damage via conversion to Met sulfoxide. Met sulfoxide can be repaired back to Met by methionine sulfoxide reductase (Msr). Catalase is an important antioxidant enzyme; we show it constitutes 4-5% of the total Helicobacter pylori protein levels. msr and katA strains were about 14- and 4-fold, respectively, more susceptible than the parent to killing by the neutrophil cell line HL-60 cells. Catalase activity of an msr strain was much more reduced by HOCl exposure than for the parental strain. Treatment of pure catalase with HOCl caused oxidation of specific MS-identified Met residues, as well as structural changes and activity loss depending on the oxidant dose. Treatment of catalase with HOCl at a level to limit structural perturbation (at a catalase/HOCl molar ratio of 1:60) resulted in oxidation of six identified Met residues. Msr repaired these residues in an in vitro reconstituted system, but no enzyme activity could be recovered. However, addition of GroEL to the Msr repair mixture significantly enhanced catalase activity recovery. Neutrophils produce large amounts of HOCl at inflammation sites, and bacterial catalase may be a prime target of the host inflammatory response; at high concentrations of HOCl (1:100), we observed loss of catalase secondary structure, oligomerization, and carbonylation. The same HOCl-sensitive Met residue oxidation targets in catalase were detected using chloramine-T as a milder oxidant.

Malate synthase contributes to the survival of Salmonella Typhimurium against nutrient and oxidative stress conditions.

To survive and replicate in the host, S. Typhimurium have evolved several metabolic pathways. The glyoxylate shunt is one such pathway that can utilize acetate for the synthesis of glucose and other biomolecules. This pathway is a bypass of the TCA cycle in which CO2 generating steps are omitted. Two enzymes involved in the glyoxylate cycle are isocitrate lyase (ICL) and malate synthase (MS). We determined the contribution of MS in the survival of S. Typhimurium under carbon limiting and oxidative stress conditions. The ms gene deletion strain (∆ms strain) grew normally in LB media but failed to grow in M9 minimal media supplemented with acetate as a sole carbon source. However, the ∆ms strain showed hypersensitivity (p < 0.05) to hypochlorite. Further, ∆ms strain has been significantly more susceptible to neutrophils. Interestingly, several folds induction of ms gene was observed following incubation of S. Typhimurium with neutrophils. Further, ∆ms strain showed defective colonization in poultry spleen and liver. In short, our data demonstrate that the MS contributes to the virulence of S. Typhimurium by aiding its survival under carbon starvation and oxidative stress conditions.

Improved quality and fertilizability of cryopreserved buffalo spermatozoa with the supplementation of methionine sulfoxide reductase A.

BACKGROUND: The excessive reactive oxygen species produced during semen-freezing and -thawing damage the macromolecules resulting in impairment of cellular functions. Proteins are the primary targets of oxidative damage, wherein methionine residues are more prone to oxidation and get converted into methionine sulfoxide, thus affecting the protein function. The methionine sulfoxide reductase A (MsrA) catalyzes the conversion of methionine sulfoxide to methionine and restores the functionality of defective proteins. OBJECTIVES: To establish the expression of MsrA in male reproductive organs, including semen and its effect on quality of cryopreserved semen upon exogenous supplementation, taking buffalo semen as a model. MATERIALS AND METHODS: The expression of MsrA was established by immunohistochemistry, PCR, and Western blots. Further, the effect of recombinant MsrA (rMsrA) supplementation on the quality of cryopreserved spermatozoa was assessed in three treatment groups containing 1.0, 1.5, and 2.0 µg of rMsrA/50 million spermatozoa in egg yolk glycerol extender along with a control group; wherein the post-thaw progressive motility, viability, membrane integrity, and zona binding ability of cryopreserved spermatozoa were studied. RESULTS: The MsrA was expressed in buffalo testis, epididymis, accessory sex glands, and spermatozoa except in seminal plasma. In group 2, the supplementation has resulted in a significant (p < 0.05) improvement as compared to the control group in mean progressive motility (47.50 ± 2.50 vs. 36.25 ± 2.63), viability (56.47 ± 1.85 vs. 48.05 ± 2.42), HOST (50.76 ± 1.73 vs. 44.29 ± 1.29), and zona binding ability of spermatozoa (149.50 ± 8.39 vs. 29.50 ± 2.85). DISCUSSION AND CONCLUSION: In the absence of native MsrA of seminal plasma, the supplementations of rMsrA may repair the oxidatively damaged seminal plasma proteins and exposed sperm plasma membrane proteins resulting in better quality with a fivefold increase in fertilizability of frozen-thawed spermatozoa. The findings can be extended to other species to improve the semen quality with the variation in the amounts of rMsrA supplementation.

Differential responses of chicken monocyte-derived dendritic cells infected with Salmonella Gallinarum and Salmonella Typhimurium.

