YjbH Requires Its Thioredoxin Active Motif for the Nitrosative Stress Response, Cell-to-Cell Spread, and Protein-Protein Interactions in Listeria monocytogenes

Abstract

Listeria monocytogenes is a model facultative intracellular pathogen. Tight regulation of virulence proteins is essential for a successful infection, and the gene encoding the annotated thioredoxin YjbH was identified in two forward genetic screens as required for virulence factor production. Accordingly, an L. monocytogenes strain lacking yjbH is attenuated in a murine model of infection. However, the function of YjbH in L. monocytogenes has not been investigated. Here, we provide evidence that L. monocytogenes YjbH is involved in the nitrosative stress response, likely through its interaction with the redox-responsive transcriptional regulator SpxA1. YjbH physically interacted with SpxA1, and our data support a model in which YjbH is a protease adaptor that regulates SpxA1 protein abundance. Whole-cell proteomics identified eight additional proteins whose abundance was altered by YjbH, and we demonstrated that YjbH physically interacted with each in bacterial two-hybrid assays. Thioredoxin proteins canonically require active motif cysteines for function, but thioredoxin activity has not been tested for L. monocytogenes YjbH. We demonstrated that cysteine residues of the YjbH thioredoxin domain active motif are essential for L. monocytogenes sensitivity to nitrosative stress, cell-to-cell spread in a tissue culture model of infection, and several protein-protein interactions. Together, these results demonstrated that the function of YjbH in L. monocytogenes requires its thioredoxin active motif and that YjbH has a role in the posttranslational regulation of several proteins, including SpxA1. IMPORTANCE The annotated thioredoxin YjbH in Listeria monocytogenes has been implicated in virulence, but its function in the cell is unknown. In other bacterial species, YjbH is a protease adaptor that mediates degradation of the transcriptional regulator Spx. Here, we investigated the function of L. monocytogenes YjbH and demonstrated its role in the nitrosative stress response and posttranslational regulation of several proteins with which YjbH physically interacts, including SpxA1. Furthermore, we demonstrated that the cysteine residues of the YjbH thioredoxin active motif are required for the nitrosative stress response, cell-to-cell spread, and some protein-protein interactions. YjbH is widely conserved among Firmicutes, and this work reveals its unique requirement of the thioredoxin-active motif in L. monocytogenes.

YjbH regulates virulence genes expression and oxidative stress resistance in Staphylococcus aureus

“…Pathogenicity was restored by the introduction of the yjbIH operon and not of the yjbI gene [14], suggesting that the yjbH gene might have a role in pathogenicity. Studies involving YjbH in Bacillus subtilis [15][16][17], S. aureus [18][19][20][21], and other Gram-positive bacteria [22][23][24] have reported it to be an adaptor protein responsible for proteolysis of Spx, a transcriptional regulator, via ClpXP protease [16][17][18][24][25][26]. However, the role of YjbH in the pathogenicity of S. aureus is obscure.…”

The transcriptional regulator SpxA1 influences the morphology and virulence of Listeria monocytogenes

“…To extend our analysis of protein turnover, mutants lacking yjbH and clpX were generated in both wt and Δ spxA1 backgrounds. SpxA1 abundance is regulated by YjbH- and ClpX-dependent degradation such that mutants deficient for yjbH or clpX have increased SpxA1 (20, 34). L. monocytogenes Δ yjbH and clpX::Tn mutants formed cells significantly smaller than wt (Fig 5).…”

“…Protein isolation was performed as previously described (34). L. monocytogenes were grown anaerobically in BHI for 2 hrs.…”

Investigating the Roles of Listeria monocytogenes Peroxidases in Growth and Virulence

“…Plaque assays were performed as previously described ( 16 , 27 , 52 , 53 ). Briefly, TC-treated 6-well dishes were seeded with 1.2 × 10 6 L2 murine fibroblasts per well.…”