In through the Out Exit: the Role of the Exocyst in Listeria monocytogenes Cell Entry

ABSTRACT

The intracellular pathogen Listeria monocytogenes is one of the leading causes of death from foodborne illness in the United States. Internalin A is the key surface protein that drives Listeria uptake by epithelial cells expressing E-cadherin. G. C. Gyanwali, T. U. B. Herath, A. Gianfelice, and K. Ireton (Infect Immun 90:e00326-22, 2022, https://doi.org/10.1128/iai.00326-22) unravel the close relationship between internalin A and the exocyst, adding another layer of complexity to the bacterial internalization process.

TEXT

Listeria monocytogenes is a ubiquitous microorganism that is present as a saprophyte in the environment. However, once it is ingested, it can survive the harsh conditions of the digestive tract and switch to an intracellular virulent form, mainly driven by the master virulence regulator, PrfA. L. monocytogenes invades cells upon interaction of the bacterial protein internalin A (InlA) with the host protein E-cadherin (1). This interaction takes place either at the basolateral membrane of extruding enterocytes at the tip of intestinal villi or the apical membrane of intestinal goblet cells (2, 3). Listeria is then endocytosed and, soon afterward, released from the endocytic vacuole by means of the enzymatic activity of phospholipases C and listeriolysin O (LLO) (1). The next stage of Listeria cellular infection is the process of cell-to-cell spread, driven by the polymerization of host cell actin. Tilney and Portnoy originally described the formation of the bacterial actin tail following L. monocytogenes infection in a seminal paper back in 1989, and at the time, this was the only known process triggered within the eukaryotic cell following L. monocytogenes infection (4). Actin-based motility is crucial for the movement of the intracellular pathogen since L. monocytogenes does not possess flagella-based motility at 37°C. In addition to actin polymerization, another important factor for moving L. monocytogenes into the neighboring cells is the relief of cortical tension at the cell membrane, a process triggered by secreted internalin InlC as reported by Keith Ireton and colleagues (5).

Target tissues for L. monocytogenes replication include the liver, where another internalin B (InlB), is involved in the uptake of the pathogen by the hepatocytes. Work also presented by the Ireton lab led to the identification of the Met protein as the receptor for InlB (6). Subsequent work discussed the possible involvement of cellular exocytosis during L. monocytogenes infection for the first time back in 2014 after postulating the involvement of the GTPase dynamin 2 in L. monocytogenes infection. A few years later, cellular exocytosis was indeed implicated in association with InlB-Met-dependent entry and the demonstration that internalin C (InlC) was also associated with the exocyst complex (7).

The present work by Ireton and colleagues featured in this issue of Infection and Immunity (12) represents another pivotal step in the understanding of L. monocytogenes cell invasion in epithelial cells expressing E-cadherin. The present work adds InlA as a key player associated with the activation of the exocyst complex. The authors used L. monocytogenes and InlA-coated latex beads, a simple and clean strategy for studying only InlA interaction and avoiding the influence of other bacterial factors. Previously, internalin-coated beads had been successfully used by the same group to link InlB with focal exocytosis (8). The use of RNA interference as a strategy for knocking down the expression of each member of the exocyst complex proved an effective technique to analyze key protein players one by one. They were thus able to show that Sec6, Exo70, and Exo84 were involved in the process of cell entry; however, a role for Sec5 or Sec8 could not be demonstrated. It was postulated that the latter two proteins are expressed at high levels such that the small interfering RNAs (siRNAs) used could not completely silence their expression. Alternatively, Sec5 and Sec8 may not be involved in L. monocytogenes infection, similar to the process shown for Legionella pneumophila, where some of the exocyst proteins, such as Exo70, Sec3, and Sec10, were not required (9). In this regard, it may be advisable to complement the siRNA-based approach with other strategies to more effectively silence exocyst protein expression using, for example, state-of-the-art inducible CRISPR systems (10) or the CRISPRoff method recently described (11). Interestingly, the fact that exocytosis favors the uptake of InlA-coated particles was additionally indicated by the addition of the TAT-NSF700 peptide, an inhibitor of the N-ethylmaleimide sensitive factor and hexamer protein that induces the dissociation of the exocyst complex once the process is finished in order to start another cycle. Finally, yet importantly, the key technique used by the authors to analyze exocytosis was the elegant strategy of using a green fluorescent protein (GFP)-labeled VAMP3 protein, allowing for the detailed study of the cellular response upon addition of InlA-coated beads. Together, these approaches paint a compelling picture of the complex process of L. monocytogenes entry into mammalian cells.

L. monocytogenes expresses more than 20 internalins, an important family of proteins defined based on the presence of the leucine-rich repeat (LRR) domain as well as C-terminal LPXTG motifs associated with covalent cross links to the bacterial cell wall or with C-terminal GW repeats or WxL domains as alternatives. A third type of internalin is those represented by the secreted proteins such as InlC. While InlA and InlB are the only proven internalin invasins, there are clues to strongly suggest that at least some of the other family members may play important roles in bacterial infection. Moreover, given that family members share significant similarities, it would be important to determine whether they can trigger, modulate, or be associated with the exocyst complex. Indeed, the direct relationship between InlA and the enhanced exocytosis at the point of entry is an exciting finding of Gyanwali et al (12). The exocyst complex has been reported to contribute to the virulence of a number of bacterial pathogens: it has been reported to be involved in the intercellular spread of Shigella flexneri (13, 14), the exit of Porphyromonas gingivalis from infected cells (15), and Salmonella enterica serovar Typhimurium cell invasion (16), and it may thus represent a common feature of intracellular pathogen invasion and spreading. It will definitely cause the scientific community to stay tuned for more exciting news in this flourishing field.

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