Signal transduction in the protozoan host Hartmannella vermiformis upon attachment to Legionella pneumophila.

Intracellular replication of the Legionnaires' disease bacterium, Legionella pneumophila, within protozoa plays a major role in bacterial ecology and pathogenesis. Invasion of the protozoan host Hartmannella vermiformis by L. pneumophila is mediated by attachment to the Gal/GalNAc lectin receptor, which is similar to the beta(2) integrin transmembrane receptors of mammalian cells. Bacterial invasion is associated with induction of a protein tyrosine phosphatase (PTPase) activity in H. vermiformis that results in tyrosine dephosphorylation of the lectin receptor and several cytoskeletal proteins. In this report, we show that entry of L. pneumophila into H. vermiformis is not required to induce tyrosine dephosphorylation of one of the cytoskeletal proteins, paxillin. Tyrosine dephosphorylation of paxillin is mediated at the level of bacterial attachment to the lectin receptor, and is blocked by inhibiting bacterial attachment to the lectin receptor. Attachment of L. pneumophila to the lectin receptor is not mediated by the type IV pilus, which is one of the bacterial ligands involved in attachment to protozoa. Interestingly, the lectin receptor in resting H. vermiformis is associated with several phosphorylated proteins that are dissociated upon bacterial attachment and invasion. We show that the L. pneumophila-induced PTPase activity in H. vermiformis and the associated tyrosine dephosphorylation of host proteins can be mimicked by the cytoskeletal disrupting agent, cytochalasin D. Taken together, our data indicate that attachment of L. pneumophila to the lectin receptor of H. vermiformis induces a PTPase activity, tyrosine dephosphorylation of the lectin and cytoskeletal proteins, dissociation of the lectin from its associated phosphorylated proteins, and most probably disassembly of the cytoskeleton. This novel L. pneumophila-protozoa interaction may be a bacterial strategy to invade protozoa and to be trafficked into a replicative 'niche', or to block differentiation of the protozoan host into a cyst in which L. pneumophila cannot replicate.

The modulation of host cell apoptosis by intracellular bacterial pathogens.

Recent years have witnessed significant advances in unraveling the elegant mechanisms by which intracellular bacterial pathogens induce and/or block apoptosis, which can influence disease progression. This intriguing aspect of the host-pathogen interaction adds another fascinating dimension to our understanding of the exploitation of host cell biology by intracellular bacterial pathogens.

The mechanism of killing and exiting the protozoan host Acanthamoeba polyphaga by Legionella pneumophila.

The ability of Legionella pneumophila to cause legionnaires' disease is dependent on its capacity to replicate within cells in the alveolar spaces. The bacteria kill mammalian cells in two phases: induction of apoptosis during the early stages of infection, followed by an independent and rapid necrosis during later stages of the infection, mediated by a pore-forming activity. In the environment, L. pneumophila is a parasite of protozoa. The molecular mechanisms by which L. pneumophila kills the protozoan cells, after their exploitation for intracellular proliferation, are not known. In an effort to decipher these mechanisms, we have examined induction of both apoptosis and necrosis in the protozoan Acanthamoeba polyphaga upon infection by L. pneumophila. Our data show that, although A. polyphaga undergoes apoptosis following treatment with actinomycin D, L. pneumophila does not induce apoptosis in these cells. Instead, intracellular L. pneumophila induces necrotic death in A. polyphaga, which is mediated by the pore-forming activity. Mutants of L. pneumophila defective in expression of the pore-forming activity are indistinguishable from the parental strain in intracellular replication within A. polyphaga. The parental strain bacteria cause necrosis-mediated lysis of all the A. polyphaga cells within 48 h after infection, and all the intracellular bacteria are released into the tissue culture medium. In contrast, all cells infected by the mutants remain intact, and the intracellular bacteria are 'trapped' within A. polyphaga after the termination of intracellular replication. Failure to exit the host cell after termination of intracellular replication results in a gradual decline in the viability of the mutant strain bacteria within A. polyphaga starting 48h after infection. Our data show that the pore-forming activity of L. pneumophila is not required for intracellular bacterial replication within A. polyphaga but is required for killing and exiting the protozoan host upon termination of intracellular replication.

