Legionella pneumophila utilizes the same genes to multiply within Acanthamoeba castellanii and human macrophages

In previous reports we described a 22-kb Legionella pneumophila chromosomal locus containing 18 genes. Thirteen of these genes (icmT, -R, -Q, -P, -O, -M, -L, -K, -E, -C, -D, -J, and -B) were found to be completely required for intracellular growth and killing of human macrophages. Three genes (icmS, -G, and -F) were found to be partially required, and two genes (lphA and tphA) were found to be dispensable for intracellular growth and killing of human macrophages. Here, we analyzed the requirement of these genes for intracellular growth in the protozoan host Acanthamoeba castellanii, a well-established important environmental host of L. pneumophila. We found that all the genes that are completely required for intracellular growth in human macrophages are also completely required for intracellular growth in A. castellanii. However, the genes that are partially required for intracellular growth in human macrophages are completely required for intracellular growth in A. castellanii. In addition, the lphA gene, which was shown to be dispensable for intracellular growth in human macrophages, is partially required for intracellular growth in A. castellanii. Our results indicate that L. pneumophila utilizes the same genes to grow intracellularly in both human macrophages and amoebae.     

Legionella pneumophila catalase-peroxidases are required for proper trafficking and growth in primary macrophages

Legionella pneumophila, a parasite of aquatic amoebae and pathogen of pulmonary macrophages, replicates intracellularly, utilizing a type IV secretion system to subvert the trafficking of Legionella-containing phagosomes. Defense against host-derived reactive oxygen species has been proposed as critical for intracellular replication. Virulence traits of null mutants in katA and katB, encoding the two Legionella catalase-peroxidases, were analyzed to evaluate the hypothesis that L. pneumophila must decompose hydrogen peroxide to establish a replication niche in macrophages. Phagosomes containing katA or katB mutant Legionella colocalize with LAMP-1, a late endosomal-lysosomal marker, at twice the frequency of those of wild-type strain JR32 and show a decreased frequency of bacterial replication, in similarity to phenotypes of mutants with mutations in dotA and dotB, encoding components of the Type IV secretion system. Quantitative similarity of the katA/B phenotypes indicates that each contributes to virulence traits largely independently of intracellular compartmentalization (KatA in the periplasm and KatB in the cytosol). These data support a model in which KatA and KatB maintain a critically low level of H(2)O(2) compatible with proper phagosome trafficking mediated by the type IV secretion apparatus. During these studies, we observed that dotA and dotB mutations in wild-type strain Lp02 had no effect on intracellular multiplication in the amoeba Acanthamoeba castellanii, indicating that certain dotA/B functions in Lp02 are dispensable in that experimental model. We also observed that wild-type JR32, unlike Lp02, shows minimal contact-dependent cytotoxicity, suggesting that cytotoxicity of JR32 is not a prerequisite for formation of replication-competent Legionella phagosomes in macrophages.     

Legionella pneumophila growth restriction in permissive macrophages cocultured with nonpermissive lipopolysaccharide-activated macrophages.

Macrophages can be activated by lipopolysaccharides (LPS) from gram-negative bacteria to evince a number of biological activities, including increased resistance to intracellular infection by opportunistic bacteria. In the present study, intraperitoneal injection of LPS into A/J mice activated peritoneal macrophages so that they resisted subsequent in vitro infection with Legionella pneumophila. Coculture of these macrophages with those from nontreated A/J mice converted the entire population of cells from permissive to nonpermissive. This effect did not appear to be mediated by soluble factors released from the LPS-treated macrophages, since the levels of interleukins-1 and -6 and tumor necrosis factor alpha produced by the macrophages were not found to be markedly elevated at the time when the macrophages from the LPS-treated mice were most effective in converting normal macrophages to nonpermissiveness. Furthermore, macrophages from mice injected intraperitoneally with either interferon or tumor necrosis factor alpha did not evince nonpermissiveness and also did not have the ability to convert normal spleen cells to nonpermissiveness. Polymyxin B, a known inactivator of LPS activity, did not inhibit the macrophages from the LPS-treated mice from inducing this resistance. It seemed unlikely that free LPS released from the macrophages mediated this effect. The results of this study thus showed that macrophages activated by LPS in vivo can evince nonpermissiveness for Legionella growth in vitro and also can induce macrophages from normal, permissive mice to become nonpermissive for Legionella growth in vitro.     

