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. multidrug efflux ͉ type I secretion ͉ bacterial pathogenesis

During the pathogenesis of Lyme disease, Borrelia burgdorferi spreads hematogenously from the site of a tick bite to several tissues throughout the body. The specific mechanism of spirochete emigration is presently unknown. Using cultured human umbilical vein endothelial cells, we found that Borrelia burgdorfen bound to the endothelial cells and to the subendothelial matrix. Low passage isolates adhered 22-30-fold greater than a strain maintained in culture continuously. Spirochete binding to subendothelial matrix was inhibited 48-63% by pretreatment of the matrix with anti-fibronectin antiserum. Spirochete migration across endothelial monolayers cultured on amniotic membrane was increased when the monolayers were damaged by chemical or physical means. Electron microscopic examination of spirochete-endothelial interactions demonstrated the presence of spirochetes in the intercellular junctions between endothelial cells as well as beneath the monolayers. Scanning electron microscopy identified a mechanism of transendothelial migration whereby spirochetes pass between cells into the amniotic membrane at areas where subendothelium is exposed.

Bovine microvascular endothelial cells (BMECs) proliferated to confluence on the stromal surface of human amniotic membrane that had been denuded of its natural epithelium . The resulting cultures had the following characteristics : (a) The endothelial cells formed a thin, continuous monolayer and, like their in vivo counterparts, contained basal adhesion plaques and large numbers of cytoplasmic vesicles and 10-nm filaments . In addition, the endothelial cells elaborated a basement membrane-like structure . The microvascular endothelium plays an important role in regulating the exchange of fluid, macromolecules, and cells between the blood and the extravascular tissue . Studies of the mechanisms that underlie these exchanges have been limited by the lack of a simple and relevant in vitro model of the microvessel wall. The basic requirements for such a model are a suitable strain of endothelial cells and a substrate on which a monolayer of the cells can be maintained in a welldifferentiated state . For these monolayers to be useful in examining the transendothelial movement of materials and cells, it is important that they possess permeability characteristics that are similar to those of endothelium in vivo. That is, the endothelial cells must form intercellular junctions that exclude appropriate macromolecular probes and resist the passage of electrical current.We have developed an in vitro model of a microvessel wall that consists of cloned bovine microvascular endothelial cells (BMECs)' cultured on connective tissue prepared from hu-'Abbreviations used in this paper: BMECs, bovine microvascular endothelial cells ; HIDCS, heat-inactivated donor calf serum ; aMEM, minimal essential medium, alpha modification ; WGA-HRP, wheat germ agglutinin coupled to horseradish peroxidase .

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.

We have developed a method for studying the permeability properties of human endothelia in vitro. Human umbilical vein endothelial cells (HUVEC) were cultured on a substrate of human amnion. Confluent monolayers of these cells demonstrated 6-12 delta.cm2 of electrical resistance (a measure of their permeability to ions) and restricted the transendothelial passage of albumin from their apical to their basal surface. To determine whether leukocyte emigration alters endothelial permeability in this model, we examined the effects of migrating human polymorphonuclear leukocytes (PMN) on these two parameters. Few PMN migrated across the HUVEC monolayers in the absence of chemoattractants. In response to chemoattractants, PMN migration through HUVEC monolayers was virtually complete within 10 minutes and occurred at random locations throughout the monolayer. PMN migrated across the monolayer via the paracellular pathway. Although one PMN migrated across the monolayer for each HUVEC, PMN migration induced no change in electrical resistance or albumin permeability of these monolayers. At this PMN:HUVEC ratio, these permeability findings were correlated morphologically to measurements that HUVEC paracellular pathway size increases by less than 0.22% with PMN migration. This increase is insufficient to effect a measurable change in the electrical resistance of the endothelial cell monolayer. These findings demonstrate that increased permeability of cultured endothelial cell monolayers is not a necessary consequence of PMN emigration.