Chlamydia trachomatis TmeA Directly Activates N-WASP To Promote Actin Polymerization and Functions Synergistically with TarP during Invasion

Abstract

Chlamydia trachomatis is a medically significant human pathogen and is an epithelial-tropic obligate intracellular parasite. Invasion of nonprofessional phagocytes represents a crucial step in the infection process and has likely promoted the evolution of a redundant mechanism and routes of entry. Like many other viral and invasive bacterial pathogens, manipulation of the host cell cytoskeleton represents a focal point in Chlamydia entry. The advent of genetic techniques in C. trachomatis, such as creation of complete gene deletions via fluorescence-reported allelic exchange mutagenesis (FRAEM), is providing important tools to unravel the contributions of bacterial factors in these complex pathways. The type III secretion chaperone Slc1 directs delivery of at least four effectors during the invasion process. Two of these, TarP and TmeA, have been associated with manipulation of actin networks and are essential for normal levels of invasion. The functions of TarP are well established, whereas TmeA is less well characterized. We leverage chlamydial genetics and proximity labeling here to provide evidence that TmeA directly targets host N-WASP to promote Arp2/3-dependent actin polymerization. Our work also shows that TmeA and TarP influence separate, yet synergistic pathways to accomplish chlamydial entry. These data further support an appreciation that a pathogen, confined by a reductionist genome, retains the ability to commit considerable resources to accomplish bottle-neck steps during the infection process. IMPORTANCE The increasing genetic tractability of Chlamydia trachomatis is accelerating the ability to characterize the unique infection biology of this obligate intracellular parasite. These efforts are leading to a greater understanding of the molecular events associated with key virulence requirements. Manipulation of the host actin cytoskeleton plays a pivotal role throughout Chlamydia infection, yet a thorough understanding of the molecular mechanisms initiating and orchestrating actin rearrangements has lagged. Our work highlights the application of genetic manipulation to address open questions regarding chlamydial invasion, a process essential to survival. We provide definitive insight regarding the role of the type III secreted effector TmeA and how that activity relates to another prominent effector, TarP. In addition, our data implicate at least one source that contributes to the functional divergence of entry mechanisms among chlamydial species.

The Chlamydia trachomatis Early Effector Tarp Outcompetes Fascin in Forming F-Actin Bundles In Vivo

“…early effector TmeA. Similar to Tarp, TmeA is important for host cell invasion by directing cytoskeleton changes via altered Arp2/3 dynamics ( McKuen et al, 2017 ; Faris et al, 2020 ; Keb et al, 2021 ). Expression of TmeA in flies via the ubi-GAL4 driver was validated by Western blot ( Figure S3A ).…”

“…The validation of Tarp’s bundling activity in vivo and its ability to compete with endogenous host bundlers during F-actin bundle formation adds to the repertoire of molecular functions that Tarp can deploy during Chlamydia infection. Promoting F-actin polymerization at the site of entry is a major Chlamydia strategy during host cell invasion ( Jewett et al, 2006 ; McKuen et al, 2017 ; Ghosh et al, 2020 ; Keb et al, 2021 ) but F-actin bundling itself has yet to be mechanistically implicated. Our finding raises the possibility that F-actin bundling and/or competition with host bundlers is a component of Tarp function in promoting Chlamydia infection.…”

Regulation of invasion-associated actin dynamics by the Chlamydia trachomatis effectors TarP and TmeA

“…Arp2/3 recruitment dynamics at entry sites displays dependence on TmeA and TarP One explanation for impaired actin kinetics following TarP and TmeA deletion is that loss of either effector prevents optimal recruitment of the actin nucleator Arp2/3. Indeed, a recent study suggested that the respective signal transduction capabilities of TarP and TmeA form a functional collaboration that enhances recruitment of Arp2/3 and increases the rate of actin polymerization [23,24]. As such, we monitored the recruitment of fluorescent Arp3 at the entry sites of wild-type, cis-complemented, or effector deletion strains in order to assess the contribution of effector collaboration on Arp2/3 recruitment during invasion (Fig.…”

“…Arp2/3 is a complex of proteins which, upon activation, enhances the rate of actin polymerization by nucleating branched actin filaments along a pre-existing “mother” filament (Bailly et al, 1999; Mullins et al, 1998), and the resulting branched network in turn increases the number of sites for Arp2/3 binding and branched nucleation (Higgs and Pollard, 1999). TarP and TmeA activate the Arp2/3 complex via downstream activation of the nucleation promoting factors WAVE2 and N-WASP, respectively (Carabeo et al, 2007; Faris et al, 2020; Keb et al, 2021; Lane et al, 2008). Both nucleation promoting factors have each been shown to be important for Chlamydia engulfment, as targeted disruption of either protein attenuated pathogen uptake (Carabeo et al, 2007; Faris et al, 2020; Keb et al, 2021).…”

“…TarP and TmeA activate the Arp2/3 complex via downstream activation of the nucleation promoting factors WAVE2 and N-WASP, respectively (Carabeo et al, 2007; Faris et al, 2020; Keb et al, 2021; Lane et al, 2008). Both nucleation promoting factors have each been shown to be important for Chlamydia engulfment, as targeted disruption of either protein attenuated pathogen uptake (Carabeo et al, 2007; Faris et al, 2020; Keb et al, 2021). Furthermore, siRNA knockdown of WAVE2 prevented the recruitment of actin at entry sites, highlighting the importance of TarP-associated Arp2/3 activation and subsequent actin remodeling in Chlamydia pathogenesis (Carabeo et al, 2007).…”