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 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.     

Intracellular growth of Legionella pneumophila within Acanthamoeba castellanii Neff

Acanthamoeba castellanii Neff supports the intracellular growth of Legionella pneumophila. When acanthamoebae were exposed to L. pneumophila for 1 h and then washed free of unassociated bacteria and placed in liquid culture, levels of viable amoeba-associated legionellae and legionellae free in the culture medium increased by three to four orders of magnitude in 48 to 72 h. However, most of the legionellae remained amoeba-associated and could be cultured only after disruption of the amoebae. Furthermore, legionella viability declined rapidly in amoeba culture medium alone or when bacteria and amoebae were separated by a microporous membrane. Therefore, direct amoeba-legionella contact is required for this growth. Infected acanthamoebae treated with cold acetone to permeabilize them to fluorescent-labeled anti-L. pneumophila antibody appeared to contain far more legionellae than amoebae fixed with glutaraldehyde so as to prevent antibody penetration. Electron micrographs of infected A. castellanii showed numerous bacteria, including some dividing forms, within vacuoles in the cytoplasm. These results together show that A. castellanii is able to provide an intracellular niche for the growth of L. pneumophila.     

Liquid medium for growth of Legionella pneumophila.

The medium described is a simple yeast extract broth capable of growing large number of Legionella neumophila, the causative organism of Legionnaires disease. Filtration was chosen as a means of sterilization, since medium that was autoclaved did not support growth without the presence of Norite A. The filtered medium gave rapid cell growth and maintained the initial antigen production. The observed generation time was 99 min with a maximum cell population of 2 X 10(2) COLONY-FORMING UNITS PER ML IN APPROXIMATELY 40 H.     

A rapid colorimetric assay for evaluating Legionella pneumophila growth in macrophages in vitro.

A rapid colorimetric technique for in vitro quantitation of Legionella pneumophila intracellular proliferation in macrophages is described. The assay is based on the electron transport activity of metabolically active L. pneumophila. The yellow tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is cleaved by the mitochondrial activity of viable L. pneumophila, forming a dark formazan derivative with an absorption spectrum different from that of the native compound. The MTT method for measuring intracellular growth of L. pneumophila closely correlated with the CFU assay. The ability of macrophages from the A/J mouse strain to support intracellular growth of L. pneumophila and the ability of desferrioxamine to restrict L. pneumophila intracellular proliferation were confirmed by both methods. The MTT assay offers the advantages of rapidity, simplicity, and cost efficiency over the CFU assay, since it can be performed in the same flat-bottom microtiter plate with measurement in an enzyme-linked immunosorbent assay reader, allowing efficient processing of large numbers of samples.     

The PmrA/PmrB two-component system of Legionella pneumophila is a global regulator required for intracellular replication within macrophages and protozoa

