is a highly infectious gram-negative coccobacillus that causes the zoonotic disease tularemia (11
). This bacterial pathogen is known for its ability to cause a fulminating disease in humans after exposure to as few as 10 cells and has raised considerable concern as a potential bioterrorist agent (10
). Because of its high infectivity and lethality, F. tularensis
is one of six types of microbes classified by the U.S. Centers for Disease Control and Prevention as a category A agent, one that poses the most serious threat as a vehicle of bioterror.
There are a variety of subspecies and biotypes of F. tularensis, but they all have greater than 95% DNA sequence identity. Although the type A and type B biotype strains are highly infectious, only type A strains, which are found exclusively in North America, cause significant mortality in infected humans. An attenuated variant of a type B biotype strain formed the basis of a live vaccine strain (LVS) of F. tularensis. Understanding the molecular basis of the differences in virulence levels of F. tularensis strains may help in the development of a rationally designed LVS.
is a facultative intracellular pathogen. The currently available evidence suggests that F. tularensis
resides inside a membrane-bound phagosome during its initial growth in a macrophage and that it may be released into the cytoplasm during a later phase of growth (3
). Very little is known about the bacterial virulence factors needed for infection, although it is clear that intracellular growth, especially in macrophages, is essential to the virulence of F. tularensis
. A biochemical study of the LVS of F. tularensis
showed that four proteins are induced after F. tularensis
entry into macrophages (15
). The gene encoding the most prominently induced protein, the 23-kDa IglC protein, has been molecularly cloned and sequenced. Recently, Golovliov et al. (16
) deleted this gene and showed that the resulting mutant was unable to grow in macrophages and unable to cause disease in mice.
Genetic approaches have also been used to discover other F. tularensis
genes needed for optimal intracellular growth. The products of mglA
, thought to be global regulators, are both required for intramacrophage growth and virulence in mice (5
). Random insertional mutagenesis revealed that inactivation of F. tularensis
genes encoding homologues of glutamine phosphoribosylpyrophosphate amidotransferase (purine biosynthesis), alanine racemase (peptidoglycan biosynthesis), and the heat shock-inducible ClpB protease reduces the ability of F. tularensis
to grow in mouse macrophages (17
). Perhaps most significantly, transposon insertion into iglB
, which are part of the pathogenicity island described in this work, profoundly affects intramacrophage growth (17
The strategy of parasitizing host cells is a common theme used by both bacterial and protozoan pathogens (2
). In many bacterial intracellular pathogens, a specific gene or set of genes that promotes entry into host cells has been identified (12
). Two general types of cell entry mechanisms have been identified. One involves the tight binding of a bacterial surface protein to a host cell receptor, followed by engulfment of the bacterial cell by a zipper-like phagocytosis. A second type of uptake uses type III secretion machinery to inject effector proteins into host cells, inducing membrane ruffling and macropinocytosis. The genetic loci in Salmonella
spp. that encode the products needed for entry into mammalian cells are pathogenicity islands of common origin (18
). The horizontal movement of this cluster of genes has enabled different species of bacteria to gain the ability to invade cells.
In this study we provide evidence for a Francisella pathogenicity island (FPI) that is required for intramacrophage growth and virulence in mice. The presumed effector proteins encoded by the FPI genes show no definitive similarity to known prokaryotic virulence proteins and thus represent novel factors required for virulence and intramacrophage growth. The gene encoding the PdpD protein appears to be absent in F. tularensis type B strains, and this absence may play a role in the wide difference in virulence of human infections between the type A and type B strains.