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

Abstract

AbstractThe obligate intracellular pathogen Chlamydia trachomatis manipulates the host actin cytoskeleton to assemble actin-rich structures that drive pathogen entry. This actin remodeling event exhibits relatively rapid dynamics that, through quantitative live-cell imaging, was revealed to consist of three phases – a fast recruitment phase which abruptly transitions to a fast turnover phase before resolving into a slow turnover of actin, indicating the end of actin remodeling. Here, we investigate Chlamydia invasion in the context of actin dynamics. Efficient invasion was associated with robust actin remodeling kinetics, which was linked to signaling from the type-III secreted effectors TarP and TmeA, and the actin nucleating activities of formin 1 (Fmn1) and Arp2/3. Stable recruitment of Fmn1 and Arp2/3 was dependent upon TarP and/or TmeA, although TarP signaling was responsible for the majority of Fmn1 and Arp2/3 recruitment. Rapid actin kinetics were due in part to a collaborative functional interaction between two different classes of actin nucleators – formins, including formin 1 and the diaphanous-related formins mDia1 and mDia2, and the Arp2/3 complex. Inhibition of either formin or Arp2/3, or deletion of TarP and TmeA, prevented this collaboration and resulted in attenuated actin kinetics and invasion efficiency. Collectively, these data support a model wherein TarP and TmeA signaling are core components of actin remodeling that operate via stable recruitment of formin and Arp2/3. At the population level, the kinetics of recruitment and turnover of actin and its nucleators were linked. However, reanalysis of the data at the level of individual elementary bodies showed significant variation and a lack of correlation between the kinetics of recruitment and turnover, suggesting that accessory factors variably modify actin kinetics at individual entry sites. In summary, efficient chlamydial invasion is an effector-driven process that requires a specific profile of actin recruitment which arises following collaboration between formin and Arp2/3.Author SummaryThe obligate intracellular pathogen Chlamydia trachomatis relies upon manipulation of the host actin cytoskeleton to drive its entry into host cells, such that impairment of actin dynamics attenuates Chlamydia invasion. Collaboration between two classes of actin nucleators, formin and Arp2/3, are known to enhance actin recruitment and turnover; we found that stable recruitment of both proteins to the signaling complex established by the type III secreted effectors, TarP and TmeA, were important for pathogen internalization. Furthermore, Formin 1 and Arp2/3 are co-recruited to sites of entry, and pharmacological inhibition of either actin nucleator impaired recruitment of the other, indicating a functional cooperation between branched and filamentous actin nucleation within pathogen entry sites. Disruption of this cooperation negatively impacted both actin dynamics and Chlamydia internalization, indicating that TarP- and TmeA-dependent entry of Chlamydia into non-phagocytic cells operates through the recruitment and activation of Arp2/3 and Formin 1. Finally, kinetic analysis of actin recruitment and turnover revealed that these processes were independently regulated, in addition to implicating the presence of local factors that fine-tune actin dynamics and subsequent invasion.