Legionella pneumophilaEntry GenertxAIs Involved in Virulence

Abstract

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...

Cloning and Characterization of the Gene Encoding the Major Cell-Associated Phospholipase A of Legionella pneumophila , plaB , Exhibiting Hemolytic Activity

“…Other bacterial factors involved in the destruction of eukaryotic cells, such as the RtxA toxin (12) or the icm/dot gene products (1,36,40), have already been characterized. However, especially in the case of the icm/dot genes, which code for a type IV protein secretion system (52,58), it is possible that these determinants do not represent the structural genes actually accounting for the cell-lysing agent.…”

“…Another hemolytic, but not cytotoxic, protein is the L. pneumophila legiolysin Lly, which is also responsible for color production and fluorescence of the bacterium (61). Pore-forming activities of L. pneumophila confer contact-dependent hemolytic and cytotoxic activities toward a variety of cells, especially at high bacterial numbers (12,31,36). The L. pneumophila RTX toxin RtxA has been shown to contribute to cytotoxicity, to pore formation, and to the initial steps of infection, namely, adherence and entry of the bacterium into host cells (12, 13).…”

“…When bacteria-laden aerosols are inhaled by humans, L. pneumophila exploits alveolar macrophages and epithelial cells for its multiplication, leading to a severe pneumonia characterized by destruction of alveolar cells (60). The cytopathology of Legionnaires' disease involves several cytotoxic or hemolytic factors produced by L. pneumophila, for example, the zinc metalloprotease ProA, the legiolysin Lly, and several pore-forming toxins, one of which is an RTX (repeats in structural toxin) protein (12,26,31,35,36,47,61). The zinc metalloprotease ProA is the major extracellular protease of L. pneumophila and its export depends on the L. pneumophila type II protein secretion system (28,37).…”

“…Pore-forming activities of L. pneumophila confer contact-dependent hemolytic and cytotoxic activities toward a variety of cells, especially at high bacterial numbers (12,31,36). The L. pneumophila RTX toxin RtxA has been shown to contribute to cytotoxicity, to pore formation, and to the initial steps of infection, namely, adherence and entry of the bacterium into host cells (12,13). Pore formation is furthermore an indispensable step in bacterial egress after bacterial intracellular multiplication in both amoebae and macrophages (1,40).…”

The amoebae plate test implicates a paralogue of lpxB in the interaction of Legionella pneumophila with Acanthamoeba castellanii

“…Additionally, the icm/ dot genes mediate cytotoxic events such as (i) contactdependent immediate cytotoxicity due to pore formation (Kirby et al, 1998;Zuckman et al, 1999), (ii) induction of apoptosis (Zink et al, 2002), and (iii) egress of the bacteria from the vacuole of the spent host cell (Molmeret et al, 2002). Other cytotoxic factors of L. pneumophila include legiolysin (Wintermeyer et al, 1991), the zinc metalloprotease Msp (Quinn & Tompkins, 1989;Szeto & Shuman, 1990), and RtxA, a member of the RTX (repeats in toxin) family of cytotoxic adhesins (Cirillo et al, 2001).…”