Legionella pneumophila htpAB heat shock operon: nucleotide sequence and expression of the 60-kilodalton antigen in L. pneumophila-infected HeLa cells

A 60-kilodalton (kDa) immunodominant antigen of Legionella pneumophila is a heat shock protein (HSP) of the GroEL class of HSPs. The gene (htpB) coding the 60-kDa protein was localized to a 3.2-kilobase DNA fragment of L. pneumophila cloned into pUC19 (pSH16) (P. S. Hoffman, C. A. Butler, and F. D. Quinn, Infect. Immun. 57:1731-1739, 1989). The nucleotide sequence of the DNA fragment cloned into M13 confirmed two open reading frames, htpA and htpB, that code for proteins of 96 and 548 amino acids, respectively. A consensus heat shock promoter sequence upstream of the start of htpA was identified, and no obvious promoter sequences were detected upstream of htpB. Amino acid sequence comparison studies revealed that the L. pneumophila HtpB protein exhibited 76% homology with the 65-kDa protein of Mycobacterium tuberculosis and 85% homology with both GroEL of Escherichia coli and HtpB of Coxiella burnetii. A comparison of the amino acid sequences among these proteins revealed several regions of nearly absolute sequence conservation, with the variable regions occurring in common areas. The purified L. pneumophila 60-kDa protein was antigenic for human T lymphocytes. Indirect fluorescent antibody studies indicated that the 60-kDa protein may be located in the periplasm or expressed on the surface by intracellular bacteria, suggesting that a stress-related mechanism may be involved in the expression of this immunodominant antigen.     

Genus-specific epitope on the 60-kilodalton Legionella heat shock protein recognized by a monoclonal antibody.

A monoclonal antibody (MAb) immunoglobulin G2a (2125) was produced against a 60-kDa Legionella heat shock protein (HSP), recognizing a unique epitope common to all species of the genus Legionella. The antibody reacted in the immunoblot with 59 Legionella species and serogroups that were tested and showed no cross-reactivity with other bacteria, including Acinetobacter spp., Bordetella spp., Pseudomonas spp., Mycobacterium spp., and Escherichia coli. Two other MAbs (2122 and 2130) reacted with the 60-kDa Legionella protein as well but showed different cross-reactivities with other gram-negative bacteria in the same molecular mass range. The genus-specific MAb 2125 as well as the cross-reacting MAbs 2122 and 2130 were shown to be reactive with the expressed protein of the cloned gene of the 60-kDa HSP of Legionella micdadei and Legionella pneumophila. These antibodies demonstrate that Legionella-specific and nonspecific epitopes are present on this protein. A sandwich enzyme-linked immunosorbent assay (ELISA) in which the genus-specific MAb is used both as a capture antibody and as a biotinylated second antibody has been established. With this test it is possible to detect Legionella whole cells, sonicated cells, and cell fractions containing the 60-kDa HSP. The main part of the 60-kDa HSP is found in the cytoplasmic fraction. The sandwich ELISA can be used to demonstrate the increased expression of the 60-kDa protein in Legionella cells following heat shock as well as marked differences in the detection of the 60-kDa HSP on whole cells of different Legionella strains. The high specificity and sensitivity of the sandwich ELISA for sonicated cells might be very useful to screen on a genus level for Legionella cells or the 60-kDa antigen in environmental isolates or body fluids of patients.     

Cloning, genetic analysis, and nucleotide sequence of a determinant coding for a 19-kilodalton peptidoglycan-associated protein (Ppl) of Legionella pneumophila.

A genomic library of Legionella pneumophila, the causative agent of Legionnaires disease in humans, was constructed in Escherichia coli K-12, and the recombinant clones were screened by immuno-colony blots with an antiserum raised against heat-killed L. pneumophila. Twenty-three clones coding for a Legionella-specific protein of 19 kDa were isolated. The 19-kDa protein, which represents an outer membrane protein, was found to be associated with the peptidoglycan layer both in L. pneumophila and in the recombinant E. coli clones. This was shown by electrophoresis and Western immunoblot analysis of bacterial cell membrane fractions with a monospecific polyclonal 19-kDa protein-specific antiserum. The protein was termed peptidoglycan-associated protein of L. pneumophila (Ppl). The corresponding genetic determinant, ppl, was subcloned on a 1.8-kb ClaI fragment. DNA sequence studies revealed that two open reading frames, pplA and pplB, coding for putative proteins of 18.9 and 16.8 kDa, respectively, were located on the ClaI fragment. Exonuclease III digestion studies confirmed that pplA is the gene coding for the peptidoglycan-associated 19-kDa protein of L. pneumophila. The amino acid sequence of PplA exhibits a high degree of homology to the sequences of the Pal lipoproteins of E. coli K-12 and Haemophilus influenzae.     

