Successful parasitism of host cells by intracellular pathogens involves adherence, entry, survival, intracellular replication, and cell-to-cell spread. Our laboratory has been examining the role of early events, adherence and entry, in the pathogenesis of the facultative intracellular pathogen Legionella pneumophila. Currently, the mechanisms used by L. pneumophila to gain access to the intracellular environment are not well understood. We have recently isolated three loci, designated enh1, enh2, and enh3, that are involved in the ability of L. pneumophila to enter host cells. One of the genes present in the enh1 locus, rtxA, is homologous to repeats in structural toxin genes (RTX) found in many bacterial pathogens. RTX proteins from other bacterial species are commonly cytotoxic, and some of them have been shown to bind to ␤ 2 integrin receptors. In the current study, we demonstrate that the L. pneumophila rtxA gene is involved in adherence, cytotoxicity, and pore formation in addition to its role in entry. Furthermore, an rtxA mutant does not replicate as well as wild-type L. pneumophila in monocytes and is less virulent in mice. Thus, we conclude that the entry gene rtxA is an important virulence determinant in L. pneumophila and is likely to be critical for the production of Legionnaires' disease in humans.Legionella pneumophila is an intracellular pathogen that causes Legionnaires' disease in humans, a potentially lethal pneumonia. The ability of L. pneumophila to enter, survive, and replicate in monocytic cells is essential for pathogenesis. Differences in the mechanisms used to enter monocytes correlate with subsequent intracellular survival and replication (13). In addition, it has recently been shown that the bacterial entry mechanism and/or factors expressed very early after entry alter intracellular trafficking (52). L. pneumophila has been shown to enter monocytes by an unusual mechanism, coiling phagocytosis (28), in addition to the conventional phagocytic mechanism observed in most other bacterial species. Although coiling phagocytosis also occurs in spirochetes (12,47,48), the bacterial factors and host cell components involved are not known. Complement (31,40,45) and antibody (30, 31) opsonization have effects on adherence of L. pneumophila to monocytes. In addition, growth conditions (14) and opsonization with complement (13) or antibodies (28) have been shown to affect the frequencies of coiling and conventional phagocytosis. However, both complement (13) and antibody (28) opsonization results in higher frequencies of conventional phagocytosis. Furthermore, conventional phagocytosis correlates with lower replication rates of L. pneumophila in monocytes (13). These data suggest that further study of the mechanisms of nonopsonic phagocytosis by monocytes is critical to obtaining a better understanding of L. pneumophila pathogenesis.Our laboratory has recently identified three chromosomal loci, designated enh1, enh2, and enh3, that affect nonopsonic entry of L. pneumophila into monocytes (15). The...

Francisella tularensis, the causative agent of the zoonotic disease tularemia, has recently gained increased attention due to the emergence of tularemia in geographical areas where the disease has been previously unknown and to the organism's potential as a bioterrorism agent. Although F. tularensis has an extremely broad host range, the bacterial reservoir in nature has not been conclusively identified. In this study, the ability of virulent F. tularensis strains to survive and replicate in the amoeba Acanthamoeba castellanii was explored. We observe that A. castellanii trophozoites rapidly encyst in response to F. tularensis infection and that this rapid encystment phenotype is caused by factor(s) secreted by amoebae and/or F. tularensis into the coculture medium. Further, our results indicate that in contrast to the live vaccine strain LVS, virulent strains of F. tularensis can survive in A. castellanii cysts for at least 3 weeks postinfection and that the induction of rapid amoeba encystment is essential for survival. In addition, our data indicate that pathogenic F. tularensis strains block lysosomal fusion in A. castellanii. Taken together, these data suggest that interactions between F. tularensis strains and amoebae may play a role in the environmental persistence of F. tularensis.

