L-cysteine acquisition mechanisms in Listeria monocytogenes

Listeria monocytogenes is a Gram-positive facultative intracellular pathogen that is the causative agent of listeriosis in humans and other mammals. L. monocytogenes leads a biphasic lifestyle, growing as a saprophyte in the environment and transitioning to an intracellular lifecycle upon ingestion by mammalian hosts. During infection, L. monocytogenes must transition its metabolism to adapt to the unique nutritional landscape of the host cytoplasm, as well as selectively induce expression of virulence genes that facilitate intracellular growth and cell-to-cell spread. L. monocytogenes is a genetically tractable, easily manipulated, and rapidly growing bacterium that is a model organism for studying bacterial pathogenesis and host cell biology alike./nVirulence gene expression in L. monocytogenes is controlled by the master virulence regulator PrfA, whose expression and activity are highly regulated at multiple levels. One of the primary regulators of PrfA activity is allosteric activation by the tripeptide glutathione (GSH), which is produced in L. monocytogenes by the bifunctional glutathione synthase GshF. GSH is an important redox-active molecule that maintains the cytoplasmic reducing environment, and the amino acid L-cysteine is the rate-limiting substrate for GSH synthesis and is also essential for bacterial growth. Unlike many bacteria, L. monocytogenes is believed to be auxotrophic for L-cysteine and must import exogenous cysteine to support growth and virulence. While much is known about L-cysteine acquisition mechanisms in L. monocytogenes, there are still many gaps in our knowledge. First, it is known that GSH is enriched in the host cytoplasm and represents an attractive source of GSH for PrfA activation and a rich source of cysteine in vivo. Previous work suggests that L. monocytogenes can utilize exogenous GSH for PrfA activation and growth, but no GSH importers or other GSH utilization mechanisms have been identified. Second, though it is known that L. monocytogenes lack the genes for sulfate assimilation and is unable to synthesize L-cysteine from sulfate, the bacteria contain a conserved CysE/CysK two-step L-cysteine biosynthetic pathway whose function in L. monocytogenes is unknown./nIn this work, we identified the first known GSH importer in L. monocytogenes and established a role for the CysE/CysK pathway in supporting bacterial growth on limited inorganic sulfur sources. Through bioinformatic analysis of known GSH importers, we identified a homolog of the GsiABCD GSH importer in E. coli that is comprised of the L. monocytogenes Ctp ABC transporter complex and the OppDF ATPases of the Opp oligopeptide importer. Here, we demonstrated that the Ctp complex is a high-affinity GSH importer that imports both reduced GSH and its oxidized counterpart GSSG and is required for growth at physiologically relevant concentrations of these substrates in the absence of other L-cysteine sources. We observed that OppDF is required for GSH import in an Opp-independent manner, and these data support a model where Ctp and OppDF form a complex for GSH import that supports L. monocytogenes growth and pathogenesis. Additionally, we demonstrated that L. monocytogenes utilizes the inorganic sulfur sources thiosulfate and H2S to support growth in a CysK-dependent manner in the absence of other L-cysteine sources./nThe results of this work open new avenues of inquiry into mechanisms of L-cysteine acquisition in L. monocytogenes and the roles of GSH and inorganic sulfur sources in pathogenesis. First, our observations suggest the role of alternative GSH utilization mechanisms that may be relevant in vivo and further work is required to identify these mechanisms and their contribution to pathogenesis. Second, even though thiosulfate and H2S are present at uniquely high concentrations in the mammalian intestine, it remains unclear if utilization of these inorganic sulfur sources is important for L. monocytogenes colonization of the host gut. Additionally, it is unclear if the ability to produce L-cysteine is the primary function of the CysE/CysK biosynthetic pathway in specific environmental and host niches, or if CysE and CysK are independently essential for other metabolic processes and L-cysteine production is an adventitious outcome. And finally, these findings suggest a pathoadaptive role for partial L-cysteine auxotrophy in L. monocytogenes, where locally high GSH or thiosulfate/H2S concentrations may signal arrival to distinct host niches.

Advisor: Portnoy, Daniel A