Chlamydia exploits filopodial capture and a macropinocytosis-like pathway for host cell entry.

Charlotte Ford,Emmanuel Boucrot,Andrea Nans,Richard D Hayward

doi:10.1371/journal.ppat.1007051

Abstract

Pathogens hijack host endocytic pathways to force their own entry into eukaryotic target cells. Many bacteria either exploit receptor-mediated zippering or inject virulence proteins directly to trigger membrane reorganisation and cytoskeletal rearrangements. By contrast, extracellular C. trachomatis elementary bodies (EBs) apparently employ facets of both the zipper and trigger mechanisms and are only ~400 nm in diameter. Our cryo-electron tomography of C. trachomatis entry revealed an unexpectedly diverse array of host structures in association with invading EBs, suggesting internalisation may progress by multiple, potentially redundant routes or several sequential events within a single pathway. Here we performed quantitative analysis of actin organisation at chlamydial entry foci, highlighting filopodial capture and phagocytic cups as dominant and conserved morphological structures early during internalisation. We applied inhibitor-based screening and employed reporters to systematically assay and visualise the spatio-temporal contribution of diverse endocytic signalling mediators to C. trachomatis entry. In addition to the recognised roles of the Rac1 GTPase and its associated nucleation-promoting factor (NPF) WAVE, our data revealed an additional unrecognised pathway sharing key hallmarks of macropinocytosis: i) amiloride sensitivity, ii) fluid-phase uptake, iii) recruitment and activity of the NPF N-WASP, and iv) the localised generation of phosphoinositide-3-phosphate (PI3P) species. Given their central role in macropinocytosis and affinity for PI3P, we assessed the role of SNX-PX-BAR family proteins. Strikingly, SNX9 was specifically and transiently enriched at C. trachomatis entry foci. SNX9-/- cells exhibited a 20% defect in EB entry, which was enhanced to 60% when the cells were infected without sedimentation-induced EB adhesion, consistent with a defect in initial EB-host interaction. Correspondingly, filopodial capture of C. trachomatis EBs was specifically attenuated in SNX9-/- cells, implicating SNX9 as a central host mediator of filopodial capture early during chlamydial entry. Our findings identify an unanticipated complexity of signalling underpinning cell entry by this major human pathogen, and suggest intriguing parallels with viral entry mechanisms.

Highlights

An essential early event in the lifecycle of many human and animal pathogens is entry into non-phagocytic host epithelial cells
Chlamydia trachomatis remains the leading bacterial agent of sexually transmitted disease worldwide and causes a form of blindness called trachoma in Developing nations, which is recognised by the World Health Organisation as a neglected tropical disease
Chlamydia must live inside human cells to survive, and here we study the mechanism of how it enters cells, which is critical to the lifecycle

Summary

Introduction

An essential early event in the lifecycle of many human and animal pathogens is entry into non-phagocytic host epithelial cells. Bacteria and parasites all engage with host cell surfaces prior to inducing the reorganisation of the plasma membrane and underlying cytoskeleton to promote their internalisation. Invasive bacteria like Salmonella and Listeria species are typically > 1 μm in diameter and promote their internalisation either by injecting virulence effector proteins that subvert host signalling to reversibly induce cytoskeletal reorganisation, or through surface ligand mimicry hijack receptor-mediated endocytosis, respectively [1]. C. trachomatis EBs exploit a T3SS and deliver effectors into the host cell that reversibly stimulate Rac. The mechanism remains incomplete, a major factor is the T3SS effector translocated actin recruiting phosphoprotein (TARP) that nucleates polymerisation directly by binding to actin, and indirectly upon tyrosine phosphorylation by acting as a scaffold for Rac guanine nucleotide exchange factors [8,9,10,11]. The role of receptors in C. trachomatis entry is less clear, as none are essential [15]

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