Surface-Associated Hsp60 Chaperonin of Legionella pneumophila Mediates Invasion in a HeLa Cell Model

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.     

Specificity of Legionella pneumophila and Coxiella burnetii vacuoles and versatility of Legionella pneumophila revealed by coinfection

Legionella pneumophila and Coxiella burnetii are phylogenetically related intracellular bacteria that cause aerosol-transmitted lung infections. In host cells both pathogens proliferate in vacuoles whose biogenesis displays some common features. To test the functional similarity of their respective intracellular niches, African green monkey kidney epithelial (Vero) cells, A/J mouse bone marrow-derived macrophages, human macrophages, and human dendritic cells (DC) containing mature C. burnetii replication vacuoles were superinfected with L. pneumophila, and then the acidity, lysosome-associated membrane protein (LAMP) content, and cohabitation of mature replication vacuoles was assessed. In all cell types, wild-type L. pneumophila occupied distinct vacuoles in close association with acidic, LAMP-positive C. burnetii replication vacuoles. In murine macrophages, but not primate macrophages, DC, or epithelial cells, L. pneumophila replication vacuoles were acidic and LAMP positive. Unlike wild-type L. pneumophila, type IV secretion-deficient dotA mutants trafficked to lysosome-like C. burnetii vacuoles in Vero cells where they survived but failed to replicate. In primate macrophages, DC, or epithelial cells, growth of L. pneumophila was as robust in superinfected cell cultures as in those singly infected. Thus, despite their noted similarities, L. pneumophila and C. burnetii are exquisitely adapted for replication in unique replication vacuoles, and factors that maintain the C. burnetii replication vacuole do not alter biogenesis of an adjacent L. pneumophila replication vacuole. Moreover, L. pneumophila can replicate efficiently in either lysosomal vacuoles of A/J mouse cells or in nonlysosomal vacuoles of primate cells.     

Comparative Analysis of Legionella pneumophila and Legionella micdadei Virulence Traits

While the majority of Legionnaire's disease has been attributed to Legionella pneumophila, Legionella micdadei can cause a similar infection in immunocompromised people. Consistent with its epidemiological profile, the growth of L. micdadei in cultured macrophages is less robust than that of L. pneumophila. To identify those features of the Legionella spp. which are correlated to efficient growth in macrophages, two approaches were taken. First, a phenotypic analysis compared four clinical isolates of L. micdadei to one well-characterized strain of L. pneumophila. Seven traits previously correlated with the virulence of L. pneumophila were evaluated: infection and replication in cultured macrophages, evasion of phagosome-lysosome fusion, contact-dependent cytotoxicity, sodium sensitivity, osmotic resistance, and conjugal DNA transfer. By nearly every measure, L. micdadei appeared less virulent than L. pneumophila. The surprising exception was L. micdadei 31B, which evaded lysosomes and replicated in macrophages as efficiently as L. pneumophila, despite lacking both contact-dependent cytopathicity and regulated sodium sensitivity. Second, in an attempt to identify virulence factors genetically, an L. pneumophila genomic library was screened for clones which conferred robust intracellular growth on L. micdadei. No such loci were isolated, consistent with the multiple phenotypic differences observed for the two species. Apparently, L. pneumophila and L. micdadei use distinct strategies to colonize alveolar macrophages, causing Legionnaire's disease.     

Molecular and cell biology of Legionella pneumophila

Legionella pneumophila is a facultative intracellular pathogen that can replicate within phagocytic host cells such as protozoa and macrophages. Evasion of phagocytic killing is mediated by the type IV Dot/Icm secretion system, which exports bacterial effectors that modulate biogenesis of the phagosome to evade endocytic fusion and also to intercept vesicles derived from the endoplasmic reticulum. Bacterial replication is associated with activation of caspase-3 in infected macrophages and is culminated in apoptosis and pore formation-mediated cytolysis of the host.     

Outer membrane proteins from Legionella pneumophila serogroups and other Legionella species.

Outer membranes were isolated from eight serogroups of L. pneumophila and five other Legionella species. The protein composition of the membranes was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single, disulfide stabilized protein with a molecular size of 29,000 to 30,000 daltons was found to be the major outer membrane protein (MOMP) of all the serogroups. The equivalent of the L. pneumophila MOMP was not observed in any of the other Legionella species examined. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels revealed distinctive patterns for each serogroup and other Legionella species that were not observed by staining with Coomassie blue and may result from the presence of lipopolysaccharide in the membrane preparations. The MOMP from serogroup 1 was isolated by exposing crude peptidoglycan to detergent in the presence of heat and reducing agent and was found to be tightly associated with lipopolysaccharide. Antibodies to this complex were used to probe the outer membranes of the remaining, L. pneumophila serogroups and other Legionella species by Western blotting. Serogroup 1 anti-MOMP antibodies were found to react with the MOMP from the remaining seven serogroups examined, whereas antibodies directed against the lipopolysaccharide of serogroup 1 only reacted with lipopolysaccharide from two of the remaining seven serogroups.     

