Molecular evolution of key genes for type II secretion in Legionella pneumophila

Given the role of type II protein secretion system (T2S) in the ecology and pathogenesis of Legionella pneumophila, it is possible that this system is a target for adaptive evolution. The population genetic structure of L.pneumophila was inferred from the partial sequences of rpoB and from the complete sequence of three T2S structural components (lspD, lspE and pilD) and from two T2S effectors critical for intracellular infection of protozoa (proA and srnA) of 37 strains isolated from natural and man-made environments and disease-related from worldwide sources. A phylogenetic analysis was obtained for the concatenated alignment and for each individual locus. Seven main groups were identified containing the same L.pneumophila strains, suggesting an ancient divergence for each cluster and indicating that the operating selective pressures have equally affected the evolution of the five genes. Although linkage disequilibrium analysis indicate a clonal nature for population structure in this sample, our results indicate that recombination is a common phenomenon among T2S-related genes on this species, as 24 of the 37 L.pneumophila isolates contained at least one locus in which recombination was identified. Furthermore, neutral selection acting on the analysed T2S-related genes emerged as a clear result, namely on T2S effectors, ProA and SrnA, indicating that they are probably implicated in conserved virulence mechanisms through legionellae hosts.     

The surfactant of Legionella pneumophila Is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent "sliding" motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment.     

The prepilin peptidase is required for protein secretion by and the virulence of the intracellular pathogen Legionella pneumophila

Prepilin peptidases cleave, among other substrates, the leader sequences from prepilin-like proteins that are required for type II protein secretion in Gram-negative bacteria. To begin to assess the importance of type II secretion for the virulence of an intracellular pathogen, we examined the effect of inactivating the prepilin peptidase (pilD) gene of Legionella pneumophila. Although the pilD mutant and its parent grew similarly in bacteriological media, they did differ in colony attributes and recoverability from late stationary phase. Moreover, at least three proteins were absent from the mutant's supernatant, indicating that PilD is necessary for the secretion of Legionella proteins. The absence of both the major secreted protein and a haemolytic activity from the mutant signalled that the L. pneumophila zinc metalloprotease is excreted via type II secretion. Most interestingly, the pilD mutant was greatly impaired in its ability to grow within Hartmannella vermiformis amoebae and the human macrophage-like U937 cells. As reintroduction of pilD into the mutant restored inefectivity and as a mutant lacking type IV pilin replicated like wild type, these data suggested that the intracellular growth of L. pneumophila is promoted by proteins secreted via a type II pathway. Intratracheal inoculation of guinea pigs revealed that the LD50 for the pilD mutant is at least 100-fold greater than that for its parent, and the culturing of bacteria from infected animals showed a rapid clearance of the mutant from the lungs. This is the first study to indicate a role for PilD and type II secretion in intracellular parasitism.     

Discovery of a nonclassical siderophore, legiobactin, produced by strains of Legionella pneumophila

The mechanisms by which Legionella pneumophila, a facultative intracellular parasite and the agent of Legionnaires' disease, acquires iron are largely unexplained. Several earlier studies indicated that L. pneumophila does not elaborate siderophores. However, we now present evidence that supernatants from L. pneumophila cultures can contain a nonproteinaceous, high-affinity iron chelator. More specifically, when aerobically grown in a low-iron, chemically defined medium (CDM), L. pneumophila secretes a substance that is reactive in the chrome azurol S (CAS) assay. Importantly, the siderophore-like activity was only observed when the CDM cultures were inoculated to relatively high density with bacteria that had been grown overnight to log or early stationary phase in CDM or buffered yeast extract. Inocula derived from late-stationary-phase cultures, despite ultimately growing, consistently failed to result in the elaboration of siderophore-like activity. The Legionella CAS reactivity was detected in the culture supernatants of the serogroup 1 strains 130b and Philadelphia-1, as well as those from representatives of other serogroups and other Legionella species. The CAS-reactive substance was resistant to boiling and protease treatment and was associated with the <1-kDa supernatant fraction. As would also be expected for a siderophore, the addition of 0.5 or 2.0 microM iron to the cultures repressed the expression of the CAS-reactive substance. Interestingly, the supernatants were negative in the Arnow, Csáky, and Rioux assays, indicating that the Legionella siderophore was not a classic catecholate or hydroxamate and, hence, might have a novel structure. We have designated the L. pneumophila siderophore legiobactin.     

