An α-Helical Core Encodes the Dual Functions of the Chlamydial Protein IncA

Erik Ronzone,Jordan Wesolowski,Laura D Bauler,Anshul Bhardwaj,Ted Hackstadt,Fabienne Paumet

doi:10.1074/jbc.m114.592063

Abstract

Chlamydia is an intracellular bacterium that establishes residence within parasitophorous compartments (inclusions) inside host cells. Chlamydial inclusions are uncoupled from the endolysosomal pathway and undergo fusion with cellular organelles and with each other. To do so, Chlamydia expresses proteins on the surface of the inclusion using a Type III secretion system. These proteins, termed Incs, are located at the interface between host and pathogen and carry out the functions necessary for Chlamydia survival. Among these Incs, IncA plays a critical role in both protecting the inclusion from lysosomal fusion and inducing the homotypic fusion of inclusions. Within IncA are two regions homologous to eukaryotic SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) domains referred to as SNARE-like domain 1 (SLD1) and SNARE-like domain 2 (SLD2). Using a multidisciplinary approach, we have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions. Circular dichroism and analytical ultracentrifugation experiments show that this core region is composed almost entirely of α-helices and assembles into stable homodimers in solution. Altogether, we propose that both IncA functions are encoded in a structured core domain that encompasses SLD1 and part of SLD2.

Highlights

The inclusion protein IncA inhibits and activates membrane fusion events during infection
We have discovered the functional core of IncA that retains the ability to both inhibit SNARE-mediated fusion and promote the homotypic fusion of Chlamydia inclusions
In order to define the mechanism of action of IncA, we have identified and characterized a trypsin-resistant region of IncA that spans the entire length of SNARE-like domain 1 (SLD1) and about one-third of SNARE-like domain 2 (SLD2)

Summary

Background

The inclusion protein IncA inhibits and activates membrane fusion events during infection. The survival of Chlamydia depends on its ability to rapidly modify the nascent inclusion in order to (i) isolate it from the canonical endolysosomal pathway that normally leads to degradation and bacterial clearance, (ii) induce inclusion-inclusion (homotypic) fusion, and (iii) generate novel fusion events with host organelles to acquire nutrients [3,4,5,6] To accomplish these modifications, bacterial effectors known as inclusion membrane proteins (Incs) are shuttled to and embedded in the inclusion membrane via a putative Type III secretion system [7, 8], where they play essential roles in pathogenicity, tissue tropism, and maintenance of inclusion integrity [9, 10]. They are located on virtually every intracellular compartment and use specific ␣-helical domains (SNARE motifs) to assemble into stable multimeric complexes and cat-

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EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION