When Legionella pneumophila grows in HeLa cells, it alternates between a replicative form and a morphologically distinct "cyst-like" form termed MIF (mature intracellular form). MIFs are also formed in natural amoebic hosts and to a lesser extent in macrophages, but they do not develop in vitro. Since MIFs accumulate at the end of each growth cycle, we investigated the possibility that they are in vivo equivalents of stationaryphase (SP) bacteria, which are enriched for virulence traits. By electron microscopy, MIFs appeared as short, stubby rods with an electron-dense, laminar outer membrane layer and a cytoplasm largely occupied by inclusions of poly-␤-hydroxybutyrate and laminations of internal membranes originating from the cytoplasmic membrane. These features may be responsible for the bright red appearance of MIFs by light microscopy following staining with the phenolic Giménez stain. In contrast, SP bacteria appeared as dull red rods after Giménez staining and displayed a typical gram-negative cell wall ultrastructure. Outer membranes from MIFs and SP bacteria were equivalent in terms of the content of the peptidoglycan-bound and disulfide bond cross-linked OmpS porin, although additional proteins, including Hsp60 (which acts as an invasin for HeLa cells), were detected only in preparations from MIFs. Proteomic analysis revealed differences between MIFs and SP forms; in particular, MIFs were enriched for an ϳ20-kDa protein, a potential marker of development. Compared with SP bacteria, MIFs were 10-fold more infectious by plaque assay, displayed increased resistance to rifampin (3-to 5-fold) and gentamicin (10-to 1,000-fold), resisted detergent-mediated lysis, and tolerated high pH. Finally, MIFs had a very low respiration rate, consistent with a decreased metabolic activity. Collectively, these results suggest that intracellular L. pneumophila differentiates into a cyst-like, environmentally resilient, highly infectious, post-SP form that is distinct from in vitro SP bacteria. Therefore, MIFs may represent the transmissible environmental forms associated with Legionnaires' disease.The genus Legionella is one of the most successful of all aquatic bacteria, consisting of over 40 named species, their numerous serogroups (7), and a collection of Legionella-like amoebal pathogens that usually exhibit an obligate intracellular lifestyle requiring a particular protozoan host (3). An obligate requirement for the amino acid cysteine (38), which cannot be substituted for by cystine (the oxidized form most commonly found in aerobic environments), conceivably limits members of the Legionella genus to an intracellular lifestyle (25,46) or to life in association with other microorganisms (46,56,63,66) that may constitute a source of cysteine. However, in a biofilm coculture model, persistence but not multiplication of legionellae could be strictly demonstrated, suggesting that natural growth may indeed require the intracellular environment of a protozoan host (52). Thus, the natural life cycle of Legionella most likely...

The freshwater ciliate Tetrahymena sp. efficiently ingested, but poorly digested, virulent strains of the gram-negative intracellular pathogen Legionella pneumophila. Ciliates expelled live legionellae packaged in free spherical pellets. The ingested legionellae showed no ultrastructural indicators of cell division either within intracellular food vacuoles or in the expelled pellets, while the number of CFU consistently decreased as a function of time postinoculation, suggesting a lack of L. pneumophila replication inside Tetrahymena. Pulsechase feeding experiments with fluorescent L. pneumophila and Escherichia coli indicated that actively feeding ciliates maintain a rapid and steady turnover of food vacuoles, so that the intravacuolar residence of the ingested bacteria was as short as 1 to 2 h. L. pneumophila mutants with a defective Dot/Icm virulence system were efficiently digested by Tetrahymena sp. In contrast to pellets of virulent L. pneumophila, the pellets produced by ciliates feeding on dot mutants contained very few bacterial cells but abundant membrane whorls. The whorls became labeled with a specific antibody against L. pneumophila OmpS, indicating that they were outer membrane remnants of digested legionellae. Ciliates that fed on genetically complemented dot mutants produced numerous pellets containing live legionellae, establishing the importance of the Dot/Icm system to resist digestion. We thus concluded that production of pellets containing live virulent L. pneumophila depends on bacterial survival (mediated by the Dot/Icm system) and occurs in the absence of bacterial replication.

