Functional similarities between the icm/dot pathogenesis systems of Coxiella burnetii and Legionella pneumophila

Coxiella burnetii, the etiological agent of Q fever, is an obligate intracellular pathogen, whereas Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen. During infection of humans both of these pathogens multiply in alveolar macrophages inside a closed phagosome. L. pneumophila intracellular multiplication was shown to be dependent on the icm/dot system, which probably encodes a type IV-related translocation apparatus. Recently, genes homologous to all of the L. pneumophila icm/dot genes (besides icmR) were found in C. burnetii. To explore the similarities and differences between the icm/dot pathogenesis systems of these two pathogens, interspecies complementation analysis was performed. Nine C. burnetii icm homologous genes (icmT, icmS, icmQ, icmP, icmO, icmJ, icmB, icmW, and icmX) were cloned under regulation of the corresponding L. pneumophila icm genes and examined for the ability to complement L. pneumophila mutants with mutations in these genes. The C. burnetii icmS and icmW homologous genes were found to complement the corresponding L. pneumophila icm mutants to wild-type levels of intracellular growth in both HL-60-derived human macrophages and Acanthamoeba castellanii. In addition, the C. burnetii icmT homologous gene was found to completely complement an L. pneumophila insertion mutant for intracellular growth in HL-60-derived human macrophages, but it only partially complemented the same mutant for intracellular growth in A. castellanii. Moreover, as previously shown for L. pneumophila, the proteins encoded by the C. burnetii icmS and icmW homologous genes were found to interact with one another, and interspecies protein interaction was observed as well. Our results strongly indicate that the Icm/Dot pathogenesis systems of C. burnetii and L. pneumophila have common features.     

The Legionella pneumophila GacA homolog (LetA) is involved in the regulation of icm virulence genes and is required for intracellular multiplication in Acanthamoeba castellanii

Legionella pneumophila, the causative agent of legionnaires' disease, is a broad-host-range facultative intracellular pathogen. Thus far, 24 genes (icm/dot genes) required for L. pneumophila intracellular growth, have been discovered. In this study, a deletion substitution was constructed in the L. pneumophila homolog of the gacA response regulator (letA) and its involvement in L. pneumophila pathogenicity and icm/dot gene expression was characterized. The letA mutant constructed had no intracellular growth defect when analyzed in HL-60 derived human macrophages, but it was found to be severely attenuated for intracellular growth in the protozoan host Acanthamoeba castellanii. The growth defect in amoebae was fully complemented by introducing the L. pneumophila letA gene on a plasmid. In addition, the LetA regulator was found to be involved in the expression of three icm genes (icmT, icmP and icmR). The level of expression of the icmT::lacZ and icmR::lacZ fusions was found to be higher, while the level of expression of the icmP::lacZ fusion was found to be lower when analyzed in the letA mutant strain, in comparison to the wild-type strain. We concluded that LetA has a moderate effect on icm/dot gene expression, but it probably plays a major role in the expression of L. pneumophila genes required for intracellular growth in protozoan hosts. A similar host specific phenotype was previously described for the RpoS sigma factor and the type II general secretion system of L. pneumophila.     

Identification of Legionella pneumophila-specific genes by genomic subtractive hybridization with Legionella micdadei and identification of lpnE, a gene required for efficient host cell entry

Legionella pneumophila is a ubiquitous environmental organism and a facultative intracellular pathogen of humans. To identify genes that may contribute to the virulence of L. pneumophila, we performed genomic subtractive hybridization between L. pneumophila serogroup 1 strain 02/41 and L. micdadei strain 02/42. A total of 144 L. pneumophila-specific clones were sequenced, revealing 151 genes that were absent in L. micdadei strain 02/42. Low-stringency Southern hybridization was used to determine the distribution of 41 sequences, representing 40 open reading frames (ORFs) with a range of putative functions among L. pneumophila isolates of various serogroups as well as strains of Legionella longbeachae, L. micdadei, Legionella gormanii, and Legionella jordanis. Twelve predicted ORFs were L. pneumophila specific, including the gene encoding the dot/icm effector, lepB, as well as several genes predicted to play a role in lipopolysaccharide biosynthesis and cell wall synthesis and several sequences with similarity to virulence-associated determinants. A further nine predicted ORFs were in all L. pneumophila serotypes tested and an isolate of L. gormanii. These included icmD, the 5' end of a pilMNOPQ locus, and two genes known to be upregulated during growth within macrophages, cadA2 and ceaA. Disruption of an L. pneumophila-specific gene (lpg2222 locus tag) encoding a putative protein with eight tetratricopeptide repeats resulted in reduced entry into the macrophage-like cell line, THP-1, and the type II alveolar epithelial cell line, A549. The gene was subsequently renamed lpnE, for "L. pneumophila entry." In summary, this investigation has revealed important genetic differences between L. pneumophila and other Legionella species that may contribute to the phenotypic and clinical differences observed within this genus.     

