IglC and PdpA are important for promoting Francisella invasion and intracellular growth in epithelial cells.

H T Law,Francis E Nano,Aarati Sriram,Charlotte Fevang,Eli B Nix,Julian Andrew Guttman

doi:10.1371/journal.pone.0104881

H T Law, Francis E Nano + Show 4 more

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https://doi.org/10.1371/journal.pone.0104881

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Abstract

The highly infectious bacteria, Francisella tularensis, colonize a variety of organs and replicate within both phagocytic as well as non-phagocytic cells, to cause the disease tularemia. These microbes contain a conserved cluster of important virulence genes referred to as the Francisella Pathogenicity Island (FPI). Two of the most characterized FPI genes, iglC and pdpA, play a central role in bacterial survival and proliferation within phagocytes, but do not influence bacterial internalization. Yet, their involvement in non-phagocytic epithelial cell infections remains unexplored. To examine the functions of IglC and PdpA on bacterial invasion and replication during epithelial cell infections, we infected liver and lung epithelial cells with F. novicida and F. tularensis ‘Type B’ Live Vaccine Strain (LVS) deletion mutants (ΔiglC and ΔpdpA) as well as their respective gene complements. We found that deletion of either gene significantly reduced their ability to invade and replicate in epithelial cells. Gene complementation of iglC and pdpA partially rescued bacterial invasion and intracellular growth. Additionally, substantial LAMP1-association with both deletion mutants was observed up to 12 h suggesting that the absence of IglC and PdpA caused deficiencies in their ability to dissociate from LAMP1-positive Francisella Containing Vacuoles (FCVs). This work provides the first evidence that IglC and PdpA are important pathogenic factors for invasion and intracellular growth of Francisella in epithelial cells, and further highlights the discrete mechanisms involved in Francisella infections between phagocytic and non-phagocytic cells.

Highlights

The intracellular bacteria Francisella tularensis cause the zoonotic disease in humans and other mammals, called tularemia
To begin to investigate whether iglC and pdpA play a role in bacterial entry into epithelial cells, we evaluated the internalization of wild-type F. novicida, deletion mutants (DiglC, DpdpA), and their respective gene complemented strains during murine liver BNL CL.2 cell infections using gentamicin protection/invasion assays
Research on the sub-cellular events underlying Francisella pathogenesis has primarily concentrated on phagocytic cell infections, while those involved in non-phagocytic epithelial cell infections have remained largely unexplored

Summary

Introduction

The intracellular bacteria Francisella tularensis cause the zoonotic disease in humans and other mammals, called tularemia. We have yet to fully understand how these proteins interact with host cells, recent evidence suggests that IglC, PdpA, and six other FPI-encoded proteins are translocated into the cytoplasm of phagocytes and their delivery is dependent on the expression of Francisella T6SS genes [37]. Despite the importance these T6SS genes play in intracellular replication, their expression does not appear to significantly influence the uptake of Francisella into macrophages [43,48,49,50]. We report that iglC and pdpA are required for efficient bacterial invasion and intracellular proliferation during non-phagocytic epithelial cell infections

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