Clinical Application of a Multiplex Real-Time PCR Assay for Simultaneous Detection of Legionella Species,Legionella pneumophila,and Legionella pneumophila Serogroup 1

We developed a single-tube multiplex real-time PCR assay capable of simultaneously detecting and discriminating Legionella spp., Legionella pneumophila, and Legionella pneumophila serogroup 1 in primary specimens. Evaluation of 21 clinical specimens and 115 clinical isolates demonstrated this assay to be a rapid, high-throughput diagnostic test with 100% specificity that may aid during legionellosis outbreaks and epidemiologic investigations.     

Compost facilities as a reservoir of Legionella pneumophila and other Legionella species

Data on the presence of Legionellae outside the aquatic environment are scarce. Alternative ecosystems that could act as a reservoir for Legionella spp. have been investigated to identify unconventional contaminated substrates that are able to produce bioaerosols. We considered eight green waste collection sites including three composting facilities. Legionella pneumophila sg 1–15, Legionella bozemanii, Legionella cincinnatiensis, Legionella jamestowniensis, Legionella micdadei and L. oakridgensis were isolated from samples taken at six of the eight sites. The degree of contamination ranged from 10³ to 10⁸ CFU/g. Compost facilities appear to comprise an important reservoir for Legionellae.     

Genetic and phenotypic differences between Legionella pneumophila strains

Legionnaires' disease is a potentially lethal pneumonia that is primarily due to infection by the species Legionella pneumophila, although more than 40 other species are known. Certain L. pneumophila subgroups, particularly serogroup 1, are associated with the majority of the epidemics. The genetic bases for these differences in virulence have not been determined. Three strains, AA100, JR32, and Lp01, have been used in many molecular pathogenesis studies of L. pneumophila. We found genetic differences between these strains by PCR and Southern analyses that may be related to their ability to cause disease. We also examined the distribution of these genetic loci in clinical and environmental isolates of Legionella and found a correlation between the presence of two of these loci, rtxA and lvh, and the ability to cause disease in humans. Examination of the interactions of these strains with host cells suggested that they differ in important phenotypic characteristics including adherence, entry, and intracellular replication. Furthermore, in the mouse model of infection they display differing levels of replication in lungs. These studies emphasize the importance of further investigation into the genetic makeup of these strains, which is likely to lead to the identification of additional factors involved in Legionella pathogenesis.     

Interactions between Naegleria fowleri and Legionella pneumophila.

Using electron microscopy we documented some of the intracellular events that occur in Naegleria fowleri suspended in Page amoeba saline after ingestion of Legionella pneumophila. Photomicrographs showed intracellular vacuoles containing bacteria in the process of binary fission that was accompanied by alignment of mitochondria and ribosome-like structures along the vacuole membrane. Although these intracellular events are remarkably similar to that seen in Legionella replication within human monocytes, we could not demonstrate an increase in the number of bacteria by CFU or dark-field microscopy. However, when the Naegleria cells were allowed to ingest Legionella cells while suspended in amoeba culture medium, the number of bacteria increased, and this was contingent upon the presence of viable amoebae.     

Serogrouping and subtyping of Legionella pneumophila with monoclonal antibodies

Monoclonal antibodies directed against Legionella pneumophila serogroups 1 to 6 were produced by fusing splenocytes of BALB/c mice with the Sp 2/0-Ag14 or the NSO mouse myeloma cell lines. Specificity of these antibodies was determined by indirect fluorescent-antibody staining: 8 reacted with L. pneumophila serogroup 1 and, respectively, 13, 6, 6, 5, and 10 reacted with serogroups 2, 3, 4, 5, and 6; all except 5 were serogroup specific, and none presented cross-reactions with six other species of Legionellaceae. Serogroup determination of 35 isolates of L. pneumophila with seven selected monoclonal antibodies resulted in correct serogrouping in all instances; a pool of the same seven monoclonal antibodies stained intensely all strains of L. pneumophila without any staining of the other species of Legionellaceae. When 24 serogroup 1 isolates of L. pneumophila were stained with eight serogroup 1-specific monoclonal antibodies, the staining patterns could be clustered in five distinct groups. These hybridomas thus represent an unlimited source of standard reagent that could be used in the detection and serogrouping of L. pneumophila; differences in staining patterns could be used as epidemiological markers for these bacteria.     