Differing macrophage and lymphocyte roles in resistance to Legionella pneumophila infection.

Similar to guinea pig macrophages and human monocytes, macrophages from the peritoneal cavity of thioglycolate pretreated A/J mice are permissive for growth of Legionella pneumophila. In contrast, macrophages from BDF1 mice are not permissive for L. pneumophila. Lymphocytes from A/J and BDF1 mice proliferated in response to Legionella Ag but guinea pig lymphocytes did not. Also, splenocyte cultures from A/J mice treated with either Con A or Legionella vaccine produced supernatants which induced A/J macrophages to restrict Legionella growth, but guinea pig splenocyte culture supernatants obtained after stimulation with L. pneumophila vaccine did not induce Legionella growth restriction activity by guinea pig macrophages. Murine rIFN-gamma but not rIFN-alpha markedly inhibited growth of Legionella in A/J mouse macrophages and monoclonal anti-IFN-gamma antibody neutralized the anti-Legionella activity of culture supernatants from A/J mouse splenocytes responding to Legionella Ag. From these data, IFN-gamma appears to be an important factor in anti-Legionella activity of Ag-activated mouse splenocyte culture supernatants. Cyclosporin A, when given to either A/J or BDF1 mice, reduced the proliferation responses of splenocytes to T cell mitogens and also decreased the IFN production of A/J spleen cells to Legionella Ag. In addition, drug treatment decreased the resistance of A/J mice to Legionella infection as shown by an increase in the number of viable bacteria in the liver. However, injection of drug treated mice with lymphokine-rich splenocyte culture supernatant reconstituted the resistance of these animals. These results suggest an important role for lymphocyte activation and lymphokine production in the resistance of A/J mice to Legionella infection. The greater resistance of BDF1 mice, however, may result from nonpermissive macrophages and responsive lymphocytes. In the case of guinea pigs, susceptibility to Legionella infections may result from both the permissive nature of the macrophages and the relatively unresponsive nature of the lymphocytes in these animals.     

The DotA protein from Legionella pneumophila is secreted by a novel process that requires the Dot/Icm transporter.

Legionella pneumophila requires the dot/icm genes to create an organelle inside eukaryotic host cells that will support bacterial replication. The dot/icm genes are predicted to encode a type IV-related secretion apparatus. However, no proteins have been identified that require the dot/icm genes for secretion. In this study we show that the DotA protein, which was previously found to be a polytopic membrane protein, is secreted by the Dot/Icm transporter into culture supernatants. Secreted DotA protein was purified and N-terminal sequencing of the purified protein revealed that a 19 amino acid leader peptide is removed from DotA prior to secretion. Extracellular DotA protein did not fractionate in membrane vesicles. Structures containing secreted DotA protein were visualized by electron microscopy and were shaped like hollow rings. These data indicate that the large poly topic membrane protein DotA is secreted from L.pneumophila by a unique process. This represents the first target secreted by the dot/icm-encoded apparatus and demonstrates that this transporter is competent for protein secretion.     

Dendritic cells pulsed with live and dead Legionella pneumophila elicit distinct immune responses

Legionella pneumophila is the causative pathogen of Legionnaires' disease, which is characterized by severe pneumonia. In regard to the pathophysiology of Legionella infection, the role of inflammatory phagocytes such as macrophages has been well documented, but the involvement of dendritic cells (DCs) has not been clarified. In this study, we have investigated the immune responses that DCs generate in vitro and in vivo after contact with L. pneumophila. Heat- and formalin-killed L. pneumophila, but not live L. pneumophila, induced immature DCs to undergo similar phenotypic maturation, but the secreted proinflammatory cytokines showed different patterns. The mechanisms of the DC maturation by heat- or formalin-killed L. pneumophila depended, at least in part, on Toll-like receptor 4 signaling or on Legionella LPS, respectively. After transfer to naive mice, DCs pulsed with dead Legionella produced serum Ig isotype responses specific for Legionella, leading to protective immunity against an otherwise lethal respiratory challenge with L. pneumophila. The in vivo immune responses required the Ag presentation of DCs, especially that on MHC class II molecules, and the immunity yielded cross-protection between clinical and environmental strains of L. pneumophila. Although the DC maturation was impaired by live Legionella, macrophages were activated by live as well as dead L. pneumophila, as evidenced by the up-regulation of MHC class II. Finally, DCs, but not macrophages, exhibited a proliferative response to live L. pneumophila that was consistent with their cell cycle progression. These findings provide a better understanding of the role of DCs in adaptive immunity to Legionella infection.     