HeLa cells have been previously used to demonstrate that virulent strains of Legionella pneumophila (but not salt-tolerant avirulent strains) efficiently invade nonphagocytic cells. Hsp60, a member of the GroEL family of chaperonins, is displayed on the surface of virulent L. pneumophila (R. A. Garduño et al., J. Bacteriol. 180:505–513, 1988). Because Hsp60 is largely involved in protein-protein interactions, we investigated its role in adherence-invasion in the HeLa cell model. Hsp60-specific antibodies inhibited the adherence and invasiveness of two virulent L. pneumophila strains in a dose-dependent manner but had no effect on the association of their salt-tolerant avirulent derivatives with HeLa cells. A monospecific anti-OmpS (major outer membrane protein) serum inhibited the association of both virulent and avirulent strains of L. pneumophila to HeLa cells, suggesting that while both Hsp60 and OmpS may mediate bacterial association to HeLa cells, only virulent strains selectively displayed Hsp60 on their surfaces. Furthermore, the surface-associated Hsp60 of virulent bacterial cells was susceptible to the action of trypsin, which rendered the bacteria noninvasive. Additionally, pretreatment of HeLa cells with purified Hsp60 or precoating of the plastic surface where HeLa cells attached with Hsp60 reduced the adherence and invasiveness of the two virulent strains. Finally, recombinant Hsp60 covalently bound to latex beads promoted the early association of beads with HeLa cells by a factor of 20 over bovine serum albumin (BSA)-coated beads and competed with virulent strains for association with HeLa cells. Hsp60-coated beads were internalized in large numbers by HeLa cells and remained in tight endosomes that did not fuse with other vesicles, whereas internalized BSA-coated beads, for which endocytic trafficking is well established, resided in more loose or elongated endosomes. Mature intracellular forms of L. pneumophila, which were up to 100-fold more efficient than agar-grown bacteria at associating with HeLa cells, were enriched for Hsp60 on the bacterial surface, as determined by immunolocalization techniques. Collectively, these results establish a role for surface-exposed Hsp60 in invasion of HeLa cells by L. pneumophila.

The intracellular bacterial pathogen Legionella pneumophila follows a developmental cycle in which replicative forms (RFs) differentiate into infectious stationary-phase forms (SPFs) in vitro and in vivo into highly infectious mature intracellular forms (MIFs). The potential relationships between SPFs and MIFs remain uncharacterized. Previously we determined that L. pneumophila survives, but does not replicate, while it transiently resides (for 1 to 2 h) in food vacuoles of the freshwater ciliate Tetrahymena tropicalis before being expelled as legionellae-laden pellets. We report here that SPFs have the ability to rapidly (<1 h) and directly (in the absence of bacterial replication) differentiate into MIFs while in transit through T. tropicalis, indicating that SPFs and MIFs constitute a differentiation continuum. Mutant RFs lacking the sigma factor gene rpoS, or the response regulator gene letA, were unable to produce normal SPFs in vitro and did not fully differentiate into MIFs in vivo, further supporting the existence of a common mechanism of differentiation shared by SPFs and MIFs. Mutants with a defective Dot/Icm system morphologically differentiated into MIFs while in transit through T. tropicalis. Therefore, T. tropicalis has allowed us to unequivocally conclude that SPFs can directly differentiate into MIFs and that the Dot/Icm system is not required for differentiation, two events that could not be experimentally addressed before. The Tetrahymena model can now be exploited to study the signals that trigger MIF development in vivo and is the only replication-independent model reported to date that allows the differentiation of Dot/Icm mutants into MIFs.

Nucleotide sequence analysis of an ϳ80-kb genomic region revealed an ϳ65-kb locus that bears hallmarks of a pathogenicity island. This locus includes homologues of a type IV secretion system, mobile genetic elements, and known virulence factors. Comparative studies with other Legionella pneumophila strains and serogroups indicated that this ϳ65-kb locus is unique to L. pneumophila serogroup 1 Philadelphia-1 strains.