Structure-Function Analysis of DipA, a Francisella tularensis Virulence Factor Required for Intracellular Replication

Audrey Chong,Robert Child,Dedeke Rockx-Brouwer,Jean Celli,Aiping Qin,Barbara J Mann,Tara D Wehrly,Martin Pavelka

doi:10.1371/journal.pone.0067965

Abstract

Francisella tularensis is a highly infectious bacterium whose virulence relies on its ability to rapidly reach the macrophage cytosol and extensively replicate in this compartment. We previously identified a novel Francisella virulence factor, DipA (FTT0369c), which is required for intramacrophage proliferation and survival, and virulence in mice. DipA is a 353 amino acid protein with a Sec-dependent signal peptide, four Sel1-like repeats (SLR), and a C-terminal coiled-coil (CC) domain. Here, we determined through biochemical and localization studies that DipA is a membrane-associated protein exposed on the surface of the prototypical F. tularensis subsp. tularensis strain SchuS4 during macrophage infection. Deletion and substitution mutagenesis showed that the CC domain, but not the SLR motifs, of DipA is required for surface exposure on SchuS4. Complementation of the dipA mutant with either DipA CC or SLR domain mutants did not restore intracellular growth of Francisella , indicating that proper localization and the SLR domains are required for DipA function. Co-immunoprecipitation studies revealed interactions with the Francisella outer membrane protein FopA, suggesting that DipA is part of a membrane-associated complex. Altogether, our findings indicate that DipA is positioned at the host–pathogen interface to influence the intracellular fate of this pathogen.

Highlights

The Gram-negative intracellular bacterium Francisella tularensis is the causative agent of tularemia, a potentially fatal zoonosis affecting a variety of mammals, including humans [1]
DipA is localized to the surface of F. tularensis subsp. tularensis SchuS4
In F. novicida, IglA has been detected in the cytoplasm, outer membrane and membrane insoluble fractions, and the ambiguity of its distribution has been ascribed to its association with a macromolecular structure spanning the periplasm from the inner membrane [36,37,38]

Summary

Introduction

The Gram-negative intracellular bacterium Francisella tularensis is the causative agent of tularemia, a potentially fatal zoonosis affecting a variety of mammals, including humans [1]. We report that DipA is a membrane-associated protein localized on the bacterial surface during macrophage infection, and show that the SLR and CC domains are functionally distinct. Under conditions that do not permeabilize bacterial membranes, we detected DipA-HA on the surface of intracellular bacteria at 10 h post infection (p.i.) in BMMs by immunofluorescence labelling (Figure 3A).

Results

Conclusion