Species-specific detection of Legionella using polymerase chain reaction and reverse dot-blotting

Abstract Legionella pneumophila and some other Legionella species are capable of causing Legionnaire's disease, a potentially fatal pneumonia. The identification of legionellae by standard laboratory techniques such as culture is difficult and time-consuming. In the present work, the DNA sequence of the 23S-5S spacer region was determined for 43 Legionella isolates, and the sequence information was used to develop a species-specific detection system using PCR and reverse dot-blotting which employs just one PCR amplicon to perform genus- and species-specific detection. L. pneumophila serogroups 1–16 as well as 21 non- pneumophila isolates could be identified and differentiated at the species level using this system.     

Two cellulases, CelA and CelC, from the polycentric anaerobic fungus Orpinomyces strain PC-2 contain N-terminal docking domains for a cellulase-hemicellulase complex.

Two cDNAs encoding two cellulases, CelA and CelC, were isolated from a cDNA library of the polycentric anaerobic fungus Orpinomyces sp. strain PC-2 constructed in Escherichia coli. Nucleotide sequencing revealed that the celA cDNA (1,558 bp) and celC cDNA (1,628 bp) had open reading frames encoding polypeptides of 459 (CelA) and 449 (CelC) amino acids, respectively. The two cDNAs were 76.9 and 67.7% identical at the nucleotide and amino acid levels, respectively. Analysis of the deduced amino acid sequences showed that starting from the N termini, both CelA and CelC had signal peptides, which were followed by noncatalytic repeated peptide domains (NCRPD) containing two repeated sequences of 33 to 40 amino acid residues functioning as docking domains. The NCRPDs and the catalytic domains were separated by linker sequences. The NCRPDs were homologous to those found in several hydrolases of anaerobic fungi, whereas the catalytic domains were homologous to the catalytic domains of fungal cellobiohydrolases and bacterial endoglucanases. The linker sequence of CelA contained predominantly glutamine and proline residues, while that of CelC contained mainly threonine residues. CelA and CelC did not have a typical cellulose binding domain (CBD). CelA and CelC expressed in E. coli rapidly decreased the viscosity of carboxymethyl cellulose (CMC), indicating that there was endoglucanase activity. In addition, they produced cellobiose from CMC, acid-swollen cellulose, and cellotetraose, suggesting that they had cellobiohydrolase activity. The optimal activity conditions with CMC as the substrate were pH 4.3 to 6.8 and 50 degrees C for CelA and pH 4.6 to 7.0 and 40 degrees C for CelC. Despite the lack of a CBD, CelC displayed a high affinity for microcrystalline cellulose, whereas CelA did not.     

Development of conventional and real-time NASBA for the detection of Legionella species in respiratory specimens

Isothermal nucleic acid sequence-based amplification (NASBA) was applied to detect Legionella 16S rRNA. The assay was originally developed as a Legionella pneumophila conventional NASBA assay with electrochemiluminescence (ECL) detection and was subsequently adapted to a L. pneumophila real-time NASBA format and a Legionella spp. real-time NASBA using molecular beacons. L. pneumophila RNA prepared from a plasmid construct was used to assess the analytical sensitivity of the assay. The sensitivity of the NASBA assay was 10 molecules of in vitro wild type L. pneumophila RNA and 0.1-1 colony-forming units (CFU) of L. pneumophila. In spiked respiratory specimens, the sensitivity of the NASBA assays was 1-10000 CFU of L. pneumophila serotype 1 depending on the background. After dilution of the nucleic acid extract prior to amplification, 1-10 CFU of L. pneumophila serotype 1 could be detected with both detection methods. Finally, 27 respiratory specimens, well characterized by culture and PCR, collected during a L. pneumophila outbreak, were tested by conventional and real-time NASBAs. All 11 PCR positive samples were positive by conventional NASBA, 9/11 and 10/11 were positive by L. pneumophila real-time NASBA and Legionella spp. real-time NASBA, respectively.     

