Chlamydia trachomatis TmeA promotes pedestal-like structure formation through N-WASP and TOCA-1 interactions

Abstract

Chlamydia trachomatis (C.t.) is the causative agent of several human diseases, including the sexually transmitted infection chlamydia and the eye infection trachoma. As an obligate intracellular bacterial pathogen, invasion is critical for establishing infection and subsequent pathogenesis. During invasion, C.t. secretes effector proteins via its type III secretion system (T3SS), which manipulate host actin cytoskeletal regulation to promote bacterial entry. Previous studies identified the T3SS effector protein TmeA as a key factor in C.t. invasion, as it recruits and activates N-WASP. This interaction, in turn, activates the Arp2/3 complex, driving cytoskeletal rearrangements at the invasion site to drive C.t. uptake. In this study, we define the role of the N-WASP CRIB domain in mediating this interaction and demonstrate that TmeA functions as a mimic of Cdc42 as part of its established role in activating N-WASP. Additionally, we identified TOCA-1 as another host protein that directly interacts with TmeA. In other bacterial pathogens, notably an enterohemorrhagic E. coli, N-WASP and TOCA-1 are hijacked to mediate pedestal formation. Using siRNA-mediated knockdown of N-WASP and TOCA-1, followed by transmission electron microscopy, we found that both proteins are important for C.t.-mediated pedestal-like structure formation. Collectively, these findings expand our understanding of the intricacies of C.t. invasion, highlighting how TmeA-mediated interactions with N-WASP and TOCA-1 contribute to pedestal-like structure formation, which may represent an early step in C.t. infection.

Importance: Chlamydia trachomatis (C.t.) is an obligate intracellular bacterial pathogen that poses a significant threat to human health, being associated with various diseases, including chlamydia-the most prevalent bacterial sexually transmitted infection-and trachoma. Although often asymptomatic, chlamydia infections can lead to severe complications, such as infertility, ectopic pregnancy, and an increased risk of cervical and ovarian cancers. As an intracellular pathogen, host cell invasion is critical for C.t. survival and pathogenesis. In this study, we provide new insights into the interactions between the C.t. invasion effector protein TmeA and the host proteins N-WASP and TOCA-1, revealing that both host proteins are involved in pedestal-like structure formation during early stages of C.t. infection. These findings deepen our understanding of the mechanisms underlying TmeA-mediated host cell invasion and highlight a key pathway contributing to C.t.-mediated pathogenesis.

Keywords: Chlamydia trachomatis; N-WASP; T3SS; TOCA-1; TmeA; invasion; pedestal.

Fig 1

TmeA binds to the Cdc42 binding site of N-WASP. (A) Schematic depicting N-WASP in its autoinhibited and activated states. (B) Schematic depicting N-WASP deletion constructs. (C and D) FLAG-tagged N-WASP constructs were co-transfected with GFP-tagged C.t. effectors in HeLa cells. The FLAG-tagged proteins were immunoprecipitated, and samples were probed with anti-GFP or anti-FLAG antibodies. Data are representative of three biological replicates. (E) HeLa cells were transfected with GFP-tagged N-WASP WT or N-WASP CRIB mutant and infected with FLAG-tagged TmeA or CT584-expressing C.t. under aTc induction. The FLAG-tagged proteins were immunoprecipitated, and samples were probed with anti-GFP or anti-FLAG antibodies. Data are representative of two biological replicates.

Fig 2

TmeA interacts with endogenous TOCA-1. (A) GFP-tagged C.t. effectors were transfected in HeLa cells. The GFP-tagged proteins were immunoprecipitated, and samples were probed with anti-GFP and anti-TOCA-1 antibodies. Data are representative of three biological replicates. (B) Schematic depicting a 15 minute infection-IP experiment. (C) HeLa cells were infected with C.t. expressing TmeA-FLAG or CT584 FLAG for 15 minutes, followed by immunoprecipitation. Samples were probed with anti-FLAG, anti-N-WASP, and anti-TOCA-1 antibodies. Data are representative of two biological replicates.

Fig 3

TmeA directly binds TOCA-1 independent of Cdc42 and N-WASP binding sites. (A and D) GST-tagged TOCA-1 constructs and MBP-tagged TmeA were expressed in E. coli and purified using GST or MBP resin on gravity columns. Recombinant protein expression was confirmed with Coomassie staining. (B and E) Recombinant MBP-tagged constructs were bound on MBP agarose. Bound proteins were incubated with GST-tagged TOCA-1 constructs and eluted, and samples were probed with anti-GST and anti-MBP antibodies. Data are representative of two replicates. (C) Schematic depicting known TOCA-1 mutants and their effect on binding to Cdc42 and N-WASP.

Fig 4

N-WASP and TOCA-1 play a role in C.t. pedestal-like structure formation. (A) N-WASP and TOCA-1 were knocked down in HeLa cells. Knockdown was verified using Western blotting, probing with anti-GAPDH, anti-N-WASP, and anti-TOCA-1 antibodies and quantified using densitometry, with relative density or adjusted relative density shown under the blots. Relative density was determined compared to mock KD, and relative density was adjusted for the N-WASP and TOCA-1 blots compared to the GAPDH standards. (B) HeLa cells were asynchronously infected with WTL2 at an MOI of 50 for 15 minutes and imaged with transmission electron microscopy; three representative images are shown. (C) Quantification of EBs associated with pedestal-like structures. A total of 100 EBs per experiment were assessed from two separate experiments, in which images were blinded and categorized as associated or not associated with pedestal-like structures. EBs associated with pedestal-like structures were compared to total EBs to determine the percentage associated with pedestal-like structures. Data represent the mean of two biological replicates. Error bars represent SD, *P < 0.05. Significance was determined using one-way ANOVA followed by Tukey’s multiple comparisons test.

Fig 5

TmeA interacts with N-WASP and TOCA-1, which leads to EB pedestal-like structure association. TmeA interacts with N-WASP via the N-WASP CRIB domain as a Cdc42 mimic. It additionally interacts with TOCA-1 independently of N-WASP. The convergent roles of N-WASP and TOCA-1 in EB pedestal-like structure association indicate that these interactions likely contribute to a TmeA-mediated invasion pathway where EBs are taken up via pedestal-like structures.