Salmonella enterica serovar Gallinarum is a host-restricted bacterial pathogen that causes a serious systemic disease exclusively in birds of all ages. Salmonella enterica serovar Typhimurium is a host-generalist serovar. Dendritic cells (DCs) are key antigen-presenting cells that play an important part in Salmonella host-restriction. We evaluated the differential response of chicken blood monocyte-derived dendritic cells (chMoDCs) exposed to S. Gallinarum or S. Typhimurium. S. Typhimurium was found to be more invasive while S. Gallinarum was more cytotoxic at the early phase of infection and later showed higher resistance against chMoDCs killing. S. Typhimurium promoted relatively higher upregulation of costimulatory and other immune function genes on chMoDCs in comparison to S. Gallinarum during early phase of infection (6 h) as analyzed by real-time PCR. Both Salmonella serovars strongly upregulated the proinflammatory transcripts, however, quantum was relatively narrower with S. Gallinarum. S. Typhimurium-infected chMoDCs promoted relatively higher proliferation of naïve T-cells in comparison to S. Gallinarum as assessed by mixed lymphocyte reaction. Our findings indicated that host restriction of S. Gallinarum to chicken is linked with its profound ability to interfere the DCs function. Present findings provide a valuable roadmap for future work aimed at improved vaccine strategies against this pathogen.

Molecular Expression of Bioactive Recombinant Methionine Sulfoxide Reductase A (MsrA).

BACKGROUND: The increase in reactive oxygen species (ROS) production during cryopreservation of semen, leads to oxidation of biomolecules affecting the functionality of spermatozoa. Methionine residues in proteins are highly prone to oxidation and get converted into methionine sulfoxide (MetO). Methionine sulfoxide reductase A (MsrA) can improve the functionality of spermatozoa by reducing the MetO to methionine restoring the lost functionality of the affected proteins. OBJECTIVE: The expression of catalytically active recombinant MsrA (rMsrA). METHODS: The msrA gene was PCR amplified, cloned and sequenced. Further, the recombinant clone was used for protein expression and purification. The protein was getting precipitated during dialysis in Tris-buffer. Hence, the purified rMsrA was dialyzed at 4°C against the Tris-buffer pH 7.5 containing MgCl2, KCl, NaCl, urea and triton X-100. During dialysis, changes of buffer were done at every 12 h interval with stepwise reduction in the concentrations of NaCl, urea and triton X-100. The final dialysis was done with buffer containing 10 mM MgCl2, 30 mM KCl, and 150 mM NaCl, 25 mM Tris-HCl pH 7.5. The activity of the rMsrA was checked spectrophotometrically. RESULTS: The protein BLAST of buffalo MsrA with bovine sequence showed 14 amino acid mismatches. The rMsrA has been purified under denaturing conditions as it was forming inclusion bodies consistently during protein expression. After renaturation, the purified 33 kDa rMsrA was catalytically active by biochemical assay. CONCLUSION: The rMsrA expressed in prokaryotic system is catalytically active and can be used for supplementation to semen extender to repair the oxidatively damaged seminal plasma proteins that occur during cryopreservation.

Peptide Methionine Sulfoxide Reductase from Haemophilus influenzae Is Required for Protection against HOCl and Affects the Host Response to Infection.

Peptide methionine sulfoxide reductases (Msrs) are enzymes that repair ROS-damage to sulfur-containing amino acids such as methionine, ensuring functional integrity of cellular proteins. Here we have shown that unlike the majority of pro- and eukaryotic Msrs, the peptide methionine sulfoxide reductase (MsrAB) from the human pathobiont Haemophilus influenzae (Hi) is required for the repair of hypochlorite damage to cell envelope proteins, but more importantly, we were able to demonstrate that MsrAB plays a role in modulating the host immune response to Hi infection. Loss of MsrAB resulted in >1000-fold increase in sensitivity of Hi to HOCl-mediated killing, and also reduced biofilm formation and in-biofilm survival. Expression of msrAB was also induced by hydrogen peroxide and paraquat, but a Hi2019ΔmsrAB strain was not susceptible to killing by these ROS in vitro. Hi2019ΔmsrAB fitness in infection models was low, with a 3-fold reduction in intracellular survival in bronchial epithelial cells, increased susceptibility to neutrophil killing, and a 10-fold reduction in survival in a mouse model of lung infection. Interestingly, infection with Hi2019ΔmsrAB led to specific changes in the antibacterial response of human host cells, with genes encoding antimicrobial peptides (BPI, CAMP) upregulated between 4 and 9 fold compared to infection with Hi2019WT, and reduction in expression of two proteins with antiapoptotic functions (BIRC3, XIAP). Modulation of host immune responses is a novel role for an enzyme of this type and provides first insights into mechanisms by which MsrAB supports Hi survival in vivo.