Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells

Legionella pneumophila expresses pili of variable lengths, either long (0.8 to 1.5 microm) or short (0.1 to 0.6 microm), that can be observed by transmission electron microscopy. We have identified a gene in L. pneumophila with homology to the type IV pilin genes (pilEL). An insertion mutation was constructed in pilEL and introduced into the L. pneumophila wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The L. pneumophila pilEL mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and Acanthamoeba polyphaga but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that the pilEL locus is present in L. pneumophila serogroups 1 through 13 but is variable in 16 other Legionella species. The presence of a type IV pilin gene and its expression by L. pneumophila may provide an advantage for colonization of lung tissues during Legionnaires' disease and invasion of amoebas in the environment.     

Cloning of an Aeromonas hydrophila type IV pilus biogenesis gene cluster: complementation of pilus assembly functions and characterization of a type IV leader peptidase/N-methyltransferase required for extracellular protein secretion

Aeromonas hydrophila secretes several extracellular proteins that are associated with virulence including an enterotoxin, a protease, and the hole-forming toxin, aerolysin. These degradative enzymes and toxins are exported by a conserved pathway found in many Gram-negative bacteria. In Pseudomonas aeruginosa this export pathway and type IV pilus biogenesis are dependent on the product of the pilD gene. PilD is a bifunctional enzyme that processes components of the extracellular secretory pathway as well as a type IV prepilin. An A. hydrophila genomic library was transferred into a P. aeruginosa pilD mutant that is defective for type IV pilus biogenesis. The A. hydrophila pilD homologue, tapD, was identified by its ability to complement the pilD mutation in P. aeruginosa. Transconjugants containing tapD were sensitive to the type IV pilus-specific phage, PO4. Sequence data revealed that tapD is part of a cluster of genes (tapABCD) that are homologous to P. aeruginosa type IV pilus biogenesis genes (pilABCD). We showed that TapB and TapC are functionally homologous to P. aeruginosa PilB and PilC, the first such functional complementation of pilus assembly demonstrated between bacteria that express type IV pili. In vitro studies revealed that TapD has both endopeptidase and N-methyltransferase activities using P. aeruginosa prepilin as substrate. Furthermore, we show that tapD is required for extracellular secretion of aerolysin and protease, indicating that tapD may play an important role in the virulence of A. hydrophila.     

Characterization of type IV pilus genes in Pseudomonas syringae pv. tomato DC3000

Many strains of Pseudomonas syringae produce retractile pili that act as receptors for lytic bacteriophage phi 6. As these are also characteristics of type IV pili, it was postulated that P. syringae may possess genes for type IV pilus biogenesis. A cosmid clone bank of P. syringae pv. tomato DC3000 genomic DNA was used to complement a mutant of Pseudomonas aeruginosa defective in the PilD (XcpA) prepilin peptidase gene by selection for restoration of extracellular protein secretion, a function also known to require PilD. A cosmid able to complement this mutant was also able to complement mutations in the pilB and pilC genes, suggesting that, if the organization of these genes is similar to that of P. aeruginosa, the cosmid may contain the P. syringae pilA. This was confirmed by sequencing a region from this plasmid that was shown to hybridize at low stringency to the P. aeruginosa pilA gene. The deduced P. syringae PilA polypeptide possesses the characteristic properties of the type IV pilins. Heterologous expression of the P. syringae pilA in P. aeruginosa was also shown, conferring not only phi 6 phage sensitivity to P. aeruginosa pilA mutants but also sensitivity to PO4, a lytic bacteriophage specific for the pilus of P. aeruginosa. This suggests that additional components might be present in the mature pilus of P. aeruginosa that are the true receptors for this phage. Chromosomal mutations in P. syringae pv. tomato DC3000 pilA and pilD genes were shown to abolish its sensitivity to bacteriophage phi 6. To determine the importance of P. syringae pilus in plant leaf interactions, these mutations were tested under laboratory and field conditions. Although little effect was seen on pathogenicity, culturable leaf-associated population sizes of the pilA mutant were significantly different from those of the wild-type parent. In addition, the expression of the DC3000 pilA gene appears to contribute to the UV tolerance of P. syringae and may play a role in survival on the plant leaf surface.     

