Thioredoxin A Is Essential for Motility and Contributes to Host Infection of Listeria monocytogenes via Redox Interactions

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.

Deletion of glutaredoxin promotes oxidative tolerance and intracellular infection in Listeria monocytogenes

“…Moreover, under oxidative stress, glutaredoxin may catalyze formation of mixed disulfides from GSSG, which may be of a protective function to avoid oxidation of a thiol to higher oxidation state [25]. Unlike with the fact that the bacterial thioredoxins have been widely shown to play major roles in protection of cells against toxic oxygen species as well as maintaining the intracellular thio-disulfide balance [8,26], limited data had been available on the role of the glutaredoxins. However, glutaredoxins from E. coli has previously been demonstrated to contribute to the defense against oxidative stress.…”

“…The presence of the Grx system in E. coli provides a strong backup for the Trx system to participate in the antioxidant process by deglutathionylation as in mammalian cells, which was nevertheless not the case in our finding on L. monocytogenes. Our previous work has collectively determined 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 correct-folding, and therefore contributes to bacterial antioxidant and virulence [8].…”

“…The ability of Grx to catalyze reduction of human insulin (sigma) in the presence of DTT was measured as described previously [17,18]. The activity has been used to determine whether the glutaredoxin and its sites mutants were able to reduce disulfide-bridged insulin, using the thioredoxin from L. monocytogenes as a positive control [8]. The rate of insulin disulfide reduction was monitored spectrophotometrically following the turbidity at 650 nm.…”

“…The Trx and the Grx systems, as the two major antioxidant systems in organisms, play roles in controlling cellular redox environment [2]. Based on our previous research, TrxA is involved in the regulation of oxidative stress and bacterial virulence [8]. Hence, the grx gene in-frame deletion mutant strain was developed for revealing the unknown biological functions.…”

“…L. monocytogenes is a gram-positive facultative bacterial pathogen that can cause serious infections leading to high mortality in the immunocompromised individuals and pregnant women [5,6]. This organism is well-adapted to various physiological environments, employing various strategies to counteract hostile acidity, osmolarity, oxygen tension, and other stress conditions present in the environment and within the vacuolar compartment of phagocytic cells [7,8].…”