A conserved and immunodominant lipoprotein of Francisella tularensis is proinflammatory but not essential for virulence.

Francisella tularensis is a highly virulent bacterium that causes tularemia, a disease that is often fatal if untreated. A live vaccine strain (LVS) of this bacterium is attenuated for virulence in humans but produces lethal disease in mice. F. tularensis has been classified as a Category A agent of bioterrorism. Despite this categorization, little is known about the components of the organism that are responsible for causing disease in its hosts. Here, we report the deletion of a well-characterized lipoprotein of F. tularensis, designated LpnA (also known as Tul4), in the LVS. An LpnA deletion mutant was comparable to the wild-type strain in its ability to grow intracellularly and cause lethal disease in mice. Additionally, mice inoculated with a sublethal dose of the mutant strain were afforded the same protection against a subsequent lethal challenge with the LVS as were mice initially administered a sublethal dose of the wild-type bacterium. The LpnA-deficient strain showed an equivalent ability to promote secretion of chemokines by human monocyte-derived macrophages as its wild-type counterpart. However, recombinant LpnA potently stimulated primary cultures of human macrophages in a Toll-like receptor 2-dependent manner. Although human endothelial cells were also activated by recombinant LpnA, their response was relatively modest. LpnA is clearly unnecessary for multiple functions of the LVS, but its inflammatory capacity implicates it and other Francisella lipoproteins as potentially important to the pathogenesis of tularemia.

Francisella tularensis has a significant extracellular phase in infected mice.

The ability of Francisella tularensis to replicate in macrophages has led many investigators to assume that it resides primarily intracellularly in the blood of mammalian hosts. We have found this supposition to be untrue. In almost all cases, the majority of F. tularensis recovered from the blood of infected mice was in plasma rather than leukocytes. This distribution was observed irrespective of size of inoculum, route of inoculation, time after inoculation, or virulence of the infecting strain. Our findings yield new insight into the pathogenesis of tularemia and may have important ramifications in the search for anti-Francisella therapies.

Mac-1+ cells are the predominant subset in the early hepatic lesions of mice infected with Francisella tularensis.

The cell composition of early hepatic lesions of experimental murine tularemia has not been characterized with specific markers. The appearance of multiple granulomatous-necrotic lesions in the liver correlates with a marked increase in the levels of serum alanine transferase and lactate dehydrogenase. Francisella tularensis, detected by specific antibodies, can be first noted by day 1 and becomes associated with the lesions by 5 days postinoculation. These lesions become necrotic, with some evidence of in situ apoptosis. The lesions do not contain B, T, or NK cells. Rather, the lesions are largely composed of two subpopulations of Mac-1(+) cells that are associated with the bacteria. Gr-1(+) Mac-1(+) immature myeloid cells and major histocompatibility complex class II-positive (MHC-II(+)) Mac-1(+) macrophages were the most abundant cell phenotypes found in the granuloma and are likely major contributors in controlling the infection in its early stages. Our findings have shown that there is an early development of hepatic lesions where F. tularensis colocalizes with both Gr-1(+) Mac-1(+) and MHC-II(+) Mac-1(+) cells.

Deletion of TolC orthologs in Francisella tularensis identifies roles in multidrug resistance and virulence.

The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia. Interest in this zoonotic pathogen has increased due to its classification as a category A agent of bioterrorism, but little is known about the molecular mechanisms underlying its virulence, and especially what secretion systems and virulence factors are present. In this study, we characterized two genes in the F. tularensis genome, tolC and a gene we term ftlC, whose products have high homology with the Escherichia coli TolC protein. TolC functions as the outer membrane channel component for both type I secretion and multidrug efflux systems. We constructed deletion mutations of these genes in the F. tularensis live vaccine strain by allelic replacement. Deletion of either tolC or ftlC caused increased sensitivity to various antibiotics, detergents, and dyes, indicating both genes are involved in the multidrug resistance machinery of F. tularensis. Complementation of the deletion mutations in trans restored drug resistance. Neither tolC nor ftlC was required for replication of the live vaccine strain in murine bone marrow-derived macrophages. However, deletion of tolC, but not ftlC, caused a significant attenuation of virulence in a mouse model of tularemia that could be complemented by addition of tolC in trans. Thus, tolC is a critical virulence factor of F. tularensis in addition to its role in multidrug resistance, which suggests the presence of a functional type I secretion system.

The live vaccine strain of Francisella tularensis replicates in human and murine macrophages but induces only the human cells to secrete proinflammatory cytokines.

Francisella tularensis is the highly infectious agent of tularemia, a disease that can prove fatal in humans. An attenuated live vaccine strain (LVS) of this bacterium is avirulent in man but produces lethal illness in mice. As a step toward understanding the species specificity of the LVS, we compared its interactions with murine and human leukocytes. The bacterium replicated within murine bone marrow-derived macrophages (muBMDM), human monocyte-derived macrophages (huMDM), and freshly isolated human monocytes. However, the murine and human phagocytes differed in their ability to secrete proinflammatory cytokines in response to the LVS. The huMDM released large amounts of CXC chemokine ligand 8 (CXCL8) and CC chemokine ligand 2 when incubated with live or killed LVS organisms, and live bacteria also elicited production of interleukin-1beta (IL-1beta). Furthermore, human monocytes secreted CXCL8, IL-1beta, and tumor necrosis factor alpha in response to various bacterial preparations. In contrast, muBMDM produced little to no proinflammatory cytokines or chemokines when treated with any preparations of the LVS. Clearly, human and murine macrophages support growth of this bacterium. However, the greater proinflammatory response of human leukocytes to F. tularensis LVS may contribute to the avirulence of this strain in the human host.

Francisella tularensis selectively induces proinflammatory changes in endothelial cells.

Naturally acquired infections with Francisella tularensis, the bacterial agent of tularemia, occur infrequently in humans. However, the high infectivity and lethality of the organism in humans raise concerns that it might be exploited as a weapon of bioterrorism. Despite this potential for illicit use, the pathogenesis of tularemia is not well understood. To examine how F. tularensis interacts with cells of its mammalian hosts, we tested the ability of a live vaccine strain (LVS) to induce proinflammatory changes in cultured HUVEC. Living F. tularensis LVS induced HUVEC to express the adhesion molecules VCAM-1 and ICAM-1, but not E-selectin, and to secrete the chemokine CXCL8, but not CCL2. Stimulation of HUVEC by the living bacteria was partially suppressed by polymyxin B, an inhibitor of LPS, but did not require serum, suggesting that F. tularensis LVS does not stimulate endothelium through the serum-dependent pathway that is typically used by LPS from enteric bacteria. In contrast to the living organisms, suspensions of killed F. tularensis LVS acquired the ability to increase endothelial expression of both E-selectin and CCL2. Up-regulation of E-selectin and CCL2 by the killed bacteria was not inhibited by polymyxin B. Exposure of HUVEC to either live or killed F. tularensis LVS for 24 h promoted the transendothelial migration of subsequently added neutrophils. These data indicate that multiple components of F. tularensis LVS induce proinflammatory changes in endothelial cells in an atypical manner that may contribute to the exceptional infectivity and virulence of this pathogen.