Legionella pneumophila feoAB promotes ferrous iron uptake and intracellular infection

In order to determine the role of ferrous iron transport in Legionella pathogenesis, we identified and mutated the feoB gene in virulent Legionella pneumophila strain 130b. As it is in Escherichia coli, the L. pneumophila feoB gene was contained within a putative feoAB operon. L. pneumophila feoB insertion mutants exhibited decreased ferrous but not ferric iron uptake compared to the wild type. Growth on standard buffered charcoal yeast extract agar or buffered yeast extract broth was unaffected by the loss of L. pneumophila FeoB. However, the L. pneumophila feoB mutant had a reduced ability to grow on buffered charcoal yeast extract agar with a reduced amount of its usual iron supplementation, a phenotype that could be complemented by the addition of feoB in trans. In unsupplemented buffered yeast extract broth, the feoB mutant also had a growth defect, which was further exacerbated by the addition of the ferrous iron chelator, 2,2'-dipyridyl. The feoB mutant was also 2.5 logs more resistant to streptonigrin than wild-type 130b, confirming its decreased ability to acquire iron during extracellular growth. Decreased replication of the feoB mutant was noted within iron-depleted Hartmannella vermiformis amoebae and human U937 cell macrophages. The reduced intracellular infectivity of the feoB mutant was complemented by the introduction of a plasmid containing feoAB. The L. pneumophila feoB gene conferred a modest growth advantage for the wild type over the mutant in a competition assay within the lungs of A/J mice. Taken together, these results indicate that L. pneumophila FeoB is a ferrous iron transporter that is important for extracellular and intracellular growth, especially in iron-limited environments. These data represent the first evidence for the importance of ferrous iron transport for intracellular replication by a human pathogen.     

The Legionella pneumophila iraAB locus is required for iron assimilation, intracellular infection, and virulence

Legionella pneumophila, a facultative intracellular parasite of human alveolar macrophages and protozoa, causes Legionnaires' disease. Using mini-Tn10 mutagenesis, we previously isolated a L. pneumophila mutant that was hypersensitive to iron chelators. This mutant, NU216, and its allelic equivalent, NU216R, were also defective for intracellular infection, particularly in iron-deficient host cells. To determine whether NU216R was attenuated for virulence, we assessed its ability to cause disease in guinea pigs following intratracheal inoculation. NU216R-infected animals yielded 1,000-fold fewer bacteria from their lungs and spleen compared to wild-type-130b-infected animals that had received a 50-fold-lower dose. Moreover, NU216R-infected animals subsequently cleared the bacteria from these sites. While infection with 130b resulted in high fever, weight loss, and ruffled fur, inoculation with NU216R did not elicit any signs of disease. DNA sequence analysis revealed that the transposon insertion in NU216R lies in the first open reading frame of a two-gene operon. This open reading frame (iraA) encodes a 272-amino-acid protein that shows sequence similarity to methyltransferases. The second open reading frame (iraB) encodes a 501-amino-acid protein that is highly similar to di- and tripeptide transporters from both prokaryotes and eukaryotes. Southern hybridization analyses determined that the iraAB locus was largely limited to strains of L. pneumophila, the most pathogenic of the Legionella species. A newly derived mutant containing a targeted disruption of iraB showed reduced ability to grow under iron-depleted extracellular conditions, but it did not have an infectivity defect in the macrophage-like U937 cells. These data suggest that iraA is critical for virulence of L. pneumophila while iraB is involved in a novel method of iron acquisition which may utilize iron-loaded peptides.     

Association of flagellum expression and intracellular growth of Legionella pneumophila.

We examined the role of the flagella of Legionella pneumophila in the infection of amoebae and human monocyte-like cells. Insertional mutants were constructed with mini-Tn10. Ten mutants (F-) which did not react with polyclonal L. pneumophila antiflagellar antisera were identified. Ten randomly selected mutants (F+) that did react with the polyclonal antiflagellar antiserum were also identified. The infectivity of these 20 mutants in Hartmannella vermiformis and human U937 cells was characterized. Seven of the 10 F- mutants were attenuated in their ability to multiply in the amoebae during the first 3 days of coincubation and failed to multiply in U937 cells. Three of the 10 F- mutants multiplied as well as the wild-type parent strain did in amoebae and to a limited degree in U937 cells. None of the 10 F+ mutants were attenuated in either the amoebae or U937 cells. While the flagellar structure is not essential for virulence, the ability of L. pneumophila to infect amoebae and human phagocytic cells appears to be linked to flagellar expression. We believe that the attenuated F- mutants contain insertions in genes critical to both flagellum expression and the infection process.     

