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

Modulation of Biofilm-Formation in <i>Salmonella enterica</i> Serovar Typhimurium by the Periplasmic DsbA/DsbB Oxidoreductase System Requires the GGDEF-EAL Domain Protein STM3615

<div><p>In <i>Salmonella enterica</i> serovar Typhimurium (<i>S</i>. Typhimurium), biofilm-formation is controlled by the cytoplasmic intracellular small-molecular second messenger cyclic 3′, 5′-di- guanosine monophosphate (c-di-GMP) through the activities of GGDEF and EAL domain proteins. Here we describe that deleting either <i>dsbA</i> or <i>dsbB</i>, respectively encoding a periplasmic protein disulfide oxidase and a cytoplasmic membrane disulfide oxidoreductase, resulted in increased biofilm-formation on solid medium. This increased biofilm-formation, defined as a <i>r</i>ed, <i>d</i>ry <i>a</i>nd <i>r</i>ough (<i>rdar</i>) colony morphotype, paralleled with enhanced expression of the biofilm master regulator CsgD and the biofilm-associated fimbrial subunit CsgA. Deleting <i>csgD</i> in either <i>dsb</i> mutant abrogated the enhanced biofilm-formation. Likewise, overexpression of the c-di-GMP phosphodiesterase YhjH, or mutationally inactivating the CsgD activator EAL-domain protein YdiV, reduced biofilm-formation in either of the <i>dsb</i> mutants. Intriguingly, deleting the GGDEF-EAL domain protein gene <i>STM3615 (yhjK)</i>, previously not connected to <i>rdar</i> morphotype development, also abrogated the escalated <i>rdar</i> morphotype formation in <i>dsb</i> mutant backgrounds. Enhanced biofilm-formation in <i>dsb</i> mutants was furthermore annulled by exposure to the protein disulfide catalyst copper chloride. When analyzed for the effect of exogenous reducing stress on biofilm-formation, both <i>dsb</i> mutants initially showed an escalated <i>rdar</i> morphotype development that later dissolved to reveal a smooth mucoid colony morphotype. From these results we conclude that biofilm-development in <i>S.</i> Typhimurium is affected by periplasmic protein disulphide bond status through CsgD, and discuss the involvement of selected GGDEF/EAL domain protein(s) as signaling mediators.</p></div

Effect of <i>ydiV</i>, <i>yhjK</i> and <i>yhjH</i> mutations on <i>dsb</i> associated biofilm-formation.

<p>Immunoblot for CsgD from overnight salt-less Luria Agar cultures shows altered level of CsgD expression for <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants by introducing mutations in <i>ydiV</i>, <i>yhjK, ydiV-yhjK</i>, and <i>yhjH</i> genes respectively.</p

Strains and Plasmids.

<p>Strains and Plasmids.</p

Effect of reducing stress on biofilm.

<p><b>A</b>) Upon reductive stress (5 mM DTT) <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants first shows an escalated <i>rdar</i> morpotype development, where after the appearance becomes smoother and mucoid. Overexpression of YhjH and deletion of <i>csgD</i> enhances slime production in <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants under DTT reductive stress. Coomassie blue stained SDS-PAGE (<b>B</b>) and immunoblot (<b>C</b>) show decrease in the amounts of respectively CsgA and CsgD under 5 mM DTT stress from 18 hours at 28°C. The numbers underneath the lanes indicate relative amounts. <b>D</b>) and <b>E</b>) The <i>csgD-lacZ</i> promoter fusion activity in <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants under 5 mM DTT stress in LB and LA cultures at 28°C. Error bars indicate SEM. ns  =  non-significant compared to respective wild type.</p

Effect of GGDEF/EAL proteins on <i>dsb</i> associated biofilm-formation.

<p>−/+/2+/3+ etc. represents the degree of the biofilm-formation compared to the WT.</p

DsbA/B regulatory pathway leading to <i>rdar</i> morphotype formation in <i>S.</i> Typhimurium.

<p>In the illustration, the OM indicates outer cell membrane and IM refers inner cell membrane. The diguanyl cyclases are marked as black and the phosphodiesterases are marked as grey.</p

Biofilm-formation on solid media and liquid media.

<p>The formation of <i>rdar</i> morphotype in wild type, single and double <i>dsb</i> mutants grown for 48 hours on Congo red plates at 28°C is illustrated in panel (<b>A</b>). This escalated rdar morphotype development can be reverted to wild type level by <i>trans</i>-complementation with corresponding cloned <i>dsb</i> genes, or by introducing a cloned <i>yhjH</i> gene or depleting <i>csgD</i> (<b>B</b>). VC indicates the vector control, pBAD30. <b>C</b>) Crystal violet staining of biofilm adherent to polystyrene tubes as an indicator of biofilm-formation at liquid-air interface. The <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants fail to make a pellicle at air-liquid interface in static LB without salt culture at 28°C 24 hours post inoculation. <b>D</b>) Quantification of adherent biofilm measured as retained Crystal violet in biofilm. Error bars indicate SEM. ***  = p≤0.001.</p

Effect of <i>ΔdsbA</i> and <i>ΔdsbB</i> mutations on the expression of CsgA and CsgD.

<p><b>A</b>) Coomassie blue stained SDS-PAGE gel revealing increased CsgA production in <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants when grown on LA without salt at 28°C at 24 hours of incubation. <b>B</b>) Immunoblot for CsgD shows increased level of CsgD for <i>ΔdsbA</i> and <i>ΔdsbB</i> mutants from the same bacterial cultures. Numbers underneath the lanes in <b>A</b>) and <b>B</b>) specifies the relative band intensities. The <i>csgD-lacZ</i> promoter fusion activity at 18 hours post inoculation from salt-less Luria Agar cultures (<b>C</b>) or from salt-less Luria broth cultures (<b>D</b>) grown at 28°C. Error bars indicate SEM. ***  = p≤0.001; **  = p≤0.01; ns  =  not significant compared to respective wild type.</p

Effect of GGDEF/EAL protein gene mutations on <i>dsb</i> associated biofilm-formation.

<p>The development of <i>rdar</i> morphotype in wild type, single and double mutants on Congo red plates at 28°C after 24 and 48 hours of incubation.</p