The Legionella pneumophila prp locus; required during infection of macrophages and amoebae.

Transposon mutagenesis was performed using mTn 10phoA to identify Legionella pneumophila genes that are expressed under certain in vitro conditions, and are required for intracellular replication. Of the 1653 PhoA fusions examined, 19 PhoA(+)fusion mutants were isolated and screened for differential expression of fusion proteins after growth at 30 or 37 degrees C, in the presence of low iron, or increased magnesium concentrations. The mutants were examined for their cytopathogenicity and intracellular replication within U937 macrophage-like cells and the protozoan Hartmannella vermiformis. One of the mutants generated, BS10, was defective in its multiplication within U937 macrophage-like cells and H. vermiformis. The defect in BS10 was complemented with a cosmid clone containing the wild type locus. The open reading frame interrupted by the insertion was homologous to prpD of Salmonella typhimurium and mmgE of Bacillus subtilis.

Phenotypic modulation by intracellular bacterial pathogens.

Microorganisms have the capacity to sense their environment and to respond to it by alteration in gene expression and protein synthesis. Two-dimensional electrophoresis (2-DE) provides a powerful tool to examine the global response in bacterial protein synthesis upon exposure to different environmental signals. One of the most complex environments encountered by facultative intracellular pathogenic bacteria is the intracellular environment of the host cell. Numerous studies have documented that intracellular bacterial pathogens that replicate within phagosomes are simultaneously exposed to multiple signals and they respond to them by a global alteration in protein synthesis that involves elevated levels of several stress-induced proteins. This stress response is manifested regardless of the nature or the stage of maturation of the phagosome of different intracellular pathogens. In contrast, intracellular bacterial pathogens that replicate within the cytoplasm undergo phenotypic modulation in response to the cytoplasmic environment, but their responses do not include elevated levels of stress-induced proteins. This review describes the use of 2-DE to examine bacterial phenotypic modulation in response to the intracellular environment and contrasts this response between three intracellular pathogens; Legionella pneumophila, Salmonella typhimurium, and Listeria monocytogenes. The Legionella pneumophila phagosome is completely blocked from maturation through the endosomal lysosomal pathway but the S. typhimurium phagosome is a specialized compartment that has partial characteristics of an acidified late endosome, while L. monocytogenes rapidly escapes from an acidified phagosome into the cytoplasm.

Natural competence for DNA transformation by Legionella pneumophila and its association with expression of type IV pili.

We have recently described the expression of two pili of different lengths on the surface of Legionella pneumophila (B. J. Stone and Y. Abu Kwaik, Infect. Immun. 66:1768-1775, 1998). Production of long pili requires a functional pilEL locus, encoding a type IV pilin protein. Since type IV pili in Neisseria gonorrhoeae are associated with competence for DNA transformation, we examined the competence of L. pneumophila for DNA transformation under conditions that allowed the expression of type IV pili. We show that L. pneumophila is naturally competent for DNA transformation by isogenic chromosomal DNA and by plasmid DNA containing L. pneumophila DNA. Many different L. pneumophila loci are able to transform L. pneumophila after addition of plasmid DNA, including gspA, ppa, asd, and pilEL. The transformation frequency is reduced when competing DNA containing either L. pneumophila DNA or vector sequences is added to the bacteria, suggesting that uptake-specific sequences may not be involved in DNA uptake. Competence for DNA transformation correlates with expression of the type IV pili, and a pilEL mutant defective in expression of type IV pili is not competent for DNA transformation. Complementation of the mutant for competence is restored by the reintroduction of a cosmid that restores production of type IV pili. Minimal competence is restored to the mutant by introduction of pilEL alone. We conclude that competence for DNA transformation in L. pneumophila is associated with expression of the type IV pilus and results in recombination of L. pneumophila DNA into the chromosome. Since expression of type IV pili also facilitates attachment of L. pneumophila to mammalian cells and protozoa, we designated the type IV pili CAP (for competence- and adherence-associated pili).