Rapid identification of legionellae by a colony blot assay based on a genus-specific monoclonal antibody.

We recently developed a monoclonal antibody immunoglobulin G2a (2125) recognizing a genus-specific epitope on the 60-kDa heat shock protein of all Legionella species. In the current study, this antibody was used in a colony blot enzyme-linked immunosorbent assay for the rapid identification of Legionella cultures on agar plates. The whole protocol was completed in less than 2 h. All 59 Legionella species and serogroups that were tested gave a positive signal. No unspecific reactions with nonlegionellae were observed. This test is a rapid procedure for the identification of legionellae growing on agar medium to the genus level.     

Cloning and expression of a common Legionella outer membrane antigen in Escherichia coli

A genomic library of Legionella pneumophila was constructed by inserting L. pneumophila knoxville-1 strain (LPK-1) chromosome fragments into cosmid vector pHC79. Screening of the library with antibodies directed against a major outer membrane protein/lipopolysaccharide complex from LPK-1 resulted in the identification of six clones that reacted with the antiserum. Western blot analysis indicated that a 19,000 dalton (19 kDa) component was the reactive antigen in all of the clones. Western blot analysis of outer membranes from L. pneumophila serogroups and other Legionella species revealed that the cloned 19 kDa antigen was common to all serogroups and all but one of the five other Legionella species examined. One of the 19 kDa expressing clones was used as an immunoabsorbent to recover antibody to the 19 kDa antigen thus confirming the surface localization of this L. pneumophila antigen in E. coli.     

Nucleotide sequence of htpB, the Legionella pneumophila gene encoding the 58-kilodalton (kDa) common antigen, formerly designated the 60-kDa common antigen.

Gene htpB, which encodes the 58-kilodalton protein of Legionella pneumophila, was cloned in Escherichia coli and its complete nucleotide sequence was determined. Analysis of this sequence revealed an open reading frame of 1,644 nucleotides encoding a protein with a predicted molecular mass of 57,952 daltons. Data obtained by amino-terminal sequencing of the purified 58-kilodalton protein agreed, except for one amino acid residue, with the predicted amino acid sequence, identifying this open reading frame as htpB. A comparison of the primary structure of this protein to other proteins of similar molecular weights from E. coli, Mycobacterium leprae, M. tuberculosis, and Coxiella burnetii revealed significant regions of sequence similarity, which are discussed.     

Isolation and sequence analysis of the Chlamydia pneumoniae GroE operon.

Chlamydia pneumoniae has emerged as an important human respiratory pathogen. From a lambda gt11 gene bank constructed from C. pneumoniae isolate AR-39 DNA, an immunoreactive plaque containing a 3.0-kb insert was purified. In immunoblots, a 60-kDa protein was recognized by anti-C. pneumoniae rabbit immune serum. The recombinant protein was reactive with a Chlamydia genus-specific monoclonal antibody recognizing a 60-kDa protein found in the Sarkosyl-soluble fraction and with rabbit immune serum prepared against the Chlamydia trachomatis 60-kDa GroEL homolog associated with the delayed-type hypersensitivity response. DNA sequence analysis confirmed that the C. pneumoniae gene product is an analog of the C. trachomatis delayed-type hypersensitivity antigen and the Escherichia coli GroEL heat shock protein.     

The 56-kilodalton major protein antigen of Rickettsia tsutsugamushi: molecular cloning and sequence analysis of the sta56 gene and precise identification of a strain-specific epitope.