Identification and subcellular localization of the Legionella pneumophila IcmX protein: a factor essential for establishment of a replicative organelle in eukaryotic host cells

The gram-negative respiratory pathogen Legionella pneumophila infects and grows within mammalian macrophages and protozoan host cells. Upon uptake into macrophages, L. pneumophila establishes a replicative organelle that avoids fusion with endocytic vesicles. There are 24 dot/icm genes on the L. pneumophila chromosome required for biogenesis of this vacuole. Many of the Dot/Icm proteins are predicted to be components of a membrane-bound secretion apparatus similar to type IV conjugal transfer systems. We have been investigating the function of L. pneumophila dot/icm gene products that do not have obvious orthologs in other type IV transfer systems, since these determinants could govern processes unique to phagosome biogenesis. The icmX gene product falls into this category. To understand the role of the IcmX protein in pathogenesis, we have detailed interactions between an L. pneumophila icmX deletion mutant and murine bone marrow-derived macrophages. These data demonstrate that icmX is required for biogenesis of the L. pneumophila replicative organelle. Immunoblot analysis indicates that the icmX gene product is a polypeptide with an estimated molecular mass of 50 kDa. The IcmX protein was localized to the bacterial periplasm, and periplasmic translocation was mediated by an N-terminal sec-dependent leader peptide. A truncated IcmX product was secreted into culture supernatants by wild-type L. pneumophila growing extracellularly in liquid media; however, transport of the IcmX protein into eukaryotic host cells was not detected. Proteins similar in molecular weight to IcmX were identified in other Legionella species by immunoblot analysis using a monoclonal antibody specific for L. pneumophila IcmX protein. From these data, we conclude that the IcmX protein is an essential component of the dot/icm secretion apparatus, and that a conserved mechanism of host cell parasitism exists for members of the Legionellaceae family.     

Processing and major histocompatibility complex class II presentation of Legionella pneumophila antigens by infected macrophages

To better understand interactions between the intracellular pathogen Legionella pneumophila and macrophages (Mphis), host and bacterial determinants important for presentation of antigens on major histocompatibility complex class II molecules (MHC-II) were investigated. It was determined that immune CD4 T-cell responses to murine bone marrow-derived Mphis (BMphis) infected with wild-type L. pneumophila were higher than the responses to avirulent dotA mutant bacteria. Although this enhanced response by immune T cells required modulation of vacuole transport mediated by the Dot/Icm system, it did not require intracellular replication of L. pneumophila. Intracellular cytokine staining identified a population of immune CD4 T cells that produced gamma interferon upon incubation with BMphis infected with wild-type L. pneumophila that did not respond to Mphi infection with dotA mutant bacteria. Endocytic processing was required for presentation of L. pneumophila antigens on MHC-II as determined by a defect in CD4 T-cell responses when the pH of BMphi endosomes was neutralized with chloroquine. Investigation of MHC-II presentation of antigens by BMphis infected with L. pneumophila icmR, icmW, and icmS mutants indicated that these mutants have an intermediate presentation phenotype relative to those of wild-type and dotA mutant bacteria. In addition, it was found that antigens from dot and icm mutants are presented earlier than antigens from wild-type L. pneumophila. Although immune CD4 T-cell responses to proteins secreted by the L. pneumophila Lsp system were not detected, it was found that the Lsp system is important for priming L. pneumophila-specific T cells in vivo. These data indicate that optimal antigen processing and MHC-II presentation to immune CD4 T cells involves synthesis of L. pneumophila proteins in an endoplasmic reticulum-derived compartment followed by transport to lysosomes.     

