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).     

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.     

Characterization of a Legionella micdadei mip mutant.

The pathogenesis of Legionella micdadei is dependent upon its ability to infect alveolar phagocytes. To better understand the basis of intracellular infection by this organism, we examined the importance of its Mip surface protein. In Legionella pneumophila, Mip promotes infection of both human macrophages and freshwater protozoa. Southern hybridization and immunoblot analyses demonstrated that mip sequences were present and expressed within a panel of virulent L. micdadei strains. Using allelic exchange mutagenesis, we then constructed an L. micdadei strain that completely and specifically lacked Mip. Although unimpaired in its ability to grow in bacteriologic media, this Mip mutant was defective in its capacity to infect U937 cells, a human macrophage-like cell line. Most significantly, the Mip- organism displayed a 24-fold reduction in survivability immediately after its entry into the phagocyte. Similarly, the mutant was less able to parasitize Hartmannella amoebae. Taken together, these data argue that Mip specifically potentiates intracellular growth by L. micdadei.     

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.     

Legionella pneumophila Mip, a surface-exposed peptidylproline cis-trans-isomerase, promotes the presence of phospholipase C-like activity in culture supernatants

The type II secretion system of Legionella pneumophila promotes pathogenesis. Among the Legionella type II-dependent exoenzymes is a p-nitrophenol phosphorylcholine (p-NPPC) hydrolase whose activity is only partially explained by the PlcA phospholipase C. In a screen to identify other factors that promote secreted hydrolase activity, we isolated a mip mutant. L. pneumophila Mip is a surface-exposed, FK506-binding protein that is needed for optimal infection and has peptidylproline cis-trans-isomerase (PPIase) activity. Since the molecular target of Mip was undefined, we investigated a possible relationship between Mip and the secreted p-NPPC hydrolase activity. In the mip mutant there was a 40 to 70% reduction in secreted activity that was successfully complemented by providing mip on a plasmid. A similar phenotype was observed when we examined four other independently derived mip mutants, and in all cases the defect was complemented by reintroduction of mip. Thus, mip promotes the presence of a p-NPPC hydrolase activity in culture supernatants. We also found that the C terminus of Mip is required for this effect. When supernatants were examined by anion-exchange chromatography, the p-NPPC hydrolase activity associated with Mip proved to be type II dependent but distinct from PlcA. This conclusion was supported by the phenotype of a newly constructed mip plcA double mutant. Thus, Mip promotes the elaboration of a new type II exoprotein. These data provide both the first evidence for a target for Mip and the first indication that a surface PPIase is involved in the secretion or activation of proteins beyond the outer membrane.     

应用单一和双重荧光定量PCR法快速检测军团菌

目的 建立单一和双重荧光定量PCR方法分别和同时进行军团菌属及嗜肺军团菌的检测.方法 利用军团菌属16 S rRNA基因和嗜肺军团菌mip基因设计引物和探针,两条基因探针分别标记FAM和HEX,并将相关反应体系和条件进行优化.分别应用单一基因探针(单一荧光定量PCR)和双重基因探针(双重荧光定量PCR)对嗜肺军团菌、非嗜肺军团菌及非军团菌进行检测,并验证两种方法的特异度、敏感度.应用双重荧光定量PCR检测空调水样滤膜样品和DNA提取样品,比较两者结果的一致性.结果 针对军团菌属及嗜肺军团菌,应用荧光定量PCR,16 S rRNA基因和mip基因均能较好的检出,16S rRNA和mip的最低检出限分别为8和10个拷贝.经优化得到了最佳反应体系.单一荧光定量PCR方法所检的8株嗜肺军用菌及4株非嗜肺军团菌16 S rRNA基因均为阳性,嗜肺军团菌mip基因阳性,非嗜肺军团菌mip基因阴性.双重荧光定量PCR方法所检的23株嗜肺军团菌中有2株为假阴性,9株非嗜肺军团菌和非军团菌属中有1株为假阳性.49份空调水样滤膜直接检测和提取DNA后检测的结果一致,其中26份水样军团菌阳性,20份为嗜肺军团菌,6份为非嗜肺军团菌;1份弗朗西斯菌检测HEX阳性(假阳性),占实际培养分离的1/26.结论 单一及双重荧光定量PCR法特异、快速、敏感,一次同时检测嗜肺与非嗜肺军团菌,满足对空调和环境水样军团菌监测的要求.     

