Abstract
Microbes employ the thioredoxin system to defend against oxidative stress and ensure correct disulfide bonding to maintain protein function. Listeria monocytogenes has been shown to encode a putative thioredoxin, TrxA, but its biological roles and underlying mechanisms remain unknown. Here, we showed that expression of L. monocytogenes TrxA is significantly induced in bacteria treated with the thiol-specific oxidizing agent, diamide. Deletion of trxA markedly compromised tolerance of the pathogen to diamide, and mainly impaired early stages of infection in human intestinal epithelial Caco-2 cells. In addition, most trxA mutant bacteria were not associated with polymerized actin, and the rare bacteria that were associated with polymerized actin displayed very short tails or clouds during infection. Deletion or constitutive overexpression of TrxA, which was regulated by SigH, severely attenuated the virulence of the pathogen. Transcriptome analysis of L. monocytogenes revealed over 270 genes that were differentially transcribed in the ΔtrxA mutant compared to the wild-type, especially for the virulence-associated genes plcA, mpl, hly, actA, and plcB. Particularly, deletion of TrxA completely reduced LLO expression, and thereby led to a thoroughly impaired hemolytic activity. Expression of these virulence factors are positively regulated by the master regulator PrfA that was found here to use TrxA to maintain its reduced forms for activation. Interestingly, the trxA deletion mutant completely lacked flagella and was non-motile. We further confirmed that this deficiency is attributable to TrxA in maintaining the reduced intracellular monomer status of MogR, the key regulator for flagellar formation, to ensure correct dimerization. In summary, we demonstrated for the first time that L. monocytogenes thioredoxin A as a vital cellular reductase is essential for maintaining a highly reducing environment in the bacterial cytosol, which provides a favorable condition for protein folding and activation, and therefore contributes to bacterial virulence and motility.
Highlights
Listeria monocytogenes is a Gram-positive foodborne pathogen that causes listeriosis leading to high mortality, especially in the aging population, infants, and immunocompromised individuals (Vazquez-Boland et al, 2001; Corr and O’Neill, 2009)
Phylogenetic analysis further revealed TrxA and its homolog from B. subtilis form a sister branch, while the other five thioredoxin homologures of L. monocytogenes form a separate clade (Figure 1B), indicating that these Trxs within this clade are distinct from the TrxA found in other bacteria species
Recombinant TrxA efficiently catalyzed the thiol-disulfide oxidoreduction of insulin in the presence of DTT as an electron donor (Figures 1C,D). Both C28A and C31A single mutations completely abolished the oxidoreductase ability of the protein to catalyze the reduction of insulin (Figure 1D), clearly indicating that L. monocytogenes trxA encodes a functional thioredoxin and the residues Cys28 and Cys31 are required for its catalytic activity
Summary
Listeria monocytogenes is a Gram-positive foodborne pathogen that causes listeriosis leading to high mortality, especially in the aging population, infants, and immunocompromised individuals (Vazquez-Boland et al, 2001; Corr and O’Neill, 2009). Oxidative stress is an imbalance in electrons that can damage DNA, iron-sulfur clusters, lipids, and proteins. Oxidative stress is both produced by the bacteria (endogenous) and encountered in the environment (exogenous; Imlay, 2003). The intracellular life cycle of L. monocytogenes begins when the bacterium is phagocytosed by a host cell, where it transiently resides within the oxidizing environment of the phagosome. The bacteria secrete the pore-forming toxin listeriolysin O to escape from the phagosome and enter into the cytosol that is a highly reducing environment (Schnupf and Portnoy, 2007). The rapid transit from the oxidizing phagosome to the reducing cytosol, making L. monocytogenes an ideal model for studying adaptive responses to redox changes during infection
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