Nucleotide sequence analysis of the Legionella micdadei mip gene, encoding a 30-kilodalton analog of the Legionella pneumophila Mip protein.

After the demonstration of analogs of the Legionella pneumophila macrophage infectivity potentiator (Mip) protein in other Legionella species, the Legionella micdadei mip gene was cloned and expressed in Escherichia coli. DNA sequence analysis of the L. micdadei mip gene contained in the plasmid pBA6004 revealed a high degree of homology (71%) to the L. pneumophila mip gene, with the predicted secondary structures of the two Mip proteins following the same pattern. Southern hybridization experiments, with the plasmid pBA6004 as the probe, suggested that the mip gene of L. micdadei has extensive homology with the mip-like genes of several Legionella species. Furthermore, amino acid sequence comparisons revealed significant homology to two eukaryotic proteins with isomerase activity (FK506-binding proteins).     

Characterization of the icmH and icmF genes required for Legionella pneumophila intracellular growth, genes that are present in many bacteria associated with eukaryotic cells

Legionella pneumophila, the causative agent of Legionnaires' disease, replicates intracellularly within a specialized phagosome of mammalian and protozoan host cells, and the Icm/Dot type IV secretion system has been shown to be essential for this process. Unlike all the other known Icm/Dot proteins, the IcmF protein, which was described before, and the IcmH protein, which is characterized here, have homologous proteins in many bacteria (such as Yersinia pestis, Salmonella enterica, Rhizobium leguminosarum, and Vibrio cholerae), all of which associate with eukaryotic cells. Here, we have characterized the L. pneumophila icmH and icmF genes and found that both genes are present in 16 different Legionella species examined. The icmH and icmF genes were found to be absolutely required for intracellular multiplication in Acanthamoeba castellanii and partially required for intracellular growth in HL-60-derived human macrophages, for immediate cytotoxicity, and for salt sensitivity. Mutagenesis of the predicted ATP/GTP binding site of IcmF revealed that the site is partially required for intracellular growth in A. castellanii. Analysis of the regulatory region of the icmH and icmF genes, which were found to be cotranscribed, revealed that it contains at least two regulatory elements. In addition, an icmH::lacZ fusion was shown to be activated during stationary phase in a LetA- and RelA-dependent manner. Our results indicate that although the icmH and icmF genes probably have a different evolutionary origin than the rest of the icm/dot genes, they are part of the icm/dot system and are required for L. pneumophila pathogenesis.     

Broad-spectrum enzyme-linked immunosorbent assay for detection of Legionella soluble antigens.

An enzyme-linked immunosorbent assay (ELISA) was developed which detected soluble antigens from culture extracts of Legionella pneumophila serogroups 1 to 8, L. micdadei, L. bozemanii serogroups 1 and 2, L. dumoffii, L. gormanii, L. longbeachae serogroups 1 and 2, L. wadsworthii, L. oakridgensis, L. anisa, L. feeleii serogroup 1, and L. jordanis. The assay was approximately 10-fold more sensitive for the eight L. pneumophila serogroups than for the other Legionella species tested. The ELISA detected Legionella antigens in the urine specimens of 25 of 35 patients with L. pneumophila serogroup 1, 3, 4, 6, and 8; L. micdadei; and L. longbeachae serogroup 1 infections. None of the 334 urine specimens from patients with either non-Legionella pneumonia or urinary tract infections was positive. For 10 patients from whom sequential urine specimens were available, Legionella antigens were not detectable from 7 to 19 days after laboratory diagnosis. Test sensitivity was not affected by heavy bacterial contamination. This ELISA offers the detection of a broad spectrum of Legionella antigens by a single test.     