应用单一和双重荧光定量PCR法快速检测军团菌

目的 建立单一和双重荧光定量PCR方法分别和同时进行军团菌属及嗜肺军团菌的检测.方法 利用军团菌属16 S rRNA基因和嗜肺军团菌mip基因设计引物和探针,两条基因探针分别标记FAM和HEX,并将相关反应体系和条件进行优化.分别应用单一基因探针(单一荧光定量PCR)和双重基因探针(双重荧光定量PCR)对嗜肺军团菌、非嗜肺军团菌及非军团菌进行检测,并验证两种方法的特异度、敏感度.应用双重荧光定量PCR检测空调水样滤膜样品和DNA提取样品,比较两者结果的一致性.结果 针对军团菌属及嗜肺军团菌,应用荧光定量PCR,16 S rRNA基因和mip基因均能较好的检出,16S rRNA和mip的最低检出限分别为8和10个拷贝.经优化得到了最佳反应体系.单一荧光定量PCR方法所检的8株嗜肺军用菌及4株非嗜肺军团菌16 S rRNA基因均为阳性,嗜肺军团菌mip基因阳性,非嗜肺军团菌mip基因阴性.双重荧光定量PCR方法所检的23株嗜肺军团菌中有2株为假阴性,9株非嗜肺军团菌和非军团菌属中有1株为假阳性.49份空调水样滤膜直接检测和提取DNA后检测的结果一致,其中26份水样军团菌阳性,20份为嗜肺军团菌,6份为非嗜肺军团菌;1份弗朗西斯菌检测HEX阳性(假阳性),占实际培养分离的1/26.结论 单一及双重荧光定量PCR法特异、快速、敏感,一次同时检测嗜肺与非嗜肺军团菌,满足对空调和环境水样军团菌监测的要求.     

Application of RNA polymerase beta-subunit gene (rpoB) sequences for the molecular differentiation of Legionella species

The nucleotide sequences of the partial rpoB gene were determined from 38 Legionella species, including 15 serogroups of Legionella pneumophila. These sequences were then used to infer the phylogenetic relationships among the Legionella species in order to establish a molecular differentiation method appropriate for them. The sequences (300 bp) and the phylogenetic tree of rpoB were compared to those from analyses using 16S rRNA gene and mip sequences. The trees inferred from these three gene sequences revealed significant differences. This sequence incongruence between the rpoB tree and the other trees might have originated from the high frequency of synonymous base substitutions and/or from horizontal gene transfer among the Legionella species. The nucleotide variation of rpoB enabled more evident differentiation among the Legionella species than was achievable by the 16S rRNA gene and even by mip in some cases. Two subspecies of L. pneumophila (L. pneumophila subsp. pneumophila and subsp. fraseri) were clearly distinguished by rpoB but not by 16S rRNA gene and mip analysis. One hundred and five strains isolated from patient tissues and environments in Korea and Japan could be identified by comparison of rpoB sequence similarity and phylogenetic trees. These results suggest that the partial sequences of rpoB determined in this study might be applicable to the molecular differentiation of Legionella species.     

Characterization of the Legionella pneumophila gene ligA

Legionella pneumophila is a bacterial pathogen that resides and multiplies in macrophages as well as in its natural aquatic hosts, the protozoa. Different bacterial factors contribute to pathogenicity and accompanying eukaryotic intracellular events. Sequencing of mip flanking regions revealed a gene of 2610 bp, ligA, that has no significant similarity to any of the genes identified previously. Epidemiological studies indicate that this gene is present in Legionella pneumophila, the species most often associated with cases of the Legionnaires' disease, but not in Legionella species other than L. pneumophila. The isogenic ligA deletion mutant was resistant to NaCl, and showed decreased cytotoxicity to human monocytes and decreased hemolytic activity to red blood cells. However, the most prominent effect of the L. pneumophila ligA mutant strain LEPF1 was the nearly completely reduced replication within the natural host Acanthamoeba castellanii. Since this gene is L. pneumophila specific and regulates numerous bacterial properties we designated this gene ligA for Legionella pneumophila infectivity gene A.     

Detection of Legionella pneumophila by real-time PCR for the mip gene

A real-time PCR assay for the mip gene of Legionella pneumophila was tested with 27 isolates of L. pneumophila, 20 isolates of 14 other Legionella species, and 103 non-Legionella bacteria. Eight culture-positive and 40 culture-negative clinical specimens were tested. This assay was 100% sensitive and 100% specific for L. pneumophila.     

Genetic, immunological, and cytotoxic comparisons of Legionella proteolytic activities.

