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.
Highlights
Many Gram-negative bacterial pathogens require a type 3 secretion system (T3SS) to deliver effector proteins into eukaryotic cells
The two previous studies that investigated IpaC topology used purified recombinant IpaC. These studies showed IpaC inserting into the membrane with its N terminus on the extracellular surface of the plasma membrane, but they came to opposing conclusions about the location of the C terminus; the study of interactions of IpaC with artificial liposomes concluded that IpaC contained a single transmembrane ␣-helix with the C terminus present in the liposome lumen [9], whereas investigation of purified IpaC incorporating into macrophage membranes concluded that IpaC contained two transmembrane ␣-helixes with the C terminus accessible on the extracellular surface of the macrophage [10]
When the T3SS was artificially activated by incubation of bacteria with the dye Congo red [14], all IpaC cysteine substitution derivatives were readily secreted through the type 3 secretion needle into the culture supernatant (Fig. 1B and C), indicating that the cysteine substitutions did not disrupt or only minimally disrupted protein conformation in the bacterium
Summary
Many Gram-negative bacterial pathogens require a type 3 secretion system (T3SS) to deliver effector proteins into eukaryotic cells. Upon contact of the tip complex with a eukaryotic cell, the T3SS secretes two bacterial proteins that embed in the plasma membrane [6], where they assemble into a heterooligomeric pore, known as the translocon pore [3]. The translocon pore is essential for T3SS activity; it functions as a conduit through which bacterial virulence proteins (“effectors”) traverse the plasma membrane to gain access to the eukaryotic cytosol [3], and it participates in defining the timing of the secretion of these effectors by the T3SS [7]. We defined the topology of the Shigella translocon protein IpaC during bacterial infection following its native delivery into the plasma membrane by the T3SS. We observed subtle differences between the two proteins in the accessibility of the transmembrane ␣-helixes and the C-terminal regions that may contribute to organism-specific functional differences of these T3SSs during infection
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