To examine the role of the PmrA/PmrB two-component system (TCS) of Legionella pneumophila in global gene regulation and in intracellular infection, we constructed pmrA and pmrB isogenic mutants by allelic exchange. Genome-wide microarray gene expression analyses of the pmrA and pmrB mutants at both the exponential and the postexponential phases have shown that the PmrA/PmrB TCS has a global effect on the expression of 279 genes classified into nine groups of genes encoding eukaryotic-like proteins, Dot/Icm apparatus and secreted effectors, type II-secreted proteins, regulators of the postexponential phase, stress response genes, flagellar biosynthesis genes, metabolic genes, and genes of unknown function. Forty-one genes were differentially regulated in the pmrA or pmrB mutant, suggesting a possible cross talk with other TCSs. The pmrB mutant is more sensitive to low pH than the pmrA mutant and the wild-type strain, suggesting that acidity may trigger this TCS. The pmrB mutant exhibits a significant defect in intracellular proliferation within human macrophages, Acanthamoeba polyphaga, and the ciliate Tetrahymena pyriformis. In contrast, the pmrA mutant is defective only in the ciliate. Despite the intracellular growth defect within human macrophages, phagosomes harboring the pmrB mutant exclude late endosomal and lysosomal markers and are remodeled by the rough endoplasmic reticulum. Similar to the dot/icm mutants, the intracellular growth defect of the pmrB mutant is totally rescued in cis within communal phagosomes harboring the wild-type strain. We conclude that the PmrA/PmrB TCS has a global effect on gene expression and is required for the intracellular proliferation of L. pneumophila within human macrophages and protozoa. Differences in gene regulation and intracellular growth phenotypes between the pmrA and pmrB mutant suggests a cross talk with other TCSs.     

Chemically defined medium for Legionella pneumophila growth.

A chemically defined medium containing 18 amino acids, inorganic salts, rhamnose, choline, and ferric pyrophosphate has been developed. The final concentrations of salts and amino acids were modeled after yeast extract. This medium supported the growth of four serogroups of Legionella pneumophila. Growth in shake cultures at 37 degrees C produced a lag time of approximately 5 h and a generation time of 4 h with a maximum growth yield of 10 9 colony-forming units per ml. A soluble brown pigment was observed in the stationary phase of growth. The optimal pH was 6.3. Rhamnose and choline were stimulatory; arginine, serine, threonine, cysteine, valine, and methionine were essential. Supplemental iron was not required to attain maximum growth, but iron deprivation caused an extended lag phase.     

Amino acid requirements for Legionella pneumophila growth.

The amino acids L-arginine, L-isoleucine, L-leucine, L-methionine, L-serine, L-threonine, and L-valine were essential for the growth of Legionella pneumophila in a chemically defined medium. A partial requirement for L-cysteine (or L-cystine) was also observed. A minimal medium containing only the eight required amino acids supported the growth of this bacterium only if the medium was supplemented with L-glutamic acid. This latter compound was the only amino acid capable of stimulating growth in the eight-amino acid medium.     

Transposon mutagenesis in Legionella pneumophila. II.--Mutants exhibiting impaired intracellular growth within cultured macrophages and reduced virulence in vivo.

Transposon Tn5 mutants of L. pneumophila were isolated and screened for loss of virulence-associated characteristics. Three mutants were found with normal ability to produce putative pathogenicity determinants and to be endocytosed by guinea pig alveolar macrophages in vitro but with a greatly reduced ability to multiply within them. These mutants showed considerable loss of virulence in an authentic animal model of the pneumonia.     

Comparison of liquid growth media for Legionella pneumophila.

Ten liquid media were compared under standard conditions for their ability to support the growth of Legionella pneumophila. Modified gonococcal-ferric cysteine broth (without sodium chloride) supplemented with 1% yeast extract yielded the best overall growth of the one strain of L. pneumophila examined. Growth rates were independent of pH changes which occurred during incubation. The growth rates of 10 different strains of L.pneumophila were compared in this medium. There appeared to be little difference in the growth rates of strains passaged frequently or infrequently, or between environmental and clinical isolates. Moderate aeration resulted in a faster growth rate and in approximately a 1 log10 higher final cell concentration as compared to a static broth culture. These experiments demonstrate that there are moderate to marked differences among the various media described in the literature and that no liquid medium yet developed supports rapid growth of L. pneumophila incubated without shaking.     

2-Deoxy-D-glucose inhibits intracellular multiplication and promotes intracellular killing of Legionella pneumophila in A/J mouse macrophages.

