Topological Analysis of the Type 3 Secretion System Translocon Pore Protein IpaC following Its Native Delivery to the Plasma Membrane during Infection

Abstract

Many Gram-negative bacterial pathogens require a type 3 secretion system (T3SS) to deliver effector proteins into eukaryotic cells. Contact of the tip complex of the T3SS with a target eukaryotic cell initiates secretion of the two bacterial proteins that assemble into the translocon pore in the plasma membrane. The translocon pore functions to regulate effector protein secretion and is the conduit for effector protein translocation across the plasma membrane. To generate insights into how the translocon pore regulates effector protein secretion, we defined the topology of the Shigella translocon pore protein IpaC in the plasma membrane following its native delivery by the T3SS. Using single cysteine substitution mutagenesis and site-directed labeling with a membrane-impermeant chemical probe, we mapped residues accessible from the extracellular surface of the cell. Our data support a model in which the N terminus of IpaC is extracellular and the C terminus of IpaC is intracellular. These findings resolve previously conflicting data on IpaC topology that were based on nonnative delivery of IpaC to membranes. Salmonella enterica serovar Typhimurium also requires the T3SS for effector protein delivery into eukaryotic cells. Although the sequence of IpaC is closely related to the Salmonella translocon pore protein SipC, the two proteins have unique functional attributes during infection. We showed a similar overall topology for SipC and IpaC and identified subtle topological differences between their transmembrane α-helixes and C-terminal regions. Together, our data suggest that topological differences among distinct translocon pore proteins may dictate organism-specific functional differences of the T3SSs during infection. IMPORTANCE The type 3 secretion system (T3SS) is a nanomachine required for virulence of many bacterial pathogens that infect humans. The system delivers bacterial virulence proteins into the cytosol of human cells, where the virulence proteins promote bacterial infection. The T3SS forms a translocon pore in the membranes of target cells. This pore is the portal through which bacterial virulence proteins are delivered by the T3SS into the eukaryotic cytosol. The pore also regulates secretion of these virulence proteins. Our work defines the topology of translocon pore proteins in their native context during infection, resolves previously conflicting reports about the topology of the Shigella translocon pore protein IpaC, and provides new insights into how interactions of the pore with the T3SS likely produce signals that activate secretion of virulence proteins.

Activation ofShigella flexneritype 3 secretion requires a host-induced conformational change to the translocon pore

“…Seven IpaC cysteine substitutions, S17C, A38C, S63C, A106C, K350C, A353C, and A363C, displayed a band that migrated at ~80 kilodaltons, more slowly than WT IpaC (Fig. 1c-d), consistent with the presence of a disulfide bond between adjacent IpaC molecules and consistent with our previous demonstration that each of these residues is accessible from the extracellular surface of host cells [16]: IpaC S17, A38, and S63 are within the extracellular N-terminal domain, A106 is within the single transmembrane span that contributes to the pore channel, and K350, A353, and A363 are within a stretch of residues near the C terminus that appears to loop back into the pore channel (Fig 1b).…”

“…We took advantage of the ability of the oxidant copper to induce disulfide crosslinks between cysteine residues that reside in an oxidative environment in close proximity to one another [9]. We screened a library of functional IpaC cysteine substitution mutants [16] for residues that within the pore lie sufficiently close to one another to form disulfide bonds; wild-type (WT) IpaC lacks cysteines. Because we hypothesized that the interaction of IpaC with intermediate filaments induces a conformational change in the translocon pore, we looked for IpaC intermolecular proximity both in the presence and in the absence of intermediate filaments.…”

“…Thus, we anticipated formation of intermolecular disulfide bonds in the subset of IpaC cysteine substitutions that are extracellular, or potentially in the pore channel, and that are oriented correctly and situated in close proximity to one another. The overall topology of IpaC is N-terminal domain extracellular and C-terminal domain cytosolic (Fig 1b, and [16]). Seven IpaC cysteine substitutions, S17C, A38C, S63C, A106C, K350C, A353C, and A363C, displayed a band that migrated at ~80 kilodaltons, more slowly than WT IpaC (Fig.…”

“…We and others hypothesize that host contact generates a series of conformational changes along the T3SS needle that constitute a signal from the extracellular environment into the bacterial cytoplasm [6, 14], where effector secretion is orchestrated [11, 15]. Because the T3SS translocon pore is situated in the host membrane, its interactions with the host could be the initial sensor that instigates this signaling cascade [9, 16]; however, how the translocon pore would generate such a signal and specifically whether conformational changes that might contribute to such a signal occur in the translocon pore and/or needle are unknown.…”

Shigella flexneridisruption of host cell-cell tension promotes intercellular spread

“…We tested the efficiency of spread for S. flexneri Δ ipaC producing WT IpaC or an IpaC derivative that contains a point mutation near the C-terminus (IpaC R362W) and is unable to interact with intermediate filaments (Harrington et al, 2006; Russo et al, 2016; Terry et al, 2008). IpaC R362W is efficiently secreted and forms normal-sized pores in the plasma membrane during invasion (Russo et al, 2019; Russo et al, 2016). Bacterial plaques formed in monolayers of mouse embryonic fibroblasts (MEFs) were significantly smaller for S. flexneri Δ ipaC producing IpaC R362W than for S. flexneri Δ ipaC producing WT IpaC (Figure 1A-B).…”