Identification of Novel Type III Secretion Chaperone-Substrate Complexes of Chlamydia trachomatis

Abstract

Chlamydia trachomatis is an obligate intracellular bacterial pathogen of humans that uses a type III secretion (T3S) system to manipulate host cells through the delivery of effector proteins into their cytosol and membranes. The function of T3S systems depends on small bacterial cytosolic chaperone-like proteins, which bind T3S substrates and ensure their appropriate secretion. To find novel T3S chaperone-substrate complexes of C. trachomatis we first searched its genome for genes encoding proteins with features of T3S chaperones. We then systematically tested for interactions between candidate chaperones and chlamydial T3S substrates by bacterial two-hybrid. This revealed interactions between Slc1 (a known T3S chaperone) or CT584 and several T3S substrates. Co-immunoprecipation after protein expression in Yersinia enterocolitica and protein overlay binding assays indicated that Slc1 interacted with the N-terminal region of the known T3S substrates Tarp (a previously described substrate of Slc1), CT694, and CT695, and that CT584 interacted with a central region of CT082, which we identified as a C. trachomatis T3S substrate using Y. enterocolitica as a heterologous system. Further T3S assays in Yersinia indicated that Slc1 or CT584 increased the amount of secreted Tarp, CT694, and CT695, or CT082, respectively. Expression of CT584 increased the intra-bacterial stability of CT082, while Slc1 did not affect the stability of its substrates. Overall, this indicated that in C. trachomatis Slc1 is a chaperone of multiple T3S substrates and that CT584 is a chaperone of the newly identified T3S substrate CT082.

A working model for the type III secretion mechanism in Chlamydia

“…CT584 [7] and Cpn0803 [66] can form hexamers, but solved structures do not resemble those of either class-1 or -2 tip proteins typical of either Salmonella SipD or Yersinia LcrV, respectively [31]. In addition, CT584 has recently been implicated as a potential chaperone [53]. Therefore, further investigation is warranted before designating CT584 as the tip protein.…”

Multipart Chaperone-Effector Recognition in the Type III Secretion System of Chlamydia trachomatis

“…There are three classes of T3S chaperones in Chlamydia spp. (8,(22)(23)(24)(25)(26)(27). The class I chaperones include class Ia chaperones (Scc1 and Scc4 or CT663), which bind to just one effector, and class Ib chaperones (Slc1, Mcsc, and CT584), which bind to multiple effectors.…”

New Frontiers in Type III Secretion Biology: the Chlamydia Perspective

“…These include the Slc1 (SycE-like chaperone 1) T3SC that binds to the TarP, CT694, and CT695 effector proteins (34,35). Slc1 has been shown to enhance the translocation of TarP via the Yersinia T3SS.…”

“…Slc1 has been shown to enhance the translocation of TarP via the Yersinia T3SS. Furthermore, TarP is present in complex with Slc1 in EBs and likely facilitates the delivery of TarP and CT694 during the invasion process (34)(35)(36). Thus, Slc1 functions to coordinate the delivery of multiple T3SS effector proteins, a function similar to class IB chaperones in other T3SSs.…”

“…Thus, Slc1 functions to coordinate the delivery of multiple T3SS effector proteins, a function similar to class IB chaperones in other T3SSs. Other less wellcharacterized chlamydial class IB T3SCs include the multiple cargo secretion chaperone T3SC, which binds and stabilizes at least two Inc proteins (Cap1 and CT618) (14), and CT584, a potential T3SC that interacts with at least six T3S substrates, including CT082 (34). CT584 secretion is possible since this protein has been suggested to act as a tip complex protein (37) and is capable of binding host lipids in vitro (38).…”