Host-dependent trigger of caspases and apoptosis by Legionella pneumophila

The Dot/Icm system of Legionella pneumophila triggers activation of caspase-3 during early stages of infection of human macrophages, but apoptosis is delayed until late stages of infection. During early stages of infection of mouse macrophages, the organism triggers rapid caspase-1-mediated cytotoxicity, which is mediated by bacterial flagellin. However, it is not known whether caspase-1 is triggered by L. pneumophila in human macrophages or whether caspase-3 is activated in permissive or nonpermissive mouse macrophages. Using single-cell analyses, we show that the wild-type strain of L. pneumophila does not trigger caspase-1 activation throughout the intracellular infection of human monocyte-derived macrophages (hMDMs), even when the flagellated bacteria escape into the cytoplasm during late stages. Using single-cell analyses, we show that the Dot/Icm system of L. pneumophila triggers caspase-3 but not caspase-1 within permissive A/J mouse bone marrow-derived primary macrophages by 2 to 8 h, but apoptosis is delayed until late stages of infection. While L. pneumophila triggers a Dot/Icm-dependent activation of caspase-1 in nonpermissive BALB/c mouse-derived macrophages, caspase-3 is not activated at any stage of infection. We show that robust intrapulmonary replication of the wild-type strain of L. pneumophila in susceptible A/J mice is associated with late-stage Dot/Icm-dependent pulmonary apoptosis and alveolar inflammation. In the lungs of nonpermissive BALB/c mice, L. pneumophila does not replicate and does not trigger pulmonary apoptosis or alveolar inflammation. Thus, similar to hMDMs, L. pneumophila does not trigger caspase-1 but triggers caspase-3 activation during early and exponential replication in permissive A/J mouse-derived macrophages, and apoptosis is delayed until late stages of infection. The Dot/Icm type IV secretion system is essential for pulmonary apoptosis in the genetically susceptible A/J mice.     

Intracellular pathogens and antigen presentation-new challenges with Legionella pneumophila

In this issue of Immunity, examine the intracellular life of Legionella pneumophila in dendritic cells (DC) and macrophages, as well as the presentation of its antigens to CD4 T cells. Legionella is a particularly interesting bacterium because of the peculiarities inherent in its intracellular sojourn in phagocytes: it resides in an unusual vesicle characterized by ribosomes studded along its walls. In this compartment, Legionella proteins encoded by the dot gene inhibit phagosome-lysosome fusion and endosomal acidification, yielding a vesicular structure conducive to the multiplication of Legionella, poor in lysosomal contents, and in MHC molecules.     

Comparative adjuvant activities of Legionella pneumophila and Mycobacterium tuberculosis

Adjuvant activity of heat-killed Legionella pneumophila was demonstrated and compared with that of inactivated Mycobacterium tuberculosis H37Rv. The two species of bacteria were suspended separately in oil and Arlacel A. Bovine serum albumin (BSA) in saline was then emulsified within the respective adjuvants and injected intradermally into guinea pigs. Antibodies to the BSA antigen in the sera of the animals were quantitated with the kinetic-dependent enzyme-linked immunosorbent assay (k-Elisa). Guinea pigs immunized with BSA in adjuvant with killed L. pneumophila produced high titers of anti-BSA antibody, which, on the average, were nearly as high as in those immunized with BSA in complete Freund's adjuvant with M. tuberculosis H37Rv, and which were much greater than in others immunized with incomplete adjuvant, lacking bacteria. Moreover, with a polypeptide hapten, the L. pneumophila evoked as much or more antibody in rabbits as the mycobacterium adjuvant. The effect of the legionella adjuvant upon the cellular immune response was examined using skin tests. For this purpose guinea pigs were immunized with picryl-guinea pig albumin in these adjuvants. 6 weeks later, they were skin-tested with that antigen. They showed reactions which appeared to have immediate as well as delayed components when examined grossly and histologically. Others, immunized with incomplete adjuvant, did not exhibit delayed reactions. Accordingly, heat-killed L. pneumophila acts as a potent adjuvant. Under the circumstances of these experiments, it was as effective as heat-killed M. tuberculosis.