Identification of putative cytoskeletal protein homologues in the protozoan host Hartmannella vermiformis as substrates for induced tyrosine phosphatase activity upon attachment to the Legionnaires' disease bacterium, Legionella pneumophila.

The Legionnaires' disease bacterium, Legionella pneumophila, is a facultative intracellular pathogen that invades and replicates within two evolutionarily distant hosts, free living protozoa and mammalian cells. Invasion and intracellular replication within protozoa are thought to be major factors in the transmission of Legionnaires' disease. We have recently reported the identification of a galactose/N-acetyl-D-galactosamine (Gal/GalNAc) lectin in the protozoan host Hartmannella vermiformis as a receptor for attachment and invasion by L. pneumophila (Venkataraman, C., B.J. Haack, S. Bondada, and Y.A. Kwaik. 1997. J. Exp. Med. 186:537-547). In this report, we extended our studies to the effects of bacterial attachment and invasion on the cytoskeletal proteins of H. vermiformis. We first identified the presence of many protozoan cytoskeletal proteins that were putative homologues to their mammalian counterparts, including actin, pp125(FAK), paxillin, and vinculin, all of which were basally tyrosine phosphorylated in resting H. vermiformis. In addition to L. pneumophila-induced tyrosine dephosphorylation of the lectin, bacterial attachment and invasion was associated with tyrosine dephosphorylation of paxillin, pp125(FAK), and vinculin, whereas actin was minimally affected. Inhibition of bacterial attachment to H. vermiformis by Gal or GalNAc monomers blocked bacteria-induced tyrosine dephosphorylation of detergent-insoluble proteins. In contrast, inhibition of bacterial invasion but not attachment failed to block bacteria-induced tyrosine dephosphorylation of H. vermiformis proteins. This was further supported by the observation that 10 mutants of L. pneumophila that were defective in invasion of H. vermiformis were capable of inducing tyrosine dephosphorylation of H. vermiformis proteins. Entry of L. pneumophila into H. vermiformis was predominantly mediated by noncoated receptor-mediated endocytosis (93%) but coiling phagocytosis was infrequently observed (7%). We conclude that attachment but not invasion by L. pneumophila into H. vermiformis was sufficient and essential to induce protein tyrosine dephosphorylation in H. vermiformis. These manipulations of host cell processes were associated with, or followed by, entry of the bacteria by a noncoated receptor-mediated endocytosis. A model for attachment and entry of L. pneumophila into H. vermiformis is proposed.

Identification of macrophage-specific infectivity loci (mil) of Legionella pneumophila that are not required for infectivity of protozoa.

