Disruption of Francisella tularensis Schu S4 iglI , iglJ , and pdpC Genes Results in Attenuation for Growth in Human Macrophages and In Vivo Virulence in Mice and Reveals a Unique Phenotype for pdpC

Abstract

e Francisella tularensis is a facultative intracellular bacterial pathogen and the causative agent of tularemia. After infection of macrophages, the organism escapes from its phagosome and replicates to high density in the cytosol, but the bacterial factors required for these aspects of virulence are incompletely defined. Here, we describe the isolation and characterization of Francisella tularensis subsp. tularensis strain Schu S4 mutants that lack functional iglI, iglJ, or pdpC, three genes of the Francisella pathogenicity island. Our data demonstrate that these mutants were defective for replication in primary human monocyte-derived macrophages and murine J774 cells yet exhibited two distinct phenotypes. The iglI and iglJ mutants were similar to one another, exhibited profound defects in phagosome escape and intracellular growth, and appeared to be trapped in cathepsin Dpositive phagolysosomes. Conversely, the pdpC mutant avoided trafficking to lysosomes, phagosome escape was diminished but not ablated, and these organisms replicated in a small subset of infected macrophages. The phenotype of each mutant strain was reversed by trans complementation. In vivo virulence was assessed by intranasal infection of BALB/c mice. The mutants appeared avirulent, as all mice survived infection with 10 8 CFU iglJ-or pdpC-deficient bacteria. Nevertheless, the pdpC mutant disseminated to the liver and spleen before being eliminated, whereas the iglJ mutant did not. Taken together, our data demonstrate that the pathogenicity island genes tested are essential for F. tularensis Schu S4 virulence and further suggest that pdpC may play a unique role in this process, as indicated by its distinct intermediate phenotype.

IglE Is an Outer Membrane-Associated Lipoprotein Essential for Intracellular Survival and Murine Virulence of Type A Francisella tularensis

“…Consistent with this result, Long et al found that while Schu S4 iglI and IglJ were essential for phagosomal escape and alteration of endosomal trafficking, a mutant lacking pdpC was only partially defective in this regard (30). This led to the conclusion that PdpC contributes to, but is not essential for, remodeling of the host phagosomal pathway (30). A similar result was reported for an LVS pdpC mutant (57).…”

“…Differences in intracellular replication and induction of host cell death pathways were also observed following infection of J774 macrophages with pdpC, iglC, iglG, or iglI mutants of LVS (56). Consistent with this result, Long et al found that while Schu S4 iglI and IglJ were essential for phagosomal escape and alteration of endosomal trafficking, a mutant lacking pdpC was only partially defective in this regard (30). This led to the conclusion that PdpC contributes to, but is not essential for, remodeling of the host phagosomal pathway (30).…”

“…novicida in the D. melanogaster fly model, it is tempting to speculate that a subset of FPI genes performs a common, nonredundant function and is essential for FPI activity and, hence, pathogenesis in multiple host species; another set may contribute to pathogenesis but is not absolutely essential for FPI activity per se. The extent of apparent phagosomal escape observed here for the Schu S4 iglE-null strain (i.e., 38% of bacteria were cytoplasmic) was higher than that reported for Schu S4 variants lacking either iglC (31), iglI, or iglJ or the FPI regulator fevR (30). Instead, the reduction in phagosomal escape observed in the Schu S4 iglE-null strain was more similar to that for Schu S4 lacking pdpC, which was only partially defective in this regard (30).…”

“…The extent of apparent phagosomal escape observed here for the Schu S4 iglE-null strain (i.e., 38% of bacteria were cytoplasmic) was higher than that reported for Schu S4 variants lacking either iglC (31), iglI, or iglJ or the FPI regulator fevR (30). Instead, the reduction in phagosomal escape observed in the Schu S4 iglE-null strain was more similar to that for Schu S4 lacking pdpC, which was only partially defective in this regard (30). In contrast to the latter study, however, our results indicate that iglE is essential for colonization and dissemination in the C3H/HeN mouse model following i.n.…”

“…novicida but is duplicated in type A and type B strains. The presence of two identical copies in the most virulent forms of F. tularensis has limited all but a few genetic studies (8,(28)(29)(30)(31)(32) to lower-virulence F. tularensis subsp. novicida (which is more easily manipulated via standard genetic techniques owing to the presence of a single copy of the FPI).…”