Francisella tularensis, the zoonotic cause of tularemia, can infect numerous mammals and other eukaryotes. Although studying F. tularensis pathogenesis is essential to comprehending disease, mammalian infection is just one step in the ecology of Francisella species. F. tularensis has been isolated from aquatic environments and arthropod vectors, environments in which chitin could serve as a potential carbon source and as a surface for attachment and growth. We show that F. tularensis subsp. novicida forms biofilms during the colonization of chitin surfaces. The ability of F. tularensis to persist using chitin as a sole carbon source is dependent on chitinases, since mutants lacking chiA or chiB are attenuated for chitin colonization and biofilm formation in the absence of exogenous sugar. A genetic screen for biofilm mutants identified the Sec translocon export pathway and 14 secreted proteins. We show that these genes are important for initial attachment during biofilm formation. We generated defined deletion mutants by targeting two chaperone genes (secB1 and secB2) involved in Sec-dependent secretion and four genes that encode putative secreted proteins. All of the mutants were deficient in attachment to polystyrene and chitin surfaces and for biofilm formation compared to wild-type F. novicida. In contrast, mutations in the Sec translocon and secreted factors did not affect virulence. Our data suggest that biofilm formation by F. tularensis promotes persistence on chitin surfaces. Further study of the interaction of F. tularensis with the chitin microenvironment may provide insight into the environmental survival and transmission mechanisms of this pathogen.

To extend our knowledge of host-cell targets of Helicobacter pylori, we characterized the interaction between H. pylori and human T84 epithelial cell polarized monolayers. Transcriptional analysis by use of human microarrays and a panel of isogenic H. pylori mutants revealed distinct responses to infection. Of the 670 genes whose expression changed, most (92%) required the cag pathogenicity island (PAI). Although altered expression of many genes was dependent on CagA (80% of the PAI-dependent genes), expression of >30% of these host genes occurred independent of the phosphorylation state of the CagA protein. Similarly, we found that injected CagA localized to the apical surface of cells and showed preferential accumulation at the apical junctions in a phosphorylation-independent manner. These data suggest the presence of distinct functional domains within the CagA protein that play essential roles in protein targeting and alteration of host-cell signaling pathways.

Mycobacterium marinum is closely related to Mycobacterium tuberculosis, the cause of tuberculosis in humans. M. marinum has become an important model system for the study of the molecular mechanisms involved in causing tuberculosis in humans. Through molecular genetic analysis of the differences between pathogenic and nonpathogenic mycobacteria, we identified two loci that affect the ability of M. marinum to infect macrophages, designated mel 1 and mel 2 . In silico analyses of the 11 putative genes in these loci suggest that mel 1 encodes secreted proteins that include a putative membrane protein and two putative transglutaminases, whereas mel 2 is involved in secondary metabolism or biosynthesis of fatty acids. Interestingly, mel 2 is unique to M. marinum and the M. tuberculosis complex and not present in any other sequenced mycobacterial species. M. marinum mutants with mutations in mel 1 and mel 2 , constructed by allelic exchange, are defective in the ability to infect both murine and fish macrophage cell lines. These data suggest that the genes in mel 1 and mel 2 are important for the ability of M. marinum to infect host cells.Although mycobacteria were among the first organisms associated with disease in humans (19, 23), they remain possibly the most important cause of death due to a single infectious agent throughout the world. Possible reasons for this are the relative difficulty of manipulating mycobacteria in the laboratory and the relatively low growth rate (ϳ20-h generation time) of the most important mycobacterial species, Mycobacterium tuberculosis. Investigators have sought appropriate models that are both relevant and easy to manipulate to speed progress in tuberculosis research. Recently, there has been a great deal of interest in Mycobacterium marinum as a model for study of M. tuberculosis pathogenesis because of its ease of genetic manipulation (2, 17, 33, 37), close genetic relationship to M. tuberculosis (36,42,46), relatively high growth rate (ϳ4-h generation time) (10), and the presence of a number of useful laboratory models for in vitro (16) and in vivo (14,34,38,45) virulence studies.Pathogenic mycobacterial species, such as M. tuberculosis, differ from nonpathogenic species, such as Mycobacterium smegmatis, in that they invade mammalian cells more efficiently (6, 13, 39), block lysosomal fusion (4, 20), and replicate well in eukaryotic cells (29,40,47,48). Investigators have taken advantage of these differences to identify the genes involved in host cell interactions by cloning genomic DNA from pathogenic species into nonpathogenic mycobacterial species (6,29,47,48). Although these studies have resulted in identification of genes that are potentially important, further analyses have been slowed by the fact that they were isolated from slowgrowing pathogenic mycobacterial species. As a result, no strains have been constructed with mutations in these genes, and it remains unclear whether mutants would be defective for host cell interactions. Our group recently found that the relatively...