We have recently shown that many mutants of Legionella pneumophila exhibit similar defective phenotypes within both U937 human-derived macrophages and the protozoan host Acanthamoeba (L.-Y. Gao, O. S. Harb, and Y. Abu Kwaik, Infect. Immun. 65:4738-4746, 1997). These observations have suggested that many of the mechanisms utilized by L. pneumophila to parasitize mammalian and protozoan cells are similar, but our data have not excluded the possibility that there are unique mechanisms utilized by L. pneumophila to survive and replicate within macrophages but not protozoa. To examine this possibility, we screened a bank of 5,280 miniTn10::kan transposon insertion mutants of L. pneumophila for potential mutants that exhibited defective phenotypes of cytopathogenicity and intracellular replication within macrophage-like U937 cells but not within Acanthamoeba polyphaga. We identified 32 mutants with various degrees of defects in cytopathogenicity, intracellular survival, and replication within human macrophages, and most of the mutants exhibited wild-type phenotypes within protozoa. Six of the mutants exhibited mild defects in protozoa. The defective loci were designated mil (for macrophage-specific infectivity loci). Based on their intracellular growth defects within macrophages, the mil mutants were grouped into five phenotypic groups. Groups I to III included the mutants that were severely defective in macrophages, while members of the other two groups exhibited a modestly defective phenotype within macrophages. The growth kinetics of many mutants belonging to groups I to III were also examined, and these were shown to have a similar defective phenotype in peripheral blood monocytes and a wild-type phenotype within another protozoan host, Hartmannella vermiformis. Transmission electron microscopy of A. polyphaga infected by three of the mil mutants belonging to groups I and II showed that they were similar to the parent strain in their capacity to recruit the rough endoplasmic reticulum (RER) around the phagosome. In contrast, infection of macrophages showed that the three mutants failed to recruit the RER around the phagosome during early stages of the infection. None of the mil mutants was resistant to NaCl, and the dot or icm NaCl(r) mutants are severely defective within mammalian and protozoan cells. Our data indicated that in addition to differences in mechanisms of uptake of L. pneumophila by macrophages and protozoa, there were also genetic loci required for L. pneumophila to parasitize mammalian but not protozoan cells. We hypothesize that L. pneumophila has evolved as a protozoan parasite in the environment but has acquired loci specific for intracellular replication within macrophages. Alternatively, ecological coevolution with protozoa has allowed L. pneumophila to possess multiple redundant mechanisms to parasitize protozoa and that some of these mechanisms do not function within macrophages.

Heterogeneity in the attachment and uptake mechanisms of the Legionnaires' disease bacterium, Legionella pneumophila, by protozoan hosts.

Invasion and intracellular replication of Legionella pneumophila within protozoa in the environment plays a major role in the transmission of Legionnaires' disease. Intracellular replication of L. pneumophila within protozoa occurs in a rough endoplasmic reticulum (RER)-surrounded phagosome (Y. Abu Kwaik, Appl. Environ. Microbiol. 62:2022-2028, 1996). Since the subsequent fate of many intracellular pathogens is determined by the route of entry, we compared the mechanisms of attachment and subsequent uptake of L. pneumophila by the two protozoa Hartmannella vermiformis and Acanthamoeba polyphaga. Our data provide biochemical and genetic evidence that the mechanisms of attachment and subsequent uptake of L. pneumophila by the two protozoan hosts are, in part, different. First, uptake of L. pneumophila by H. vermiformis is completely blocked by the monovalent sugars galactose and N-acetyl-D-galactosamine, but these sugars partially blocked A. polyphaga. Second, attachment of L. pneumophila to H. vermiformis is associated with a time-dependent and reversible tyrosine dephosphorylation of multiple host proteins. In contrast, only a slight dephosphorylation of a 170-kDa protein of A. polyphaga is detected upon infection. Third, synthesis of H. vermiformis proteins but not of A. polyphaga proteins is required for uptake of L. pneumophila. Fourth, we have identified L. pneumophila mutants that are severely defective in attachment to A. polyphaga but which exhibit minor reductions in attachment to H. vermiformis and, thus, provide a genetic basis for the difference in mechanisms of attachment to both protozoa. The data indicate a remarkable adaptation of L. pneumophila to attach and invade different protozoan hosts by different mechanisms, yet invasion is followed by a remarkably similar intracellular replication within a RER-surrounded phagosome and subsequent killing of the host cell.

Fatal attraction of mammalian cells to Legionella pneumophila.

Legionella pneumophila is a protozoan parasite that causes Legionnaires' disease. Its ability to do so is dependent on its capacity to replicate intracellularly within a phagosome that is not trafficked through the endosomal-lysosomal pathway and is surrounded by the rough endoplasmic reticulum. Within this unique niche, the bacterium undergoes alterations in gene expression. In addition, many virulence-related phenotypes that are induced in vitro by starvation are expressed intracellularly as the bacteria exit the logarithmic growth phase. (p)ppGpp appears to signal expression of the virulence-related genes in L. pneumophila upon starvation. This growth phase-dependent phenotypical transition is concomitant with lysis of the host cell, in which both necrosis and apoptosis seem to play roles. Many genetic loci that are required for intracellular replication within mammalian and protozoan cells have been identified, and the majority of them are novel. Two secretion systems have been identified, one of which may be distantly related to type IV secretion systems. The other is a type II secretion system similar to the PilBCD piliation system of Pseudomonas aeruginosa.