Several strains of Legionella pneumophila and other species of Legionella with proteolytic activities were compared by assays, including Southern hybridizations and Western immunoblots, to determine their proteolytic, hemolytic, and cytotoxic activities. Only proteases from strains of L. pneumophila were both hemolytic and cytotoxic, and proteolytic activities extracted from other species of Legionella possessed only hemolytic activity. A 4.0-kilobase DNA sequence encoding the 38-kilodalton metalloprotease from L. pneumophila Philadelphia 1 that we showed previously was responsible for the observed hemolytic and cytotoxic phenotypes (F. D. Quinn and L. S. Tompkins, Mol. Microbiol., 3:797-805, 1989) was used in Southern hybridizations to probe chromosomal DNA from several strains of L. pneumophila and other Legionella species. The probe hybridized to the chromosomal DNA of all serogroups of L. pneumophila but not to any strains of L. dumoffii, L. micdadei, L. feeleii, or L. jordanis that we examined. Additionally, Western immunoblots done with rabbit antisera made to the cloned L. pneumophila protease demonstrated cross-reactions among 38-kilodalton proteins from strains of L. pneumophila, but no reactions were observed with proteins from other species of Legionella. Similarly, the cloned protease from L. pneumophila reacted with convalescent-phase sera from patients infected with L. pneumophila, but not with antisera isolated from patients infected with other Legionella species. Thus, despite some similarities among the proteolytic activities of members of the genus Legionella, including proteolytic and hemolytic phenotypes, metal requirements for zinc or iron, sensitivity to EDTA, and temperature and pH optima, we documented distinct genetic, immunological, and cytotoxicity differences among the proteolytic activities produced by Legionella species.     

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.     

实时荧光PCR快速检测嗜肺军团菌的研究

目的 建立TaqMan-MGB探针实时荧光PCR快速检测嗜肺军团菌技术,为临床和环境样品检测嗜肺军团菌提供可实用工具.方法 在对嗜肺军团菌mip序列进行分析、比较基础上,设计一对特异性引物和TaqMan-MGB探针,通过实时荧光PCR反应条件和反应体系的优化,实现对嗜肺军团菌的快速检测;用克隆到pMD-19T载体上的嗜肺军团菌mip基因阳参片段和不同菌株验证方法的敏感性和特异性.结果 当用热裂解法提取DNA,25μl的反应体系中包括上、下游引物(20μmol/L)各0.6μl,探针(20μmol/L)0.4μl,模板DNA 6.0μl,反应条件为预变95℃20 S,变性95℃10 s,退火50℃ 40 s,40个循环时,TaqMan-MGB探针实时荧光PCR技术对嗜肺军团菌mip基因阳参片段最低检测浓度为0.71拷贝/μl,其循环阈值(Ct值)与模板浓度具有极好的对应关系(r=0.999);1株嗜肺军团菌标准株、12株嗜肺军团菌分离株的Ct值在13.23～16.04之间,而包括金黄葡萄球菌、鼠伤寒沙门菌、副溶血性弧菌、大肠埃希菌、铜绿假单胞菌、痢疾志贺菌共计76株其他菌PCR Ct值均大于30;整个检测过程仅需1.5 h.结论 TaqMan-MGB探针的嗜肺军团菌实时荧光PCR检测方法具有特异性和敏感性、易操作、结果准确可靠等优点,可用于嗜肺军团菌检测.     

Purification and characterization of a UDP-glucosyltransferase produced by Legionella pneumophila

Legionella pneumophila is the agent of Legionnaires' disease. It invades and replicates within eukaryotic cells, including aquatic protozoans, mammalian macrophages, and epithelial cells. The molecular mechanisms of the Legionella interaction with target cells are not fully defined. In an attempt to discover novel virulence factors of L. pneumophila, we searched for bacterial enzymes with transferase activity. Upon screening ultrasonic extracts of virulent legionellae, we identified a uridine diphospho (UDP)-glucosyltransferase activity, which was capable of modifying a 45-kDa substrate in host cells. An approximately 60-kDa UDP-glucosyltransferase was purified from L. pneumophila and subjected to microsequencing. An N-terminal amino acid sequence, as well as the sequence of an internal peptide, allowed us to identify the gene for the enzyme within the unfinished L. pneumophila genome database. The intact gene was cloned and expressed in Escherichia coli, and the recombinant protein was purified and confirmed to possess an enzymatic activity similar to that of the native UDP-glucosyltransferase. We designated this gene ugt (UDP-glucosyltransferase). The Legionella enzyme did not exhibit significant homology with any known protein, suggesting that it is novel in structure and, perhaps, in function. Based on PCR data, an enzyme assay, and an immunoblot analysis, the glucosyltransferase appeared to be conserved in L. pneumophila strains but was absent from the other Legionella species. This study represents the first identification of a UDP-glucosyltransferase in an intracellular parasite, and therefore modification of a eukaryotic target(s) by this enzyme may influence host cell function and promote L. pneumophila proliferation.     

