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

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.

Deep mutational scanning of the Neisseria meningitidis major pilin reveals the importance of pilus tip-mediated adhesion.

Type IV pili (TFP) are multifunctional micrometer-long filaments expressed at the surface of many prokaryotes. In Neisseria meningitidis, TFP are crucial for virulence. Indeed, these homopolymers of the major pilin PilE mediate interbacterial aggregation and adhesion to host cells. However, the mechanisms behind these functions remain unclear. Here, we simultaneously determined regions of PilE involved in pilus display, auto-aggregation, and adhesion by using deep mutational scanning and started mining this extensive functional map. For auto-aggregation, pili must reach a minimum length to allow pilus-pilus interactions through an electropositive cluster of residues centered around Lys140. For adhesion, results point to a key role for the tip of the pilus. Accordingly, purified pili interacting with host cells initially bind via their tip-located major pilin and then along their length. Overall, these results identify functional domains of PilE and support a direct role of the major pilin in TFP-dependent aggregation and adhesion.

Listeria monocytogenes utilizes glutathione and limited inorganic sulfur compounds as a source of essential L-cysteine.

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 host- and bacterially-derived glutathione (GSH). The amino acid L-cysteine is the rate-limiting substrate for GSH synthesis in bacteria and is essential for bacterial growth. Unlike many bacteria, Lm is auxotrophic for L-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. Further, we demonstrated that OppDF are 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. Additionally, we show that Lm utilizes the inorganic sulfur sources thiosulfate and H 2 S for growth in a CysK-dependent manner in the absence of other L-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.