Cloning and expression in Escherichia coli of a Haemophilus influenzae type b lipooligosaccharide synthesis gene(s) that encodes a 2-keto-3-deoxyoctulosonic acid epitope.

The composition of lipooligosaccharide (LOS) can modify the virulence of Haemophilus influenzae type b (Hib). A genomic library of Hib strain A2 was constructed in the lambda bacteriophage EMBL3. Twenty-six phage clones expressed a Hib LOS oligosaccharide epitope in Escherichia coli that was detected by the monoclonal antibody (MAb) 6E4. None of the clones bound a polyclonal sera specific for Hib A2 LOS or an anti-H. influenzae lipid A MAb. One clone, designated EMBLOS-1, assembled an oligosaccharide with an apparent molecular weight of 1,400 (the 1.4K oligosaccharide) on a 4.1K lipopolysaccharide (LPS) species in E. coli LE392 and produced a novel 5.5K LPS that bound 6E4. Binding of 6E4 to the 5.5K EMBLOS-1 LPS band was abolished by treatment with sodium metaperiodate but was not affected by digestion with proteinase K, confirming the carbohydrate nature of the epitope. The EMBLOS-1 Haemophilus insert hybridized to similar restriction fragments in type b and nontypeable strains regardless of whether they expressed the 6E4 epitope. The 6E4 epitope did not undergo phase variation in Hib strain A2 at a frequency of greater than 10(-3). The oligosaccharide of the Salmonella minnesota Re mutant and 2-keto-3-deoxyoctulosonic acid (KDO) inhibited binding of 6E4 to Hib A2 LOS. We conclude that a gene(s) encoding an enzyme(s) that assembles a stable Hib LOS epitope containing KDO is conserved in H. influenzae and that the cloned Hib LOS synthesis gene products assemble a Hib LOS epitope on an E. coli K-12 LPS core.

Lipooligosaccharide epitopes shared among gram-negative non-enteric mucosal pathogens.

The non-enteric Gram-negative human pathogens, B. catarrhalis, H. ducreyi, H. influenzae, N. gonorrhoeae and N. meningitidis, do not have repeating O-antigens as part of their principle surface glycolipid, the lipooligosaccharide (LOS). Because they have similar LOS structures, we studied the conservation of LOS oligosaccharide epitopes among these organisms. Twenty-one monoclonal antibodies (mAbs) generated by immunizing mice with H. influenzae, N. gonorrhoeae and N. meningitidis were studied for cross reactivity. Five mAbs generated against non-typable H. influenzae were the only strain-specific antibodies. Ten mAbs reacted to LOS epitope(s) common to a genera or species, and six mAbs bound to epitope(s) on the LOS of strains from different genera. Some cross reactive mAbs bound to LOS bands of similar molecular weights, while others bound to bands of varying molecular weights. mAb 3F11, whose epitope mimics a human blood-group antigen, bound to a 4.8 kDa LOS band in N. gonorrhoeae and H. ducreyi, two pathogens that infect genital epithelium. mAb 3D9, whose epitope consists of 2-keto-3-deoxyoctulosonic acid (KDO), reacted with different LOS bands in N. gonorrhoeae, H. influenzae and some R mutants of S. minnesota. A 14 kb restriction fragment containing lipooligosaccharide synthesis genes responsible for the assembly of the 3D9 epitope in H. influenzae hybridized to all H. influenzae strains tested but did not hybridize to gonococcal and S. minnesota strains that expressed this epitope. These studies demonstrate that conserved LOS epitope(s) exist among different species and genera of non-enteric human pathogens and that different genetic mechanisms may have evolved in these pathogens to assemble some of these conserved epitopes.