Cloning and temperature-dependent expression in Escherichia coli of a Legionella pneumophila gene coding for a genus-common 60-kilodalton antigen.

All Legionella species express a 60-kilodalton (kDa) protein which contains a genus-specific epitope recognized by murine monoclonal antibody GW2X4B8B2H6. A genomic cosmid library of Legionella pneumophila chromosomal DNA was constructed in pHC79 and screened for 60-kDa antigen-expressing clones with the monoclonal antibody. A 3.2-kilobase EcoRI fragment from cosmid 14B11 expressing a 60-kDa protein was subcloned into pUC19 (pSH16), and deletion of a 1.2-kilobase HindIII fragment (pSH16A) generated a 33-kDa truncated polypeptide no longer reactive with the monoclonal antibody. Southern blot analysis of chromosomal DNA from selected Legionella species restricted with EcoRI and probed with the 1.2-kilobase fragment coding for the carboxyl region of the protein revealed DNA homology which was not observed with DNA from Escherichia coli. Maxicell analysis of pSH16 identified a second polypeptide of approximately 15 kDa expressed from a gene (htpA) upstream of the gene coding the 60-kDa protein (htpB). Both proteins were preferentially synthesized by L. pneumophila following heat shock (temperature shift from 25 to 42 degrees C), and under steady-state growth conditions the relative level of 60-kDa protein was unaffected by temperature. In E. coli, expression of a 60-kDa protein from pSH16 also increased following heat shock (25 to 42 degrees C), but under steady-state conditions expression was temperature dependent. Temperature-dependent expression from pSH16 was not observed in an rpoH (htpR) mutant strain of E. coli. The Legionella 60-kDa protein appears to be a heat shock protein which shares cross-reactive epitopes with the GroEL homolog of E. coli. In addition, a region of htpB encoding the 27-kDa carboxyl portion of the protein containing the monoclonal antibody-reactive epitope also contains DNA sequences unique to and conserved within the genus.     

Multigenome analysis identifies a worldwide distributed epidemic Legionella pneumophila clone that emerged within a highly diverse species

Genomics can provide the basis for understanding the evolution of emerging, lethal human pathogens such as Legionella pneumophila, the causative agent of Legionnaires' disease. This bacterium replicates within amoebae and persists in the environment as a free-living microbe. Among the many Legionella species described, L. pneumophila is associated with 90% of human disease and within the 15 serogroups (Sg), L. pneumophila Sg1 causes over 84% of Legionnaires' disease worldwide. Why L. pneumophila Sg1 is so predominant is unknown. Here, we report the first comprehensive screen of the gene content of 217 L. pneumophila and 32 non-L. pneumophila strains isolated from humans and the environment using a Legionella DNA-array. Strikingly, we uncovered a high conservation of virulence- and eukaryotic-like genes, indicating strong environmental selection pressures for their preservation. No specific hybridization profile differentiated clinical and environmental strains or strains of different serogroups. Surprisingly, the gene cluster coding the determinants of the core and the O side-chain synthesis of the lipopolysaccaride (LPS cluster) determining Sg1 was present in diverse genomic backgrounds, strongly implicating the LPS of Sg1 itself as a principal cause of the high prevalence of Sg1 strains in human disease and suggesting that the LPS cluster can be transferred horizontally. Genomic analysis also revealed that L. pneumophila is a genetically diverse species, in part due to horizontal gene transfer of mobile genetic elements among L. pneumophila strains, but also between different Legionella species. However, the genomic background also plays a role in disease causation as demonstrated by the identification of a globally distributed epidemic strain exhibiting the genotype of the sequenced L. pneumophila strain Paris.     

Multicenter comparison of molecular methods for detection of Legionella spp. in sputum samples

Legionellosis can be diagnosed by PCR using sputum samples. In this report, the methods of nine laboratories for 12 sputum samples with Legionella pneumophila and Legionella longbeachae are compared. We conclude that (i) liquefaction prevents PCR inhibition, (ii) the employed mip gene PCRs detected L. pneumophila only, and (iii) the 16S rRNA gene PCR detected both Legionella species and is preferred for the diagnosis of legionellosis.