A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

Abstract

The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ⌬secA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ⌬secA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis.T he essential general secretory (Sec) pathway is responsible for exporting the majority of secreted proteins across the bacterial cell membrane (1-3). Much of what we know about this pathway was discovered in Escherichia coli by using genetic screens to identify loss-of-function mutations in components of the Sec system. This led to the identification of components of the SecYEG complex that forms the translocation channel and the SecA ATPase that binds to signal sequences to drive the export of precursor proteins across the channel (3-9). In contrast to loss-of-function sec mutations, prl alleles are gain-of-function mutations, which expand the repertoire of substrates being exported across the SecYEG channel (6-8, 10, 11). The most dominant prl variants are in secY, known as prlA mutations, which allow for the secretion of proteins with altered signal sequence (7, 9, 11) without significantly altering the secretion of other proteins (6,12).In addition to the canonical SecA, an accessory cytosolic ATPase, SecA2, has been identified in Mycobacterium and a subset of other Gram-positive bacteria. Unlike SecA, SecA2 is not essential for cell viability but is required for virulence in some pathogenic bacteria (13-15). SecA1 and SecA2 are homologous but are not interchangeable (16)...

c‐di‐AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation

“…Disk diffusions were performed as previously described (Durack et al ., ), with the following changes. Approximately 10 7 CFU from an overnight culture were diluted in 3 ml of molten top‐agar (0.8% agarose, 0.8% NaCl) and evenly spread over LSM‐agar plates.…”

Structural Similarities and Differences between Two Functionally Distinct SecA Proteins, Mycobacterium tuberculosis SecA1 and SecA2

“…This is consistent with SecA2 working with SecYEG. In a recent study of the SecA2-only system of L. monocytogenes, suppressors of a secA2 mutation also mapped to secY (74). Furthermore, the behavior of a dominant-negative SecA1 mutant in the C. difficile system is consistent with the SecYEG translocase used by SecA1 also being used by SecA2 (18).…”

EslB is required for cell wall biosynthesis and modification inListeria monocytogenes

“…Using microbroth dilution assays, we observed a 40-fold lower MIC for lysozyme for L. monocytogenes strain 10403SDeslB(1) as compared to the wildtype strain ( Fig. 2B and S1A) (18,19). This phenotype could be complemented and strain 10403SDeslB(1) compl., in which eslB is expressed from an IPTG-inducible promoter, is even slightly more resistant to lysozyme as compared to the wildtype strain (Fig.…”

“…Deletion of eslB in L. monocytogenes leads to lysozyme sensitivity and an altered peptidoglycan structure. An eslA in-frame deletion mutant and an eslB transposon insertion mutant were shown to be more sensitive to lysozyme compared to the wild-type strain (18,19). However, it is still unknown how the function of an ABC transporter is linked to this phenotype.…”

“…Furthermore, components of a predicted ABC transporter encoded by the lmo2769-6 operon in L. monocytogenes and here referred to as eslABCR for elongation, sugar-and lysozyme sensitive phenotype (Fig. 1), have been associated with lysozyme resistance (18)(19)(20). An eslB transposon insertion mutant was also shown to be more sensitive to cefuroxime and cationic antimicrobial peptides (18).…”