Lasting immunity against Rickettsia tsutsugamushi, the causative agent of scrub typhus fever, has been demonstrated to be strain specific. Two protein antigens of 110 and 56 kilodaltons (kDa) have been shown to exhibit strain-specific epitopes. The 56-kDa scrub typhus antigen (Sta56) is an abundant outer membrane protein of R. tsutsugamushi and is an antigen often recognized by humans infected with this obligate intracellular bacterium. In this study the complete gene encoding Sta56 (strain Karp) was cloned into pBR322 on a 2.3-kilobase genomic HindIII DNA fragment and the complete 56-kDa polypeptide was expressed in Escherichia coli. DNA sequence analysis of the 2.3-kilobase HindIII fragment revealed an open reading frame large enough to encode a 56-kDa polypeptide. A putative signal sequence was identified at the deduced amino terminus of the Sta56 polypeptide, and pulse-chase analysis of maxicells labeled with [35S]methionine demonstrated that a higher-molecular-weight precursor matures into the 56-kDa polypeptide. Epitope scanning analysis with synthetic peptides derived from the deduced amino acid sequence identified an octapeptide (located from amino acid residues 117 to 124) that was reactive with a Karp strain-specific monoclonal antibody (K13F88A). Other epitopes recognized by different monoclonal antibodies, including another Karp strain-specific monoclone (K1E106), were localized to different regions of the protein based on their reactivities with lambda gt11 recombinants expressing various portions of the sta56 gene.     

The 75-kilodalton protein of Chlamydia trachomatis: a member of the heat shock protein 70 family?

The gene encoding a 75-kilodalton (kDa) protein of Chlamydia trachomatis was cloned, expressed, and sequenced. Genomic libraries from C. trachomatis serovar D DNA were constructed in vectors pUC18 and lambda gt11 and were screened with a panel of monoclonal antibodies against C. trachomatis antigens. The only recombinants identified were those that reacted with antibody UM-13, which has specificity for a genus-specific epitope on the 75-kDa protein. The gene was localized to a 2.9-kilobase DNA fragment and sequenced. The gene consists of a long open reading frame of 1,956 nucleotides, which translates into 652 amino acids totalling 70,558 daltons in mass. Putative promoter elements and a ribosome binding site were identified within 5'-flanking sequences, and a typical rho-independent terminator was identified within 3'-flanking sequences. Screening of the GenBank nucleic acid sequence data bank revealed extensive similarity between the chlamydial 75-kDa gene and the heat shock protein 70 (hsp70) family or proteins. In particular, 71 and 69% amino acid sequence similarities were identified with hsp70 of Escherichia coli and Bacillus megaterium, respectively. Polyclonal antibodies were produced to the recombinant antigen in rabbits and detected epitopes on elementary bodies in enzyme-linked immunosorbent and indirect microimmunofluorescence assays. Antibodies reacted with an antigen of identical molecular mass in L2 and C serovars in an immunoblot assay and neutralized these serovars in cell culture. The 75-kDa protein appears to be a chlamydial homolog of hsp70, is immunoaccessible on native elementary bodies, and is a target for neutralization.     

Cloning and sequencing of the genes coding for the 10- and 60-kDa heat shock proteins from Pseudomonas aeruginosa and mapping of a species-specific epitope.

A genomic library of Pseudomonas aeruginosa DNA was screened with a monoclonal antibody (MAb 2528) specific for the P. aeruginosa 60-kDa heat shock protein. A positive clone, pAS-1, was isolated. The gene coding for P. aeruginosa chaperonin (hsp60) was localized to a 2-kb EcoRI fragment subcloned in pAS-2. A sequence analysis of pAS-2 and parts of pAS-1 identified two open reading frames that encoded proteins with calculated molecular masses of 10 and 57 kDa. In amino acid sequence comparison studies the sequences of these proteins, which were designated GroES and GroEL, exhibited up to 78% homology with known prokaryotic sequences of 10- and 60-kDa heat shock proteins (hsp10 and hsp60). In order to map the epitope recognized by MAb 2528, a series of GroEL nested carboxy-terminal deletion clones were tested with MAb 2528. We identified the clone with the shortest insertion that was still recognized by MAb 2528 and the clone with the largest insertion that was not recognized by MAb 2528. The 3' ends of the insertions were determined by sequencing and were found to delimit a region that encoded 25 amino acid residues. Synthetic oligonucleotides that coded for peptides possibly resembling the epitope within this region were ligated into expression vector pGEX-3X, and fusion proteins expressed by these clones were tested for reactivity with MAb 2528. By using this method we determined that the decapeptide QADIEARVLQ (positions 339 to 348 on GroEL) was responsible for the binding of P. aeruginosa-specific MAb 2528.     

