Listeria monocytogenes utilizes glutathione and limited inorganic sulfur compounds as sources of essential cysteine

Abstract

Listeria monocytogenes (Lm) is a Gram-positive facultative intracellular pathogen that leads a biphasic lifecycle, transitioning its metabolism and selectively inducing virulence genes when it encounters mammalian hosts. Virulence gene expression is controlled by the master virulence regulator PrfA, which is allosterically activated by the host- and bacterially derived glutathione (GSH). The amino acid cysteine is the rate-limiting substrate for GSH synthesis in bacteria and is essential for bacterial growth. Unlike many bacteria, Lm is auxotrophic for cysteine and must import exogenous cysteine for growth and virulence. GSH is enriched in the host cytoplasm, and previous work suggests that Lm utilizes exogenous GSH for PrfA activation. Despite these observations, the import mechanism(s) for GSH remains elusive. Analysis of known GSH importers predicted a homologous importer in Lm comprised of the Ctp ABC transporter and the OppDF ATPases of the Opp oligopeptide importer. Here, we demonstrated that the Ctp complex is a high-affinity GSH/GSSG importer that is required for Lm growth at physiologically relevant concentrations. Furthermore, we demonstrated that OppDF is required for GSH/GSSG import in an Opp-independent manner. These data support a model where Ctp and OppDF form a unique complex for GSH/GSSG import that supports growth and pathogenesis. In addition, we show that Lm utilizes the inorganic sulfur sources thiosulfate and H₂S for growth in a CysK-dependent manner in the absence of other cysteine sources. These findings suggest a pathoadaptive role for partial cysteine auxotrophy in Lm, where locally high GSH/GSSG or inorganic sulfur concentrations may signal arrival to distinct host niches.

Keywords: CtaP; Ctp; CysK; GSH; GSSG; Opp; OppABCDF; auxotrophy; pathoadaptation; thiosulfate.

Fig 1

Lm utilizes diverse cysteine sources for growth in vitro. Broth growth of WT Lm in cysteine-free media supplemented with the indicated cysteine source. Bacteria were grown overnight in LSM supplemented with 0.5 mM cystine. Cultures were washed once in LSM lacking cysteine and inoculated into media supplemented with various physiologically relevant cysteine sources. Growth was determined by OD₆₀₀ at the indicated timepoints. Data representative of three independent replicates, and error bars represent SD.

Fig 2

CysK is required for Lm growth on thiosulfate and H₂S. (A) Schematic of the two-step cysteine biosynthetic pathway in Lm from serine and inorganic sulfur, including notable non-functional pathways in Lm. (B) Genomic organization of genes of the two-step biosynthetic pathway. (C) Broth growth of the cysK mutant and complement in LSM supplemented with either thiosulfate or the H₂S generator NaSH. Strains were grown overnight in LSM media containing 0.5 mM cystine, washed once in LSM lacking cysteine, and inoculated into media containing either cystine or the indicated inorganic sulfur source as the sole sulfur source. Growth was determined by OD₆₀₀ at the indicated timepoints. Data representative of three independent replicates, and error bars represent SD.

Fig 3

The Ctp operon and OppDF are required for growth on GSSG. (A) Genomic organization of genes of the ctp and opp operons. Dashed lines denote specific mutants constructed. (B) Broth growth of the ctp and opp mutants in LSM supplemented with GSSG. Strains were grown overnight in LSM media containing 0.5 mM cystine, washed once in LSM lacking cysteine, and inoculated into media containing either cystine or GSSG as the sole cysteine source. Growth was determined by OD₆₀₀ at the indicated timepoints. Data representative of three independent replicates, and error bars represent SD.

Fig 4

The Ctp operon and OppDF are required for growth on GSH and GSSG. Endpoint saturation broth growth of mutants and complements in cysteine-free LSM media supplemented with the indicated cysteine source. (A) Broth growth of ctp mutants and complement. (B) Broth growth of the oppDF mutant and complements. (C) Broth growth of the oppB mutant and complement. (D) Broth growth of the ctpP1/oppB::Tn double mutant and complements. Strains were grown overnight in LSM containing 0.5 mM cystine, washed once in LSM lacking cysteine, and inoculated into media containing the indicated cysteine source as the sole cysteine source. Growth was determined by OD₆₀₀ at the indicated timepoints. Data representative of three independent replicates, and error bars represent SD.

Fig 5

The Ctp complex is a high-affinity GSH/GSSG importer. Endpoint saturation broth growth of mutants and complements in cysteine-free LSM media supplemented with the indicated cysteine source. (A) Broth growth of the ctp mutant in indicated concentrations of GSSG. (B) Broth growth of the ctp mutant in indicated concentrations of GSH. All GSH media supplemented with 5 mM TCEP. (C) Broth growth of the ctpP1, oppDF, and ctpP1/oppB::Tn mutants on indicated concentrations of GSH. Strains were grown overnight in LSM containing 0.5 mM cystine, washed once in LSM lacking cysteine, and inoculated into media containing the indicated cysteine source as the sole cysteine source. Growth was determined by OD₆₀₀ at the indicated timepoints. Data representative of three independent replicates, and error bars represent SD. cLSM: LSM containing 0.5 mM cystine as a sole cysteine source.

Fig 6

Oligopeptide import via the Opp complex is required for full virulence in a murine intravenous infection model. (A) Intravenous injections of ctp and opp mutants. (B) Intravenous injection of ctp and oppDF mutants in the PrfA* background. 8-week-old female CD-1 mice were intravenously infected with 10⁵ CFU of indicated Lm strains. Livers and spleens were harvested at 48 hours post-infection and bacterial burden was measured by CFU. Data representative of at least two independent experiments. Statistical significance determined by Kruskal-Wallis test; **P < 0.0021, ***P < 0.0002, ****P < 0.0001.

Fig 7

Model of mechanisms for Lm growth on GSH, GSSG, thiosulfate, and H₂S. (A) Cysteine-containing oligopeptides are imported by the OppABCDF complex. GSH and GSSG are imported by the Ctp complex with high affinity, likely in complex with the OppDF ATPases, and small cysteine-containing oligopeptides are likely imported with low affinity. An unknown GSH importer and/or a secreted GSH-specific ɣ-glutamyl transpeptidase (GGT) and cysteine importer likely also exist that support growth on higher concentrations of GSH. (B) H₂S freely diffuses through the plasma membrane and/or thiosulfate is imported by an unknown mechanism, where they serve as sulfide donors for the production of cysteine by the CysE/CysK two-step biosynthetic pathway. It is unclear if H₂S or thiosulfate is the preferred substrate for CysK. OAS: O-acetylserine, S₂O₃²⁻: thiosulfate, H₂S: hydrogen sulfide.