bioRxiv2021
The 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 that indicates the end of actin remodeling. Here, we investigate Chlamydia invasion in the context of actin dynamics. Efficient invasion is associated with robust actin remodeling kinetics that results from 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. Recruitment of these nucleators requires the presence of the chlamydial type III effector TarP, which enables the respective nucleating activities of formin and Arp2/3 to collaboratively generate a robust actin network. A collaborative model is supported by the observation that co-inhibition of Fmm1 and Arp2/3 further reduced both actin dynamics and invasion efficiency than either treatment alone. Furthermore, inhibition of recruitment of Fmn1 and/or Arp2/3 by deleting TarP was sufficient to similarly attenuated actin kinetics and invasion efficiency, supporting a model wherein TarP is the major contributor to robust actin remodeling via its recruitment of the two classes of actin nucleators. At the population level, the kinetics of recruitment and turnover of actin and its nucleators were linked. However, a more detailed analysis 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 requires a specific profile of actin dynamics which are coordinated by TarP-dependent recruitment of two classes of actin nucleators. 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 recruitment of both proteins to the signaling complex established by the type III secreted effector, TarP, was 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-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.