In vitro activity of macrolides against intracellular Legionella pneumophila

The antibacterial effects of clarithromycin, azithromycin, and erythromycin were determined against five strains of Legionella pneumophila including L. pneumophila ATCC 33823 and four clinical isolates. Extracellular minimum inhibitory concentrations (MICs) and MBCs were determined by a microdilution method. Clarithromycin was the most active drug (MIC < or = 0.015-0.06), followed by azithromycin (MIC 0.03-0.12) and erythromycin (MIC 0.06-0.25). The antibacterial effect of these macrolides was then determined against L. pneumophila grown intracellularly in MRC-5 human fetal lung fibroblast cells. At two and eight times the extracellular MBC, erythromycin, azithromycin, and clarithromycin were equally effective in inhibiting growth of these five strains of intracellular L. pneumophila.     

Cell signaling by the type IV pili of pathogenic Neisseria

Neisseria gonorrhoeae and Neisseria meningitidis are Gram-negative bacterial pathogens that infect human mucosal epithelia. Type IV pilus-mediated adherence of these bacteria is a crucial early event for establishment of infection. In this work, we show that the type IV pili transduce a signal into the eucaryotic host cell. Purified adherent pili, but not pili from a low binding mutant, trigger an increase in the cytosolic free calcium ([Ca2+]i) in target epithelial cells, a signal known to control many cellular responses. The [Ca2+]i increase was blocked by antibodies against CD46, a putative pilus receptor, suggesting a role for this protein in signal transduction. Pilus-mediated attachment was inhibited by depletion of host cell intracellular Ca2+ stores but not by removal of extracellular Ca2+. Further, kinase inhibition studies showed that pilus-mediated adherence is dependent on casein kinase II. In summary, these data reveal a novel function of the type IV pili, namely induction of signal transduction pathways in host cells.     

Medicine, population and war

The colonization mechanisms of enteropathogenic Aeromonas strains are poorly characterized, but recent studies indicate that some filamentous structures are intestinal adhesins. This study describes the purification and characterization of a long, flexible pilus from a gastroenteritis-associated strain of Aeromonas veronii biovar sobria. SDS-PAGE analysis (various conditions) of pili preparations yielded a pilin protein band of approximately 21 kDa. Its N-terminal amino acid sequence was unambiguous and homologous with those of type IV pilins. Immunogold electron microscopy with rabbit antisera produced against this pilin protein (SFP) decorated single pili and rope-like bundles of pili on the bacterial surface. These were seen more frequently on strains grown at 22 degrees C compared with 37 degrees C and in liquid rather than on solid medium. SFP was not detected on any of 104 strains of Aeromonas (different species and sources) from our culture collection, although morphologically similar structures were seen on a number of these strains. This finding and differences among other published amino acid sequences show that Aeromonas type IV pili are antigenically diverse. Bundle-forming type IV 'class B' pili are important in the virulence of other enteropathogenic bacteria. The N-terminal amino acid sequence of the Aeromonas SFP, however, showed closer homology to the type IV 'class A' pilins. Studies are in progress to investigate the role of SFP in Aeromonas virulence.     

Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome

A 22-kb DNA locus of Legionella pneumophila is described that contains 18 genes, 16 of which are required for macrophage killing (icm genes). In this paper two previously described icm loci were linked by the discovery of five genes located between the two loci. Four of the newly described genes are required for macrophage killing (icmMLKE) and one is dispensable. The 16 icm genes appeared to be organized as six individual genes (icmR, icmQ, icmG, icmC, icmD, and icmF), and four operons (icmTS, icmPO, icmMLKE, and icmJB). Four icm genes (icmP, icmO, icmL, and icmE) show significant sequence similarity to plasmid genes involved in conjugation, whereas the other icm genes were found not to bear any sequence similarity to database entries. We found that L. pneumophila can mediate plasmid DNA transfer at a frequency of 10(-3) to 10(-4) per donor. Strains containing null mutations in two icm genes (icmT and icmR) showed a severe reduction in conjugation frequency and macrophage killing. Strains containing an insertion in four other icm genes (icmF, icmE, icmC, and dotA) were shown to have a less severe defect in conjugation. Mutations in the other 11 icm genes had no effect on conjugation frequency. We currently do not know whether conjugation itself plays a role in macrophage killing. It is possible either that small plasmids can take advantage of an existing secretion system to be mobilized or that DNA transfer is required for human macrophage killing by L. pneumophila.     

Influence of methylprednisolone on the intracellular antimicrobial activity of erythromycin and clindamycin against Legionella pneumophila

We have investigated the effect of methylprednisolone on the intracellular activity of erythromycin and clindamycin in vitro. An assay system was developed for the determination of intracellular activity of antibiotics against Legionella pneumophila using guinea pig resident alveolar macrophages. Erythromycin at a concentration of 0.625 mg/L (5 x MIC) and clindamycin at a concentration of 8 mg/L (MIC) inhibited the growth of a single strain of L. pneumophila in macrophages, whilst ceftizoxime at a concentration of 0.625 mg/L (5 x MIC) did not. Methylprednisolone at therapeutic concentrations did not affect the intracellular antibacterial activity of either erythromycin or clindamycin against L. pneumophila. We found no direct effect of methylprednisolone on the intracellular antibacterial activity of either erythromycin or clindamycin.     

Early events in phagosome establishment are required for intracellular survival of Legionella pneumophila

During infection, the Legionnaires' disease bacterium, Legionella pneumophila, survives and multiplies within a specialized phagosome that is near neutral pH and does not fuse with host lysosomes. In order to understand the molecular basis of this organism's ability to control its intracellular fate, we have isolated and characterized a group of transposon-generated mutants which were unable to kill macrophages and were subsequently found to be defective in intracellular multiplication. These mutations define a set of 20 genes (19 icm [for intracellular multiplication] genes and dotA [for defect in organelle trafficking]). In this report, we describe a quantitative assay for phagosome-lysosome fusion (PLF) and its use to measure the levels of PLF in cells that have been infected with either wild-type L. pneumophila or one of several mutants defective in different icm genes or dotA. By using quantitative confocal fluorescence microscopy, PLF could be scored on a per-bacterium basis by determining the extent to which fluorescein-labeled L. pneumophila colocalized with host lysosomes prelabeled with rhodamine-dextran. Remarkably, mutations in the six genes that were studied resulted in maximal levels of PLF as quickly as 30 min following infection. These results indicate that several, and possibly all, of the icm and dotA gene products act at an early step during phagosome establishment to determine whether L. pneumophila-containing phagosomes will fuse with lysosomes. Although not ruled out, subsequent activity of these gene products may not be necessary for successful intracellular replication.     

Analysis of the Legionella pneumophila fliI gene: intracellular growth of a defined mutant defective for flagellum biosynthesis

Using a PCR-based strategy and degenerate oligonucleotides, we isolated a Legionella pneumophila gene that showed high sequence similarity to members of the fliI gene family. An insertion mutation that disrupted the fliI open reading frame was recombined onto the L. pneumophila chromosome and analyzed for its effects on production of flagella and intracellular growth. The mutation resulted in loss of surface-localized flagellin protein but had no effect on the ability of the bacteria to grow within cultured cells. Therefore, in spite of the fact that some aflagellar mutations render L. pneumophila unable to grow within macrophages, the isolation of this defined mutant confirms that production of flagella is not required for intracellular growth.     