Legionella pneumophila DotU and IcmF are required for stability of the Dot/Icm complex

Legionella pneumophila utilizes a type IV secretion system (T4SS) encoded by 26 dot/icm genes to replicate inside host cells and cause disease. In contrast to all other L. pneumophila dot/icm genes, dotU and icmF have homologs in a wide variety of gram-negative bacteria, none of which possess a T4SS. Instead, dotU and icmF orthologs are linked to a locus encoding a conserved cluster of proteins designated IcmF-associated homologous proteins, which has been proposed to constitute a novel cell surface structure. We show here that dotU is partially required for L. pneumophila intracellular growth, similar to the known requirement for icmF. In addition, we show that dotU and icmF are necessary for optimal plasmid transfer and sodium sensitivity, two additional phenotypes associated with a functional Dot/Icm complex. We found that these effects are due to the destabilization of the T4SS at the transition into the stationary phase, the point at which L. pneumophila becomes virulent. Specifically, three Dot proteins (DotH, DotG, and DotF) exhibit decreased stability in a DeltadotU DeltaicmF strain. Furthermore, overexpression of just one of these proteins, DotH, is sufficient to suppress the intracellular growth defect of the DeltadotU DeltaicmF mutant. This suggests a model where the DotU and IcmF proteins serve to prevent DotH degradation and therefore function to stabilize the L. pneumophila T4SS. Due to their wide distribution among bacterial species and their genetic linkage to known or predicted cell surface structures, we propose that this function in complex stabilization may be broadly conserved.     

Monoclonal antibodies to Legionella Mip proteins recognize genus- and species-specific epitopes.

Monoclonal antibodies (MAbs) against the virulence-associated Mip protein of Legionella spp. were raised by immunizing BALB/c mice with (i) Legionella pneumophila, (ii) Legionella micdadei, and (iii) purified recombinant native Mip protein cloned from L. pneumophila Philadelphia 1. Following screening of seeded wells by immunoblot analysis with homologous antigens, eight Mip-specific MAbs were found. These MAbs were chosen to investigate the antigenic diversity of Mip proteins in the genus Legionella. Mip was detected in 82 Legionella strains representing all 34 species tested. One of these MAbs, obtained from immunization with L. micdadei, recognized an epitope common to all Legionella species tested by immunoblot analysis. Another MAb was discovered to be specific for the Mip protein of L. pneumophila. The remaining six MAbs recognized 18 to 79% of Legionella species included in this study. By making use of the MAbs introduced in this study, it could be shown that, based on Mip protein epitope expression, Legionella species can be divided into at least six antigenetically distinct groups. As demonstrated by 43 L. pneumophila strains representing all serogroups, no antigenic diversity of Mip proteins was found for this species. In addition, 18 non-Legionella species, including Chlamydia trachomatis, Neisseria meningitidis, Pseudomonas aeruginosa, and Saccharomyces cerevisiae, all of which are known to carry genes homologous to the Legionella mip genes, were reacted against all eight MAbs. No cross-reactivity was detectable in any of those strains.     

Icm/dot-independent entry of Legionella pneumophila into amoeba and macrophage hosts

Legionella pneumophila, the causative agent of Legionnaires' disease, expresses a type IVB secretion apparatus that translocates bacterial proteins into amoeba and macrophage hosts. When stationary-phase cultures are used to infect hosts, the type IVB apparatus encoded by the icm/dot genes is required for entry, delay of phagosome-lysosome fusion, and intracellular multiplication within host cells. Null mutants with mutations in icm/dot genes are defective in these phenotypes. Here a new model is described in which hosts are infected with stationary-phase cultures that have been incubated overnight in pH 6.5 buffer. This model is called Ers treatment because it enhances the resistance to acid, hydrogen peroxide, and antibiotic stress beyond that of stationary-phase cultures. Following Ers treatment entry into amoeba and macrophage hosts does not require dotA, which is essential for Legionella virulence phenotypes when hosts are infected with stationary-phase cultures, dotB, icmF, icmV, or icmX. Defective host entry is also suppressed for null mutants with mutations in the KatA and KatB catalase-peroxidase enzymes, which are required for proper intracellular growth in amoeba and macrophage hosts. Ers treatment-induced suppression of defective entry is not associated with increased bacterial adhesion to host cells or with morphological changes in the bacterial envelope but is dependent on protein expression during Ers treatment. By using proteomic analysis, Ers treatment was shown to induce a protein predicted to contain eight tetratricopeptide repeats, a motif previously implicated in enhanced entry of L. pneumophila. Characterization of Ers treatment-dependent changes in expression is proposed as an avenue for identifying icm/dot-independent factors that function in the entry of Legionella into amoeba and macrophage hosts.     