Legionella pneumophila major acid phosphatase and its role in intracellular infection

Legionella pneumophila is an intracellular pathogen of protozoa and alveolar macrophages. This bacterium contains a gene (pilD) that is involved in both type IV pilus biogenesis and type II protein secretion. We previously demonstrated that the PilD prepilin peptidase is crucial for intracellular infection by L. pneumophila and that the secreted pilD-dependent proteins include a metalloprotease, an acid phosphatase, an esterase/lipase, a phospholipase A, and a p-nitrophenyl phosphorylcholine hydrolase. Since mutants lacking type IV pili, the protease, or the phosphorylcholine hydrolase are not defective for intracellular infection, we sought to determine the significance of the secreted acid phosphatase activity. Three mutants defective in acid phosphatase activity were isolated from a population of mini-Tn10-mutagenized L. pneumophila. Supernatants as well as cell lysates from these mutants contained minimal acid phosphatase activity while possessing normal levels of other pilD-dependent exoproteins. Genetic studies indicated that the gene affected by the transposon insertions encoded a novel bacterial histidine acid phosphatase, which we designated Map for major acid phosphatase. Subsequent inhibitor studies indicated that Map, like its eukaryotic homologs, is a tartrate-sensitive acid phosphatase. The map mutants grew within macrophage-like U937 cells and Hartmannella amoebae to the same degree as did wild-type legionellae, indicating that this acid phosphatase is not essential for L. pneumophila intracellular infection. However, in the course of characterizing our new mutants, we gained evidence for a second pilD-dependent acid phosphatase activity that, unlike Map, is tartrate resistant.     

A Legionella pneumophila gene encoding a species-specific surface protein potentiates initiation of intracellular infection.

To investigate the pathogenesis of Legionnaires disease at a molecular level, we mutated by directed allelic exchange a gene encoding a Legionella pneumophila-specific 24,000-dalton (Da) surface protein. Southern hybridization and immunoblot analyses demonstrated that the predicted DNA rearrangement occurred in L. pneumophila with a specific loss of 24-kDa antigen expression. Compared with its isogenic parent, the mutant was significantly impaired in its ability to infect transformed U937 cells, a human macrophagelike cell line; i.e., the bacterial inoculum of the mutant strain that was required to initiate infection of the macrophage monolayer was ca. 80-fold greater than that of the isogenic parent strain. The mutant strain regained full infectivity on reintroduction of a cloned 24-kDa protein gene, indicating that the reduced infectivity was due specifically to the mutation in that gene. Compared with the parent strain, the mutant strain was recovered at titers that were ca. 10-fold lower shortly after infection, but it exhibited a similar intracellular growth rate over the next 40 h, indicating that the mutant was defective in its ability to initiate macrophage infection rather than in its ability to replicate intracellularly. When opsonized, the mutant strain was still significantly less infectious than the parent strain, despite equivalent macrophage association, suggesting that the mutant was not merely missing a ligand for macrophage attachment. The mutant also exhibited reduced infectivity in explanted human alveolar macrophages, demonstrating the relevance of the U937 cell model for analyzing this mutant phenotype. These results represent the first identification of a cloned L. pneumophila gene that is necessary for optimal intracellular infection; we designate this gene mip, for macrophage infectivity potentiator.     

Type II protein secretion is a subset of the PilD-dependent processes that facilitate intracellular infection by Legionella pneumophila

Previously, we had demonstrated that a Legionella pneumophila prepilin peptidase (pilD) mutant does not produce type IV pili and shows reduced secretion of enzymatic activities. Moreover, it displays a distinct colony morphology and a dramatic reduction in intracellular growth within amoebae and macrophages, two phenotypes that are not exhibited by a pilin (pilE(L)) mutant. To determine whether these pilD-dependent defects were linked to type II secretion, we have constructed two new mutants of L. pneumophila strain 130b. Mutations were introduced into either lspDE, which encodes the type II outer membrane secretin and ATPase, or lspFGHIJK, which encodes the pseudopilins. Unlike the wild-type and pilE(L) strains, both lspDE and lspG mutants showed reduced secretion of six pilD-dependent enzymatic activities; i.e., protease, acid phosphatase, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A. However, they exhibited a colony morphology different from that of the pilD mutant, suggesting that their surfaces are distinct. The pilD, lspDE, and lspG mutants were similarly and greatly impaired for growth within Hartmannella vermiformis, indicating that the intracellular defect of the peptidase mutant in amoebae is explained by the loss of type II secretion. When assessed for infection of U937 macrophages, both lsp mutants exhibited a 10-fold reduction in intracellular multiplication and a diminished cytopathic effect. Interestingly, the pilD mutant was clearly 100-fold more defective than the type II secretion mutants in U937 cells. These results suggest the existence of a novel pilD-dependent mechanism for promoting L. pneumophila intracellular infection of human cells.     