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 expression of the Legionella micdadei "common antigen" in Escherichia coli

To study individual Legionella antigens, a Legionella micdadei genomic library in Escherichia coli SC181 was established. Partially Sau3A digested L. micdadei DNA fragments (15-25 kilobase pairs (kb] were cloned into the tetracycline resistance gene of the cosmid vector pHC79. Four thousand ampicillin resistant recombinants were obtained; seven hundred were screened for expression of Legionella antigens in Western blot analysis with a polyspecific E. coli-absorbed anti-L. micdadei rabbit antibody. One of the positive clones expressed a 60 kilodalton (K) antigen, which reacted strongly with a monospecific rabbit antiserum raised against L. micdadei "common antigen" (CA), and an additional 13 K L. micdadei protein. The region encoding these two proteins from the 17 kb recombinant plasmid (pBA 2) was subcloned in pBGS18+. The DNA sequence of the CA encoding region in the 2.7 kb subcloned fragment will provide important information with respect to genetic vs. antigenic relatedness among Legionellae and other Gram-negative species, as well as to CA structure and possible function.     

Phenotype versus genotype of the 19 kD peptido-glycan associated protein of Legionella (PpIA), among Legionellae and other gram-negative bacteria.

The protein PpIA (19 kD) cloned from a genomic library of Legionella pneumophila, Philadelphia 1, represents a peptido-glycan associated outer membrane protein in recombinant E. coli K-12 and L. pneumophila. It exhibits distinct sequence homology to lipoproteins of Haemophilus influenzae and E. coli. A ppIA specific DNA probe generated by PCR was used in Southern hybridizations of chromosomal DNA of Legionella strains and other Gram-negative pathogens. Under conditions of high stringency, hybridization could only be observed in L. pneumophila isolates, but all other Legionella strains tested displayed hybridization under lower stringency. No signals appeared after hybridization of chromosomal DNA from a variety of other bacteria. Using anti-PpIA monospecific polyclonal antibodies in Western blots, it was demonstrated that PpIA related proteins of nearly the same size are found in all L. pneumophila isolates and in a variety of, but not all, the Legionella species analysed here.     

Identification and purification of a cpn60 heat shock protein homolog from Helicobacter pylori.

Helicobacter pylori is associated with gastritis and peptic ulcer disease in humans. We have identified a homolog of the chaperonin cpn60 family of heat shock proteins in H. pylori, referred to as Hp54K. Hp54K, purified from water-extractable H. pylori proteins, migrated as a single band at 54 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its native molecular mass was 740 kDa; thus, Hp54K apparently comprises a 14-mer. The N-terminal 33 residues of Hp54K exhibited 60.6, 57.6, 54.5, 54.5, 51.5, and 51.5% identity with corresponding sequences in the following cpn60 homologs: HtpB (Legionella pneumophila), P1 (human mitochondria), GroEL (Escherichia coli), BA60K (Brucella abortus), HypB (Chlamydia trachomatis), and the 65-kDa immunodominant protein of Mycobacterium bovis BCG, respectively. Hp54K was the only protein recognized in whole-cell preparations of H. pylori by immunoblotting using monospecific antisera against cpn60 homologs from L. pneumophila, E. coli, C. trachomatis, and M. bovis BCG. Antiserum against Hp54K recognized proteins with molecular masses of 50 to 60 kDa in a large number of gram-negative bacteria, consistent with the known highly conserved nature of cpn60 proteins. Hp54K is a major protein and is immunogenic in humans infected with H. pylori. Thus, Hp54K shares many similarities with known cpn60 homologs. On the basis of the proposed role of other cpn60 proteins in induction of chronic inflammation, immune cross-reactivity between Hp54K and gastric tissue may provide an important link between H. pylori infection and gastritis.     

Common epitope on the lipopolysaccharide of Legionella pneumophila recognized by a monoclonal antibody.