Determination of catalase, peroxidase, and superoxide dismutase within the genus Legionella.

We examined 40 strains of Legionella for reduced-oxygen scavenging enzymes. Using a simple reaction chamber with a Swinney filter for the Beers and Sizer assay, we determined the catalase activity of live cells grown on buffered charcoal-yeast extract agar. For 29 strains of Legionella pneumophila, the apparent first-order rate constants for catalase ranged from 0.000 to 0.005. Similarly, low values ranging from 0.001 to 0.005 were observed for Legionella wadsworthii, Legionella oakridgensis, and Legionella gormanii. High catalase activities were found for Legionella jordanis, Legionella longbeachae, Legionella micdadei, and Legionella bozemanii, with first-order rate constant values of 0.010 to 0.035. Cell-free extracts were analyzed for catalase, peroxidase, and superoxide dismutase. Cell-free extracts of all strains had superoxide dismutase levels ranging from 8.2 to 30.5 U per mg of protein. The species could be characterized by their catalase and peroxidase since L. pneumophila and L. gormanii had only peroxidase (relative molecular weight [Mr], 150,000); L. dumoffii had a peroxidase (Mr, 150,000) plus a catalase (Mr, 174,000); and all remaining species had catalase only (Mr, 300,000, 220,000, or 150,000).     

Identification of mip-like genes in the genus Legionella.

The mip gene of Legionella pneumophila serogroup 1 strain AA100 encodes a 24-kilodalton surface protein (Mip) and enhances the abilities of L. pneumophila to parasitize human macrophages and to cause pneumonia in experimental animals. To determine whether this virulence factor is conserved in the genus Legionella, a large panel of Legionella strains was examined by Southern hybridization and immunoblot analyses for the presence and expression of mip-related sequences. Strains representing all 14 serogroups of L. pneumophila contained a mip gene and expressed a 24-kilodalton Mip protein. Although the isolates of the 29 other Legionella species did not hybridize with mip DNA probes under high-stringency conditions, they did so at reduced stringency. In support of the notion that these strains possess mip-like genes, these species each expressed a protein (24 to 31 kilodaltons in size) that reacted with specific Mip antisera. Moreover, the cloned mip analog from Legionella micdadei encoded the cross-reactive protein. Thus, mip is conserved and specific to L. pneumophila, but mip-like genes are present throughout the genus, perhaps potentiating the intracellular infectivity of all Legionella species.     

Severe Legionnaire's disease caused by Legionella longbeachae in a long-term renal transplant patient: the importance of safe living strategies after transplantation

Legionella species are intracellular gram-negative bacilli that require specific culture media for growth. Transplant recipients with impaired cellular immunity are at particular risk for infection with this pathogen. Most human disease is caused by Legionella pneumophila; disease caused by non-L. pneumophila species is reported mainly in immunosuppressed patients with the exception of Legionella longbeachae. L. longbeachae is a common cause of Legionnaires' disease in Australia and New Zealand, and is associated with exposure to potting soil. We report the case of a patient, 26?years post kidney transplant, who presented with severe and rapidly progressive respiratory illness. L. longbeachae serogroup 1 was isolated from respiratory cultures. Further investigation revealed that she had significant soil exposure before the onset of illness. We highlight the importance of following safe living strategies to prevent exposure-related illness even in long-term transplant recipients.     

Comparative Analysis of Legionella pneumophila and Legionella micdadei Virulence Traits

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

Legionella jordanis: a new species of Legionella isolated from water and sewage.

Legionella jordanis sp. nov., as found in two cultures, is described. One isolate was from river water in Indiana and the other isolate was from sewage in DeKalb County, Ga. The former is the type strain of the species, and is designated BL-540 (ATCC 33623). L. jordanis had a partial relationship to L. bozemanii by direct fluorescent-antibody tests but was unrelated to L. pneumophila, L. dumoffii, L. micdadei, L. gormanii, or L. longbeachae. Legionella phenotypic characteristics, including large amounts of branched-chain cellular fatty acids, were shown by the isolates. Studies of DNA relatedness showed that the two cultures of L. jordanis were only slightly related to the six previously described species of Legionella but were more than 90% related to each other. Indirect fluorescent-antibody tests with human sera suggested that unrecognized human infections with L. jordanis may be occurring.     

