The type 3 secretion system (T3SS) is employed by various Gram-negative pathogens to promote infection of host cells. Central to the function of the T3SS is the injectisome, a syringe-like proteinaceous apparatus that spans both bacterial membranes and protrudes into the extracellular space. When a complex at the tip of the needle detects contact with a host cell membrane, this promotes attachment of the bacteria to the host and secretion of translocation machinery through the injectisome. In Pseudomonas aeruginosa, the proteins PopB (SctE) and PopD (SctB) move through the injectisome and insert into the host membrane, forming a heterooligomeric, membrane-spanning translocon pore. This membrane-spanning pore complex provides direct access to the host cell cytoplasm and allows the attached bacterium to translocate several cytotoxic effector proteins into the host. The structural and topological arrangements of PopB and PopD when assembled into a functional translocon are currently unknown.
In this work, we mapped out the topology of PopD in the translocon pore through use of a cysteine-specific labeling assay. Initial results showed the presence of several loop segments in the PopD structure which are exposed to the host cell cytoplasm. To determine which regions of PopD were exposed to the extracellular space, several single cysteine PopD variants were produced with cysteines inserted within the putative extracellular loop domains. The location of inserted cysteine residues was assessed based on their accessibility to a membrane-impermeable thiol-labeling reagent. By combining the cysteine mutant labeling data with previous data confirming regions exposed to the host cell cytoplasm, we here propose a novel topology for the transmembrane structure of translocon-associated PopD protein. In the future this labeling method could be used to study the topology of PopB, as well as similar translocon proteins from the T3SS of related bacterial species.