Legionella pneumophila can grow intracellularly in A/J mouse macrophages. 2-Deoxy-D-glucose (2dG) (0.1, 1, and 10 mM) inhibited intracellular multiplication and promoted intracellular killing of L. pneumophila dose dependently when it was added to the culture medium of macrophage monolayers, whereas it did not inhibit the bacterial growth in buffered yeast extract broth, which was used for an L. pneumophila culture. The effect of 2dG was reversible because the surviving bacteria resumed intracellular multiplication after the washing away of 2dG from the culture. The effect of 2dG was also competitively inhibited by high concentrations of glucose. The inhibitory effect of 2dG was not attributed to the inhibition of bacterial phagocytosis by macrophages. Furthermore, sodium fluoride (0.1 and 1 mM), cycloheximide (0.1 and 1 microgram/ml), and tunicamycin (1, 2, and 5 micrograms/ml) did not promote the killing of L. pneumophila in macrophages, implying that the inhibitory effect of 2dG cannot be attributed to the inhibition of glycolysis, protein synthesis, and protein glycosylation in macrophages. We suggest that 2dG promotes intracellular killing of L. pneumophila by activating some novel killing mechanism of macrophages.     

Identification of Legionella pneumophila by latex agglutination.

A latex agglutination test for the identification of Legionella pneumophila serogroups 1 through 6 is described. The reagent is specific for L. pneumophila and enables the ready identification of L. pneumophila colonies on agar plates. Preliminary evidence suggests that latex agglutination enables the detection of soluble L. pneumophila antigens in respiratory secretions of patients suspected of having legionellosis.     

Phenotypic modulation by Legionella pneumophila upon infection of macrophages.

Since many pathogenic bacteria manifest a coordinate regulation of gene expression in response to different environmental stimuli, we examined the phenotypic response of Legionella pneumophila to infection of macrophage-like U937 cells. Intracellular L. pneumophila was radiolabeled, and cell extracts were subjected to two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. At least 35 Legionella proteins were selectively induced during infection of macrophages, and one of these proteins was not detected in organisms grown in vitro. Expression of at least 32 proteins was selectively repressed during infection of macrophages, and 9 of these proteins were undetectable in intracellularly grown organisms. Thirteen of the macrophage-induced proteins were also induced by one or more of several stress conditions in vitro, and two of these proteins were the heat shock GroEL- and GroES-like proteins. Nineteen of the macrophage-repressed proteins were also repressed by one or more of the stress conditions in vitro. Our data showed that intracellular L. pneumophila manifested a phenotypic modulation and a global stress response to the intracellular environment of the macrophage. The data suggested that multiple regulons are involved in this modulation, which may contribute to the survival of L. pneumophila within alveolar macrophages.     

Identification of a cytotoxin produced by Legionella pneumophila.

Culture filtrates of Legionella pneumophila were cytotoxic for Chinese hamster ovary cells. The cytotoxin was found to be methanol soluble, heat stable, and stable from pH 5 through 8.     

Macrophage permissiveness for Legionella pneumophila growth modulated by iron.

We have investigated the modulation of iron in two populations of macrophages which differ in susceptibility to Legionella pneumophila intracellular proliferation. Previously, we reported that thioglycolate-elicited peritoneal macrophages obtained from the inbred A/J mouse strain readily support the intracellular growth of L. pneumophila, while resident macrophages from the same strain do not. In this study, we show that A/J elicited macrophages exhibit markedly higher expression of transferrin receptor and intracellular iron content than A/J resident macrophages. Furthermore, apotransferrin and desferrioxamine inhibited the intracellular proliferation of L. pneumophila in elicited macrophages, and this suppression was reversed by the additions of Fe-transferrin or ferric nitrilotriacetate. Fe-transferrin and ferric nitrilotriacetate did not further increase the intracellular proliferation of L. pneumophila in thioglycolate-elicited macrophages. However, ferric citrate and ferric nitrilotriacetate stimulated in a dose-dependent manner the growth of L. pneumophila in resident macrophages. Furthermore, equimolar concentrations of desferrioxamine reversed the stimulatory effect of iron in these resident cells. These data provide evidence supporting the hypothesis that differences in susceptibility to L. pneumophila growth between permissive elicited macrophages and nonpermissive resident macrophages from the A/J mouse strain are due to intracellular availability of iron.