Detection and identification of Legionella pneumophila by PCR-restriction fragment length polymorphism analysis of the RNA polymerase gene (rpoB)

The partial RNA polymerase beta-subunit coding gene (rpoB) sequences of 38 Legionella species (59 reference strains) were used to select both Legionella genus-specific and Legionella pneumophila species-specific primers to amplify the 347-bp and 217-bp DNAs, respectively. Enzyme restriction sites for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis were also generated by a computer program. Thirty-eight Legionella species were well differentiated by the identification scheme for Legionella genus-specific PCR-RFLP using HaeIII, AluI, CfoI, PstI, and MaeII. The most common and important pathogenic species, L. pneumophila, was differentiated into two subspecies (L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri) by both Legionella genus-specific PCR-RFLP and L. pneumophila species-specific PCR-RFLP using BamHI. Eighty-two Korean culture isolates could also be easily identified by both PCR-RFLP methods as 68 strains of L. pneumophila subsp. pneumophila, 11 strains of L. pneumophila subsp. fraseri, and three novel strains that were separately confirmed by 16S rDNA and rpoB sequence analysis. These results suggest that the rpoB PCR-RFLP for Legionella is a simple and convenient method, not only for specific detection, but also for the rapid identification of Legionella species.     

The DotL protein, a member of the TraG-coupling protein family, is essential for Viability of Legionella pneumophila strain Lp02

Legionella pneumophila is able to survive inside phagocytic cells by an internalization route that bypasses fusion of the nascent phagosome with the endocytic pathway to allow formation of a replicative phagosome. The dot/icm genes, a major virulence system of L. pneumophila, encode a type IVB secretion system that is required for intracellular growth. One Dot protein, DotL, has sequence similarity to type IV secretion system coupling proteins (T4CPs). In other systems, coupling proteins are not required for viability of the organism. Here we report the first example of a strain, L. pneumophila Lp02, in which a putative T4CP is essential for viability of the organism on bacteriological media. This result is particularly surprising since the majority of the dot/icm genes in Lp02 are dispensable for growth outside of a host cell, a condition that does not require a functional Dot/Icm secretion complex. We were able to isolate suppressors of the Delta dotL lethality and found that many contained mutations in other components of the Dot/Icm secretion system. A systematic analysis of dot/icm deletion mutants revealed that the majority of them (20 of 26) suppressed the lethality phenotype, indicating a partially assembled secretion system may be the source of Delta dotL toxicity in the wild-type strain. These results are consistent with a model in which the DotL protein plays a role in regulating the activity of the L. pneumophila type IV secretion apparatus.     

PCR-酶切技术快速鉴定军团菌

[目的]建立一种新型的军团菌鉴定方法,并探讨该法在鉴定环境水源和临床标本军团菌菌株中的应用价值.[方法]根据军团菌16S rRNA基因保守序列设计引物,以分离培养得到的可疑军团菌菌株作为模板,采用PCR法对模板扩增,并用限制性内切酶对PCR产物进行酶切分析,建立一种嗜肺军团菌及非嗜肺军团菌的鉴定方法.对16株嗜肺军团菌、22株非嗜肺军团菌及12株其他细菌标准菌株进行检测,验证该方法的可靠性,最后用该法检测广州地区分离的169株可疑军团菌菌株并进行基因测序.[结果]该PCR方法检测嗜肺军团菌及非嗜肺军团菌所有标准菌株均为阳性,非军团菌检测结果均为阴性;进一步的Hinf Ⅰ酶切分析可准确的区分嗜肺军团菌标准菌株;广州地区分离的169株可疑军团菌菌株经该法检测发现160株为军团菌,其中79株为嗜肺军团菌,与基因测序检测结果一致.[结论]PCR-酶切技术可快速、特异地检测军团菌及嗜肺军团菌,适用于环境水源和临床标本可疑军团菌菌株的检测.     

Novel lysophospholipase A secreted by Legionella pneumophila

We show that Legionella pneumophila possesses lysophospholipase A activity, which releases fatty acids from lysophosphatidylcholine. The NH2-terminal sequence of the enzyme contained FGDSLS, corresponding to a catalytic domain in a recently described group of lipolytic enzymes. Culture supernatants of a L. pneumophila pilD mutant lost the ability to cleave lysophosphatidylcholine.     