Identification of Legionella pneumophila rcp, a pagP-like gene that confers resistance to cationic antimicrobial peptides and promotes intracellular infection

In the course of characterizing a locus involved in heme utilization, we identified a Legionella pneumophila gene predicted to encode a protein with homology to the product of the Salmonella enterica serovar Typhimurium pagP gene. In Salmonella, pagP increases resistance to the bactericidal effects of cationic antimicrobial peptides (CAMPs). Mutants with insertions in the L. pneumophila pagP-like gene were generated and showed decreased resistance to different structural classes of CAMPs compared to the wild type; hence, this gene was designated rcp for resistance to cationic antimicrobial peptides. Furthermore, Legionella CAMP resistance was induced by growth in low-magnesium medium. To determine whether rcp had any role in intracellular survival, mutants were tested in the two most relevant host cells for Legionnaires' disease, i.e., amoebae and macrophages. These mutants exhibited a 1,000-fold-decreased recovery during a Hartmannella vermiformis coculture. Complementation of the infectivity defect could be achieved by introduction of a plasmid containing the intact rcp gene. Mutations in rcp consistently reduced both the numbers of bacteria recovered during intracellular infection and their cytopathic capacity for U937 macrophages. The rcp mutant was also more defective for lung colonization of A/J mice. Growth of rcp mutants in buffered yeast extract broth was identical to that of the wild type, indicating that the observed differences in numbers of bacteria recovered from host cells were not due to a generalized growth defect. However, in low-Mg(2+) medium, the rcp mutant was impaired in stationary-phase survival. This is the first demonstration of a pagP-like gene, involved in resistance to CAMPs, being required for intracellular infection and virulence.     

A quantitative model of intracellular growth of Legionella pneumophila in Acanthamoeba castellanii.

A model of intracellular growth for Legionella pneumophila in Acanthamoeba castellanii has been developed and provides a quantitative measure of survival and replication after entry. In this model, Acanthamoeba monolayers were incubated with bacteria in tissue culture plates under nutrient-limiting conditions. Gentamicin was used to kill extracellular bacteria following the period of incubation, and the number of intracellular bacteria was determined following lysis of amebae. Intracellular growth of virulent L. pneumophila and other wild-type Legionella species was observed when the assay was performed at 37 degrees C. At room temperature, none of the Legionella strains tested grew intracellularly, while an avirulent L. pneumophila strain was unable to replicate in this assay at either temperature. The effect of nutrient limitation on A. castellanii during the assay prevented multiplication of the amebae and increased the level of infection by Legionella spp. The level of infection of the amebae was directly proportional to the multiplicity of infection with bacteria; at an inoculum of 1.03 x 10(7) bacteria added to wells containing 1.10 x 10(5) amebae (multiplicity of infection of 100), approximately 4.4% of A. castellanii cells became infected. Cytochalasin D reduced the uptake of bacteria by the amebae primarily by causing amebae to lift off the culture dish, reducing the number of target hosts; methylamine also reduced the level of initial infection, yet neither inhibitor was able to prevent intracellular replication of Legionella spp. Consequently, once the bacteria entered the cell, only lowered temperature could restrict replication. This model of intracellular growth provides a one-step growth curve and should be useful to study the molecular basis of the host-parasite interaction.     

Legionella pneumophila mutants that are defective for iron acquisition and assimilation and intracellular infection.