Rapid escape of the dot/icm mutants of Legionella pneumophila into the cytosol of mammalian and protozoan cells

The Legionella pneumophila-containing phagosome evades endocytic fusion and intercepts endoplasmic reticulum (ER)-to-Golgi vesicle traffic, which is believed to be mediated by the Dot/Icm type IV secretion system. Although phagosomes harboring dot/icm mutants are thought to mature through the endosomal-lysosomal pathway, colocalization studies with lysosomal markers have reported contradictory results. In addition, phagosomes harboring the dot/icm mutants do not interact with endocytosed materials, which is inconsistent with maturation of the phagosomes in the endosomal-lysosomal pathway. Using multiple strategies, we show that the dot/icm mutants defective in the Dot/Icm structural apparatus are unable to maintain the integrity of their phagosomes and escape into the cytoplasm within minutes of entry into various mammalian and protozoan cells in a process independent of the type II secretion system. In contrast, mutants defective in cytoplasmic chaperones of Dot/Icm effectors and rpoS, letA/S, and letE regulatory mutants are all localized within intact phagosomes. Importantly, non-dot/icm L. pneumophila mutants whose phagosomes acquire late endosomal-lysosomal markers are all located within intact phagosomes. Using high-resolution electron microscopy, we show that phagosomes harboring the dot/icm transporter mutants do not fuse to lysosomes but are free in the cytoplasm. Inhibition of ER-to-Golgi vesicle traffic by brefeldin A does not affect the integrity of the phagosomes harboring the parental strain of L. pneumophila. We conclude that the Dot/Icm transporter is involved in maintaining the integrity of the L. pneumophila phagosome, independent of interception of ER-to-Golgi vesicle traffic, which is a novel function of type IV secretion systems.     

Characterization of Legionella pneumophila pmiA, a gene essential for infectivity of protozoa and macrophages

The ability of Legionella pneumophila to cause pneumonia is dependent on intracellular replication within alveolar macrophages. The Icm/Dot secretion apparatus is essential for the ability of L. pneumophila to evade endocytic fusion, to remodel the phagosome by the endoplasmic reticulum (ER), and to replicate intracellularly. Protozoan and macrophage infectivity (pmi) mutants of L. pneumophila, which include 11 dot/icm mutants, exhibit defects in intracellular growth and replication within both protozoa and macrophages. In this study we characterized one of the pmi loci, pmiA. In contrast to the parental strain, the pmiA mutant is defective in cytopathogenicity for protozoa and macrophages. This is a novel mutant that exhibits a partial defect in survival within U937 human macrophage-like cells but exhibits a severe growth defect within Acanthamoeba polyphaga, which results in elimination from this host. The intracellular defects of this mutant are complemented by the wild-type pmiA gene on a plasmid. In contrast to phagosomes harboring the wild-type strain, which exclude endosomal-lysosomal markers, the pmiA mutant-containing phagosomes acquire the late endosomal-lysosomal markers LAMP-1 and LAMP-2. In contrast to the parental strain-containing phagosomes that are remodeled by the ER, there was a decrease in the number of ER-remodeled phagosomes harboring the pmiA mutant. Among several Legionella species examined, the pmiA gene is specific for L. pneumophila. The predicted amino acid sequence of the PmiA protein suggests that it is a transmembrane protein with three membrane-spanning regions. PmiA is similar to several hypothetical proteins produced by bacteria with a type IV secretion apparatus. Importantly, the defect in pmiA abolishes the pore-forming activity, which has been attributed to the Icm/Dot type IV secretion system. However, the mutant is sensitive to NaCl, and this sensitivity is abrogated in the icm/dot mutants. These results suggest that PmiA is a novel virulence factor that is involved in intracellular survival and replication of L. pneumophila in macrophages and protozoan cells.