Identification of the aspartate-beta-semialdehyde dehydrogenase gene of Legionella pneumophila and characterization of a null mutant

The ability of Legionella pneumophila to cause Legionnaires' disease is dependent on its capacity to survive in the intracellular environment of its host cells. Furthermore, outbreaks of this disease have been associated with contaminated water sources where L. pneumophila survives as a parasite of protozoa. In this study, we determined the effect of nutritional auxotrophy on the ability of L. pneumophila to survive in the intracellular environment of its host cells. We generated a diaminopimelic acid (DAP) auxotroph (AA400) of L. pneumophila by disruption of the aspartate-beta-semialdehyde (asd) gene. The ability of AA400 to survive within macrophages and protozoa was found to be defective. This defect was due solely to the asd disruption since complementation of the mutant with the wild-type asd gene restored its capacity for intracellular survival. Furthermore, the defect was not completely complemented by DAP supplementation to the culture media. Thus, our results suggest that disruption of the asd gene may prove to be useful in the design of attenuated vaccines against Legionnaires' disease.     

Apoptosis in macrophages and alveolar epithelial cells during early stages of infection by Legionella pneumophila and its role in cytopathogenicity

The hallmark of Legionnaires' disease is intracellular replication of Legionella pneumophila within cells in the alveolar spaces. Cytopathogenicity of this bacterium to the host cell has been well demonstrated, but the mechanisms of host cell death due to infection by L. pneumophila are not well understood. In this study, induction of apoptosis in macrophages and alveolar epithelial cells by L. pneumophila during early stages of infection was confirmed by using multiple criteria, including DNA fragmentation by agarose gel electrophoresis, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, surface exposure of phosphatidylserine, and cellular morphology by transmission electron microscopy. Induction of nuclear apoptosis in L. pneumophila-infected macrophages is mediated by activation of the caspase cascade death machinery. We provide genetic and biochemical evidence that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells does not require intracellular bacterial replication or new protein synthesis. In addition, extracellular L. pneumophila is capable of inducing apoptosis. Furthermore, induction of apoptosis by L. pneumophila correlates with cytopathogenicity. We conclude that L. pneumophila-induced apoptosis in macrophages and alveolar epithelial cells plays an important role in cytopathogenicity to the host cell during early stages of infection.     

Phosphatase-negative mutants of Legionella pneumophila and their behavior in mammalian cell infection

Microbial phosphatases are known or suspected to play a role in the pathogenesis of several intracellular pathogens, including Legionella micdadei. Legionella pneumophila also possess phosphatase activities, but their possible roles in cellular infection are unknown. We generated mutants of a serogroup 1 isolate of L. pneumophila that lack the major phosphatase. Isolation of a Pho- mutant after random mutagenesis with transposon MudII4041 allowed us to dissociate the major alkaline phosphatase (pH optimum approximately 8) from a minor acid phosphatase activity. Both activities were concentrated in the bacterial periplasm. The gene encoding the major alkaline phosphatase (pho) was cloned by expression in E. coli and used to generate a site directed mutation in two L. pneumophila strains. Each parent-mutant pair was compared in a U937 cell tissue culture assay for capacity to infect, lyse, and grow within mammalian cells. Although the parental stains differed in their U937 cell cytopathicity, neither was significantly more infective than its Pho- derivative, suggesting that the alkaline phosphatase activity is not essential for cellular infection. Because they are not attenuated, Pho- mutants can be used to generate gene fusions with E. coli alkaline phosphatase to study and secretion and cellular infectivity in L. pneumophila.     

Purification and characterization of Eikenella corrodens type IV pilin

Eikenella corrodens is a gram-negative human pathogen associated with periodontal diseases and soft-tissue infections. Pilin was purified by association-dissociation and fast protein liquid chromatography; it had an apparent molecular mass of about 14.8 kDa and an N-terminal amino acid sequence reflective of type IV pilins. Antibodies to the purified protein reacted with pili on whole cells. This is the first report of purification of type IV pili/pilin from this organism. Other type IV pili are important virulence factors; we are currently investigating the biological role of pili in E. corrodens.     