Legionella pneumophila, a parasite of macrophages and protozoa, requires iron for optimal extracellular and intracellular growth. However, its mechanisms of iron acquisition remain uncharacterized. Using mini-Tn10 mutagenesis, we isolated 17 unique L. pneumophila strains which appeared to be defective for iron acquisition and assimilation. Eleven of these mutants were both sensitive to the iron chelator ethylenediamine di(o-hydroxyphenylacetic acid) and resistant to streptonigrin, an antibiotic whose lethal effect requires high levels of intracellular iron. Six mutants were also defective for the infection of macrophage-like U937 cells. Although none were altered in entry, mutants generally exhibited prolonged lag phases and in some cases replicated at slower rates. Overall, the reduced recoveries of mutants, relative to that of the wild type, ranged from 3- to 1,000-fold. Strain NU216, the mutant displaying the most severe lag phase and the slowest rate of replication, was studied further. Importantly, within U937 cells, NU216 was approximately 100-fold more sensitive than the wild type was to treatment with the Fe3+ chelator deferoxamine, indicating that it is defective for intracellular iron acquisition and assimilation. Furthermore, this strain was unable to mediate any cytopathic effect and was impaired for infectivity of an amoebal host. Taken together, the isolation of these mutants offers genetic proof that iron acquisition and assimilation are critical for intracellular infection by L. pneumophila.     

De novo synthesis of Legionella pneumophila antigens during intracellular growth in phagocytic cells.

Legionella pneumophilia is a gram-negative rod which is able to multiply within phagocytic cells. The process of phagocytosis leads to a rapid environmental change that might require a coordinate regulation of gene expression to ensure intracellular survival. Since there is little information on up- and downregulation of genes during the early phases of phagocytosis, we radiolabeled intracellular L. pneumophila at different times after phagocytosis by macrophages of the Mono Mac 6 cell line and immunoprecipitated antigens with antilegionella sera or monoclonal antibodies. We could identify two antigens which were upregulated, one of which was the Mip protein, three antigens which were downregulated, and three antigens which were not detectable in extracellularly grown L. pneumophila. The Mip protein was stained most intensively 4 to 8 h after intracellular infection, suggesting that it is needed during intracellular multiplication rather than initiation of infection. A 44-kDa antigen which was not detectable during extracellular growth was most prominent from 2 to 4 h postinfection when Mono Mac 6 cells were used as phagocytic cells. The 44-kDa antigen was also expressed during growth with Acanthamoeba castelanii, MRC-5, and U937 cells but with different kinetics. Synthesis of this antigen was not dependent on protein synthesis of the host cell. Since the 44-kDa antigen could be precipitated by an antiserum produced against a recombinant Escherichia coli harboring a plasmid with an L. pneumophila insert which also codes for the mip gene, we believe that the corresponding gene is within the vicinity of the mip gene. We named this protein legionella intracellular growth antigen (LIGA), since it could be found exclusively in intracellularly grown L. pneumophila.     

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.     

The Legionella pneumophila major secretory protein, a protease, is not required for intracellular growth or cell killing.

The Legionella pneumophila major secretory protein (Msp) is a Zn2+ metalloprotease whose function in pathogenesis is unknown. The structural gene for the Msp protease, mspA, was isolated from an L. pneumophila genomic library. In Escherichia coli which contain plasmids with the mspA gene, Msp protein and activity are found in the periplasmic space and the cytoplasm. Transposon mutagenesis with Tn9 of an mspA-containing plasmid in E. coli yielded mutants which no longer expressed protease activity and others with increased protease activity. These results suggested that mspA expression might be regulated. Msp was shown to be produced at a much higher level in L. pneumophila grown in rich compared to semidefined media. A Tn9 insertion which abolishes Msp expression was introduced into the L. pneumophila genome. This mspA::Tn9 L. pneumophila strain showed no detectable production of Msp by immunoblot analysis, and it had less than 0.1% of the protease activity found in the wild-type strain. This mutant was fully capable of growing within and killing human macrophages derived from the HL-60 cell line.     

The intracellular multiplication of Legionella pneumophila in protozoa from hospital plumbing systems