Legionella pneumophila EnhC is required for efficient replication in tumour necrosis factor alpha-stimulated macrophages

Legionella pneumophila enhC ^- mutants were originally identified as being defective for uptake into host cells. In this work, we found that the absence of EnhC resulted in defective intracellular growth when dissemination of intracellular bacteria to neighbouring cells was expected to occur. No such defect was observed during growth within the amoeba Dictyostelium discoideum. Culture supernatants containing the secreted products of infected macrophages added to host cells restricted the growth of the Δ enhC strain, while tumour necrosis factor α (TNF-α), at concentrations similar to those found in macrophage culture supernatants, could reproduce the growth restriction exerted by culture supernatants on L. pneumophila Δ enhC . The absence of EnhC also caused defective trafficking of the Legionella- containing vacuole in TNF-α-treated macrophages. EnhC was shown to be an envelope-associated protein largely localized to the periplasm, with its expression induced in post-exponential phase, as is true for many virulence-associated proteins. Furthermore, the absence of EnhC appeared to affect survival under stress conditions, as the Δ enhC mutant was more susceptible to H₂O₂ treatment than the wild-type strain. EnhC therefore is a unique virulence factor that is required for growth specifically when macrophages have heightened potential to restrict microbial replication.     

lbtA and lbtB are required for production of the Legionella pneumophila siderophore legiobactin

Under iron stress, Legionella pneumophila secretes legiobactin, a nonclassical siderophore that is reactive in the chrome azurol S (CAS) assay. Here, we have optimized conditions for legiobactin expression, shown its biological activity, and identified two genes, lbtA and lbtB, which are involved in legiobactin production. lbtA appears to be iron repressed and encodes a protein that has significant homology with siderophore synthetases, and FrgA, a previously described iron-regulated protein of L. pneumophila. lbtB encodes a protein homologous with members of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtA or lbtB were defective for legiobactin, producing 40 to 70% less CAS reactivity in deferrated chemically defined medium (CDM). In bioassays, mutant CDM culture supernatants, unlike those of the wild type, did not support growth of iron-limited wild-type bacteria in 2',2'-dipyridyl-containing buffered charcoal yeast extract (BCYE) agar and a ferrous iron transport mutant on BCYE agar without added iron. The lbtA mutant was modestly defective for growth in deferrated CDM containing the iron chelator citrate, indicating that legiobactin is required in conditions of severe iron limitation. Complementation of the lbt mutants restored both siderophore expression, as measured by the CAS assay and bioassays, and bacterial growth in deferrated, citrate-containing media. The lbtA mutant replicated as the wild type did in macrophages, amoebae, and the lungs of mice. However, L. pneumophila expresses lbtA in the macrophage, suggesting that legiobactin, though not required, may play a dispensable role in intracellular growth. The discovery of lbtAB represents the first identification of genes required for L. pneumophila siderophore expression.     

Mutations from a family-shuffling-library reveal amino acid residues responsible for the thermostability of endoglucanase CelA from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

We constructed a library of chimeras from the major endoglucanase, CelA, of Clostridium thermocellum and a less stable endoglucanase CelB from Clostridium josui with multiple point mutations using low-fidelity family-shuffling method. Mutations that inactivated the enzyme were rapidly eliminated with high-throughput screening. The activities and thermostabilities of selected variants were evaluated, and four amino acid substitutions, K249R, P258S, S329N and E355G, were identified as having significant impact on the thermostability of CelA without affecting enzymatic activity. In the crystal structure of CelA, most of them are away from the activity cleft and are responsible for the stabilization of secondary structures.     

Site-specific mutagenesis in Legionella pneumophila by allelic exchange using counterselectable ColE1 vectors

Abstract To study the molecular pathogenesis of infection by Legionella pneumophila , a technique of site-specific mutagenesis by allelic exchange was evaluated. To develop this system, we optimized conjugal DNA transfer by isolating a mutant that functions 1000-fold more efficiently as a recipient than the wild type strain, identified two counter-selectable markers, rpsL and sacB , that function in L. pneumophila , and constructed a counterselectable Co1E1 vector. Allelic exchange of a L. pneumophila chrosomal gene was achieved at a frequency of 10⁻⁵ per transconjugant. The allelic exchange procedure itself did not alter the ability of L. pneumophila to infect macrophages, indicating that the system can be used to study this aspect of virulence.