The role of Legionella pneumophila-infected Hartmannella vermiformis as an infectious particle in a murine model of Legionnaire's disease

Legionella pneumophila is a bacterial parasite of many species of freshwater protozoa and occasionally an intracellular pathogen of humans. While protozoa are known to play a key role in the persistence of L. pneumophila in the environment, there has been limited research addressing the potential role of L. pneumophila-infected protozoa in the pathogenesis of human infection. In this report, the potential role of an L. pneumophila-infected amoeba as an infectious particle in replicative L. pneumophila lung infection was investigated in vivo with the amoeba Hartmannella vermiformis, a natural reservoir of L. pneumophila in the environment. L. pneumophila-infected H. vermiformis organisms were prepared by coculture of the amoebae and virulent L. pneumophila cells in vitro. A/J mice, which are susceptible to replicative L. pneumophila lung infection, were subsequently inoculated intratracheally with L. pneumophila-infected H. vermiformis organisms (10(6) amoebae containing 10(5) bacteria), and intrapulmonary growth of the bacteria was assessed. A/J mice inoculated intratracheally with L. pneumophila-infected H. vermiformis organisms developed replicative L. pneumophila lung infections. Furthermore, L. pneumophila-infected H. vermiformis organisms were more pathogenic than an equivalent number of bacteria or a coinoculum of L. pneumophila cells and uninfected amoebae. These results demonstrate that L. pneumophila-infected amoebae are infectious particles in replicative L. pneumophila infections in vivo and support the hypothesis that inhaled protozoa may serve as cofactors in the pathogenesis of pulmonary disease induced by inhaled respiratory pathogens.     

Resuscitation of viable but nonculturable Legionella pneumophila Philadelphia JR32 by Acanthamoeba castellanii

Legionella pneumophila is an aquatic bacterium and is responsible for Legionnaires' disease in humans. Free-living amoebae are parasitized by legionellae and provide the intracellular environment required for the replication of this bacterium. In low-nutrient environments, however, L. pneumophila is able to enter a non-replicative viable but nonculturable (VBNC) state. In this study, L. pneumophila Philadelphia I JR 32 was suspended in sterilized tap water at 10(4) cells/ml. The decreasing number of bacteria was monitored by CFU measurements, acridine orange direct count (AODC), and hybridization with 16S rRNA-targeted oligonucleotide probes. After 125 days of incubation in water, the cells were no longer culturable on routine plating media; however, they were still detectable by AODC and by in situ hybridization. The addition of Acanthamoeba castellanii to the dormant bacteria resulted in the resuscitation of L. pneumophila JR 32 to a culturable state. A comparison of plate-grown legionellae and reactivated cells showed that the capacity for intracellular survival in human monocytes and intraperitoneally infected guinea pigs, which is considered a parameter for virulence, was not reduced in the reactivated cells. However, reactivation of dormant legionellae was not observed in the animal model.     

Back pain in young athletes. Significant differences from adults in causes and patterns

The ubiquitous occurrence of Legionellae requires an exact typing of isolated strains in order to demonstrate the source of infection. Monoclonal antibodies, analysis of genomic and plasmid DNAs, and the typing of alloenzymes are suitable for this purpose. Typing of Legionella pneumophila serogroup 1 strains by using monoclonal antibodies was found to be a rapid and adequate method. Other serogroups of L. pneumophila and non-pneumophila species are of considerably less antigenic diversity, so that the use of monoclonal antibodies is not particular profitable. In such cases, genotypic methods are needed to discriminate between unrelated strains. There are no changes in the genome structure, defined as restriction patterns, during passages on artificial media and cultured Acanthamoeba. The possibility that different species, serogroups and monoclonal or genomic subtypes can be isolated in a given water supply points to necessity to test a sufficiently large number of colonies grown from the water samples. A clonal distribution of some Legionella strains has been observed.