Between October 1987 and March 1989, we tested 144 water samples obtained from the plumbing and cooling tower systems of 5 Paris hospitals for the presence of legionellae and amoebae. Of the samples tested for Legionella, 67 out of 144 (46.5%) were positive, and 82 out of 116 tested for amoebae (70.7%) were positive. The ability of protozoa to support the multiplication of legionella was shown by incubating samples at 35.5 degrees C for 7-15 days. Prior to determining the presence of legionellae and amoebae, 51 of the 144 samples were incubated. After incubation, 22 out of 25 (88%) samples which were positive for the presence of both Legionella and amoebae showed multiplication of Legionella. In 3 out of the 25 (12%) samples containing Legionella and amoebae, Legionella failed to multiply. Six out of the 51 (11.8%) samples which were negative in direct culture for Legionella but positive for amoebae, became positive after incubation. Legionella did not multiply in samples negative for amoebae, nor was there proliferation in samples after filtration through a 1.2-microns membrane followed by incubation for the same period and temperature. Strains of Legionella pneumophila serogroup 1 and serogroup 6 (SG1 and SG6), including 3 patient isolates and 2 environmental isolates, were cocultured with 2 strains of amoebae and Tetrahymena pyriformis. Plate counts, Gimenez staining and electron microscopy demonstrated that intracellular legionellae proliferation occurred.     

Essential role for the Legionella pneumophila rep helicase homologue in intracellular infection of mammalian cells

We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (mil) loci. In this study we characterized the mil mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar to rep helicase genes of other bacteria. Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the L. pneumophila phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the L. pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in the rep helicase homologue mutant, rendering it defective in intracellular replication.     

Macrophage-induced genes of Legionella pneumophila: protection from reactive intermediates and solute imbalance during intracellular growth

A promoter-probe strategy was devised to identify genes specifically expressed by Legionella pneumophila during growth within the macrophage. Random fragments from the L. pneumophila chromosome were inserted upstream of a promoterless phage T4 td gene, and fragments that led to complementation of thymine auxotrophy during intracellular growth of the bacterium were identified. Two different selection strategies were employed to eliminate promoters that were also active during extracellular growth of the bacterium. Some of these genes were identified independently by using both of the selection strategies. The factors identified include orthologs of efflux-mediated resistance determinants and transporters, a transporter involved in protection from osmotic stress, a stress response GTP-binding protein, a response regulator, a sensor kinase, and two systems that increase the reducing potential of the bacterium, one of which encodes the L. pneumophila ortholog of ahpC. Five of the clones analyzed here were fusions to promoters that were closely linked to genes encoding three-component chemiosmotic efflux pumps that export heavy metals or toxic organic compounds. Analysis of ahpC gene expression indicates that levels increased at least sevenfold during intracellular growth of the bacterium. Inactivation of several of the genes at their chromosomal loci had no effect on the intracellular growth rate of L. pneumophila in cultured macrophages. This suggests that a number of genes with increased expression during intracellular growth may be part of redundant systems that allow survival and growth under the conditions encountered within host cells.     

Cloning and characterization of the gene encoding the major cell-associated phospholipase A of Legionella pneumophila, plaB, exhibiting hemolytic activity

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular pathogen of amoebae, macrophages, and epithelial cells. The pathology of Legionella infections involves alveolar cell destruction, and several proteins of L. pneumophila are known to contribute to this ability. By screening a genomic library of L. pneumophila, we found an additional L. pneumophila gene, plaB, which coded for a hemolytic activity and contained a lipase consensus motif in its deduced protein sequence. Moreover, Escherichia coli harboring the L. pneumophila plaB gene showed increased activity in releasing fatty acids predominantly from diacylphospho- and lysophospholipids, demonstrating that it encodes a phospholipase A. It has been reported that culture supernatants and cell lysates of L. pneumophila possess phospholipase A activity; however, only the major secreted lysophospholipase A PlaA has been investigated on the molecular level. We therefore generated isogenic L. pneumophila plaB mutants and tested those for hemolysis, lipolytic activities, and intracellular survival in amoebae and macrophages. Compared to wild-type L. pneumophila, the plaB mutant showed reduced hemolysis of human red blood cells and almost completely lost its cell-associated lipolytic activity. We conclude that L. pneumophila plaB is the gene encoding the major cell-associated phospholipase A, possibly contributing to bacterial cytotoxicity due to its hemolytic activity. On the other hand, in view of the fact that the plaB mutant multiplied like the wild type both in U937 macrophages and in Acanthamoeba castellanii amoebae, plaB is not essential for intracellular survival of the pathogen.     

Influence of intra-amoebic and other growth conditions on the surface properties of Legionella pneumophila.