Serogroup-specificity of Legionella pneumophila is related to lipopolysaccharide (LPS), and few cross-reactions between serogroups have been observed with rabbit or monkey antisera. C57BL/6 mice were sequentially immunized with crude outer membrane fractions of L. pneumophila serogroups 1, 5, and 7, Legionella bozemanii, and Legionella micdadei. Spleen cells from these mice were then fused with the Sp2-0/Ag14 mouse myeloma cell line. Outer membrane-rich fractions and LPS were prepared from L. pneumophila serogroups 1 to 8 and other Legionella and non-Legionella species. Immunoblots of these extracts were performed with monoclonal antibody obtained from these fusions. One of these monoclonal antibodies recognized an epitope common to all tested serogroups of L. pneumophila and attached to the major constituent of the outer membrane, LPS. This antibody did not react with other Legionella species and numerous gram-negative rods other than Pseudomonas fluorescens CDC93. This monoclonal antibody may be useful in preliminary identification of L. pneumophila as an alternative to direct fluorescent-antibody testing.     

Cloning of the filamentous hemagglutinin of Bordetella pertussis and its expression in Escherichia coli.

Bordetella pertussis UT25 DNA was cloned into the kanamycin resistance gene of cosmid pCP13 to construct a genomic library in Escherichia coli LE392. One clone containing plasmid pDB441 expressed the filamentous hemagglutinin (FHA) as identified by protein immunoblots with the use of rabbit anti-B. pertussis antiserum, rabbit anti-FHA antiserum, and a monoclonal antibody to FHA. FHA is a protein of 220 to 210 kilodaltons, but the immunoreactive FHA, as expressed in E. coli, was larger than that expressed in B. pertussis, suggesting that there was a difference in the processing of this protein between these two bacteria. The fha gene was mapped to a 6.5-kilobase pair DNA fragment by the use of various restriction endonucleases. The kanamycin resistance gene of pCP13 was found to provide the promoter function but probably not the translation start signal for the fha gene. Conjugative transfer of pDB441 to B. pertussis BP353, a transposon Tn5-induced FHA mutant, increased the expression of the FHA over that seen with wild-type B. pertussis.     

Human and guinea pig immune responses to Legionella pneumophila protein antigens OmpS and Hsp60.

We studied the immune responses of guinea pigs and humans to two Legionella pneumophila antigens. Guinea pigs surviving a lethal intraperitoneal challenge dose of virulent L. pneumophila exhibited strong cutaneous delayed-type hypersensitivity (DTH) reactions to purified OmpS (28-kDa major outer membrane protein) and Hsp60 (heat shock protein or common antigen), while weak DTH reactions were noted for extracellular protease (major secretory protein [MSP] [ProA]) and no reaction was observed with an ovalbumin (OA) control. Lymphocyte proliferation responses (LPRs) were measured for peripheral blood and spleen lymphocytes from guinea pigs surviving sublethal and lethal challenge doses of L. pneumophila. Lymphocytes from uninfected animals showed no proliferation to Hsp60 or OmpS, while lymphocytes from sublethally and lethally challenged animals exhibited strong proliferative responses to Hsp60 and OmpS. Guinea pigs vaccinated with purified OmpS exhibited low antibody titers and strong DTH and LPRs to OmpS, whereas lymphocytes from animals vaccinated with Hsp60 exhibited weak DTH responses and high antibody titers to Hsp60. All guinea pigs immunized with OmpS survived experimental challenge with L. pneumophila (two of two in a pilot study and seven of seven in trial 2) versus zero of seven OA-immunized controls (P = 0.006 by Fisher's exact test). In three vaccine trials in which animals were vaccinated with Hsp60, only 1 guinea pig of 15 survived lethal challenge. Peripheral blood lymphocytes (PBLs) from humans with legionellosis showed stronger LPRs to OmpS than PBLs from humans with no history of legionellosis (P = 0.0002 by Mann-Whitney test). PBLs of humans surviving legionellosis exhibited a lower but highly significant proliferative response to Hsp60 (P < 0.0001 compared with controls by Mann-Whitney test). These studies indicate that OmpS and Hsp60 are important antigens associated with the development of protective cellular immunity. However, as determined in vaccine trial studies in the guinea pig model for legionellosis, the species-specific antigen OmpS proved much more effective than the genus-common Hsp60 antigen.