Actin Recruitment to the Chlamydia Inclusion Is Spatiotemporally Regulated by a Mechanism That Requires Host and Bacterial Factors

Abstract

The ability to exit host cells at the end of their developmental growth is a critical step for the intracellular bacterium Chlamydia. One exit strategy, extrusion, is mediated by host signaling pathways involved with actin polymerization. Here, we show that actin is recruited to the chlamydial inclusion as a late event, occurring after 20 hours post-infection (hpi) and only within a subpopulation of cells. This event increases significantly in prevalence and extent from 20 to 68 hpi, and actin coats strongly correlated with extrusions. In contrast to what has been reported for other intracellular pathogens, actin nucleation on Chlamydia inclusions did not ‘flash’, but rather exhibited moderate depolymerization dynamics. By using small molecule agents to selectively disrupt host signaling pathways involved with actin nucleation, modulate actin polymerization dynamics and also to disable the synthesis and secretion of chlamydial proteins, we further show that host and bacterial proteins are required for actin coat formation. Transient disruption of either host or bacterial signaling pathways resulted in rapid loss of coats in all infected cells and a reduction in extrusion formation. Inhibition of Chlamydia type III secretion also resulted in rapid loss of actin association on inclusions, thus implicating chlamydial effector proteins(s) as being central factors for engaging with host actin nucleating factors, such as formins. In conclusion, our data illuminate the host and bacterial driven process by which a dense actin matrix is dynamically nucleated and maintained on the Chlamydia inclusion. This late stage event is not ubiquitous for all infected cells in a population, and escalates in prevalence and extent throughout the developmental cycle of Chlamydia, culminating with their exit from the host cell by extrusion. The initiation of actin recruitment by Chlamydia appears to be novel, and may serve as an upstream determinant of the extrusion mechanism.

In Contrast to Chlamydia trachomatis, Waddlia chondrophila Grows in Human Cells without Inhibiting Apoptosis, Fragmenting the Golgi Apparatus, or Diverting Post-Golgi Sphingomyelin Transport

“…Microtubules are used for initial trafficking of the nascent inclusion (45), and defined microdomains on the inclusion appear to be responsible for this interaction (46). Actin recruitment has been investigated with small-molecule inhibitors, and a contribution of bacterial activities and a number of host cell networks involved have been identified (47). Intermediate filaments have been suggested to convey stability to the inclusion (23).…”

Using Fluorescent Proteins to Visualize and Quantitate <em>Chlamydia </em>Vacuole Growth Dynamics in Living Cells

“…A novel application of this approach is for derivation of meaningful quantitative data about the inclusion, for example inclusion area, volume and shape. Finally, this visualization method is highly adaptable and can be used in combination with other fluorescent markers to reveal additional insight into the biology of Chlamydia infected host cells, for example GFP-or mKate2-expressing Chlamydia 6,11 , fluorescent actin 12 or GFP tagged Inc proteins 13 .…”

Chlamydia trachomatis Cellular Exit Alters Interactions with Host Dendritic Cells

“…Sloughing of infected epithelial cells, sometimes driven by polymorphonuclear leukocytes (PMNs), has been observed in vivo and may precede lysis (16). In contrast, extrusion begins with the contraction of the plasma and inclusion membranes, resulting in the detachment of a separate compartment of Chlamydia enclosed in a double host membrane (14,17). This is a controlled process dependent on both bacterial and host factors, which leaves the host cell intact and often residually infected (14,17).…”

“…In contrast, extrusion begins with the contraction of the plasma and inclusion membranes, resulting in the detachment of a separate compartment of Chlamydia enclosed in a double host membrane (14,17). This is a controlled process dependent on both bacterial and host factors, which leaves the host cell intact and often residually infected (14,17). The role of extrusion during human infection is unknown, but it occurs in approximately 50% of infected cells in vitro and has been observed in an animal model of in vivo infection (14,17,18).…”

“…This is a controlled process dependent on both bacterial and host factors, which leaves the host cell intact and often residually infected (14,17). The role of extrusion during human infection is unknown, but it occurs in approximately 50% of infected cells in vitro and has been observed in an animal model of in vivo infection (14,17,18). The potential infectious advantages of extrusion for immune evasion, bacterial survival, and dissemination are striking.…”