The surface properties of Legionella pneumophila were examined by analyzing outer membrane (OM) proteins, lipopolysaccharides (LPS), and cellular fatty acids after growth within Acanthamoeba polyphaga and in vitro under various nutrient-depleted conditions. Intra-amoeba-grown legionellae were found to differ in several respects from cells grown in vitro; most notably, they contained a 15-kDa OM protein and a monounsaturated straight-chain fatty acid (18:1(9)). These compounds were also found in abundant quantities in the host amoeba. Immunoblot analysis of intra-amoeba-grown legionellae with antiacanthamoebic serum revealed that both the bacterial whole cells and Sarkosyl-extracted OMs contained amoebic antigens. The findings suggest that the 15-kDa OM protein is likely to be of amoebic origin and associates with the OM of the bacterium. It is proposed that disruption of amoebic membranes, as a result of intra-amoebic infection, may liberate macromolecules, including a 15-kDa polypeptide, a major constituent of the amoebic membrane, which adhere to the surface of the legionellae. Growth under specific nutrient depletions also had a significant effect on the surface composition of L. pneumophila. Cells grown under phosphate depletion were markedly sensitive to protease K digestion and contained lower levels of LPS, as observed by silver staining of the digests on polyacrylamide gels. Intra-amoeba-grown cells contained more bands than the in vitro-grown organisms, reflecting further differences in the nature of the LPS. The whole-cell fatty acids of the phosphate-depleted cells were appreciably different from those of cells grown under other nutritional conditions. We found no evidence for expression of iron-regulated OM proteins under iron depletion.     

Induction and protective role of antibodies in Legionella pneumophila infection

Legionella pneumophila (Lpn) is a ubiquitous Gram-negative bacterium found in aquatic environments and is the causative agent of Legionnaires' disease, a severe form of pneumonia. We have used Lpn-permissive A/J mice as a model to analyze the B cell response upon intravenous (i.v.) and intranasal (i.n.) infection with Lpn. A strong antibody (Ab) response was observed upon i.v. infection with wild-type (WT) Lpn and an icmT mutant strain, which is unable to replicate within permissive host cells. In contrast to i.v. infection, only WT but not icmT mutant Lpn was able to induce specific Ab responses upon i.n. infection. After primary i.n. infection with WT Lpn, a strict compartmentalization of Lpn-specific Ab isotypes was observed, as IgG was found exclusively systemically, while IgA was detectable only locally in the lung. Regardless of the infection route, isotype switching to IgG and to IgA was strictly dependent on CD4+ T cells, whereas IgM production was completely Th-independent. Finally, we analyzed the protective capacity of the Lpn-specific Ab response. Actively or passively immunized mice or mice that were infected with opsonized Lpn had 50-100-fold reduced bacterial titers compared to naive animals, clearly demonstrating the capacity of Ab to protect against infection with Lpn.     

The cytochrome c maturation locus of Legionella pneumophila promotes iron assimilation and intracellular infection and contains a strain-specific insertion sequence element

Previously, we obtained a Legionella pneumophila mutant, NU208, that is hypersensitive to iron chelators when grown on standard Legionella media. Here, we demonstrate that NU208 is also impaired for growth in media that simply lack their iron supplement. The mutant was not, however, impaired for the production of legiobactin, the only known L. pneumophila siderophore. Importantly, NU208 was also highly defective for intracellular growth in human U937 cell macrophages and Hartmannella and Acanthamoeba amoebae. The growth defect within macrophages was exacerbated by treatment of the host cells with an iron chelator. Sequence analysis demonstrated that the transposon disruption in NU208 lies within an open reading frame that is highly similar to the cytochrome c maturation gene, ccmC. CcmC is generally recognized for its role in the heme export step of cytochrome biogenesis. Indeed, NU208 lacked cytochrome c. Phenotypic analysis of two additional, independently derived ccmC mutants confirmed that the growth defect in low-iron medium and impaired infectivity were associated with the transposon insertion and not an entirely spontaneous second-site mutation. trans-complementation analysis of NU208 confirmed that L. pneumophila ccmC is required for cytochrome c production, growth under low-iron growth conditions, and at least some forms of intracellular infection. Although ccm genes have recently been implicated in iron assimilation, our data indicate, for the first time, that a ccm gene can be required for bacterial growth in an intracellular niche. Complete sequence analysis of the ccm locus from strain 130b identified the genes ccmA-H. Interestingly, however, we also observed that a 1.8-kb insertion sequence element was positioned between ccmB and ccmC. Southern hybridizations indicated that the open reading frame within this element (ISLp 1) was present in multiple copies in some strains of L. pneumophila but was absent from others. These findings represent the first evidence for a transposable element in Legionella and the first identification of an L. pneumophila strain-specific gene.     

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.