Purification, partial characterization, and seroreactivity of a genuswide 60-kilodalton Legionella protein antigen

Pau, Chou‐Pong; Plikaytis, Bonnie B.; Carlone, George M.; Warner, Isiah M.

doi:10.1128/jcm.26.1.67-71.1988

Cited by 14 publications

(9 citation statements)

References 12 publications

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“…The deduced amino acid sequence was 547 amino acids long with a computed molecular mass of 57,952 daltons. This compares favorably with the previously reported molecular masses of 58 and 60 kDa (determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [19,20]) and 63 kDa (chromatographically determined [18]). Hydrophobicity analysis as described by Hopp and Woods (12) revealed no significant areas of hydrophobicity within the sequence.…”

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confidence: 87%

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

et al. 1990

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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.

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confidence: 87%

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

et al. 1990

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“…Escherichia coli PSH16 (strain JM109 harboring the L. pneumophila htpAB operon in pUC19), as well as its growth and overexpression of Hsp60, has been previously described (30). Recombinant Hsp60 was purified from a crude lysate of PSH16 by ammonium sulfate precipitation and ion-exchange chromatography in a DE-52 (Whatman) column (44,57), followed by dialysis, concentration by ultrafiltration (Amicon), and sterilization by filtration through a 0.2-m-pore-size membrane (Nalgene). Purity of the preparation was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), Coomassie blue R-250 staining, and immunoblotting as outlined previously (21).…”

Section: Methodsmentioning

confidence: 99%

Surface-Associated Hsp60 Chaperonin of Legionella pneumophila Mediates Invasion in a HeLa Cell Model

1998

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HeLa cells have been previously used to demonstrate that virulent strains of Legionella pneumophila (but not salt-tolerant avirulent strains) efficiently invade nonphagocytic cells. Hsp60, a member of the GroEL family of chaperonins, is displayed on the surface of virulent L. pneumophila (R. A. Garduño et al., J. Bacteriol. 180:505–513, 1988). Because Hsp60 is largely involved in protein-protein interactions, we investigated its role in adherence-invasion in the HeLa cell model. Hsp60-specific antibodies inhibited the adherence and invasiveness of two virulent L. pneumophila strains in a dose-dependent manner but had no effect on the association of their salt-tolerant avirulent derivatives with HeLa cells. A monospecific anti-OmpS (major outer membrane protein) serum inhibited the association of both virulent and avirulent strains of L. pneumophila to HeLa cells, suggesting that while both Hsp60 and OmpS may mediate bacterial association to HeLa cells, only virulent strains selectively displayed Hsp60 on their surfaces. Furthermore, the surface-associated Hsp60 of virulent bacterial cells was susceptible to the action of trypsin, which rendered the bacteria noninvasive. Additionally, pretreatment of HeLa cells with purified Hsp60 or precoating of the plastic surface where HeLa cells attached with Hsp60 reduced the adherence and invasiveness of the two virulent strains. Finally, recombinant Hsp60 covalently bound to latex beads promoted the early association of beads with HeLa cells by a factor of 20 over bovine serum albumin (BSA)-coated beads and competed with virulent strains for association with HeLa cells. Hsp60-coated beads were internalized in large numbers by HeLa cells and remained in tight endosomes that did not fuse with other vesicles, whereas internalized BSA-coated beads, for which endocytic trafficking is well established, resided in more loose or elongated endosomes. Mature intracellular forms of L. pneumophila, which were up to 100-fold more efficient than agar-grown bacteria at associating with HeLa cells, were enriched for Hsp60 on the bacterial surface, as determined by immunolocalization techniques. Collectively, these results establish a role for surface-exposed Hsp60 in invasion of HeLa cells by L. pneumophila.

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“…SDS-PAGE and immunobloting. Recombinant Hsp60 was purified from PSH16 by a combination of (NH 4 ) 2 SO 4 precipitation and ion exchange chromatography as described previously (39,55). Approximately 30 g of purified Hsp60, or cell pellets of L. pneumophila 2064 or E. coli PSH16 (from 1 ml of a suspension with an OD 620 of 1.0), was solubilized in 100 l of sample buffer, and 30 l per lane was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (32) in a 7.5 to 15% (wt/vol) polyacrylamide gradient gel.…”

Section: Methodsmentioning

confidence: 99%

Immunolocalization of Hsp60 in Legionella pneumophila

et al. 1998

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One of the most abundant proteins synthesized by Legionella pneumophila, particularly during growth in a variety of eukaryotic host cells, is Hsp60, a member of the GroEL family of molecular chaperones. The present study was initiated in response to a growing number of reports suggesting that for some bacteria, includingL. pneumophila, Hsp60 may exist in extracytoplasmic locations. Immunolocalization techniques with Hsp60-specific monoclonal and polyclonal antibodies were used to define the subcellular location and distribution of Hsp60 in L. pneumophila grown in vitro, or in vivo inside of HeLa cells. For comparative purposesEscherichia coli, expressing recombinant L. pneumophila Hsp60, was employed. In contrast to E. coli, where Hsp60 was localized exclusively in the cytoplasm, inL. pneumophila Hsp60 was predominantly associated with the cell envelope, conforming to a distribution pattern typical of surface molecules that included the major outer membrane protein OmpS and lipopolysaccharide. Interestingly, heat-shocked L. pneumophila organisms exhibited decreased overall levels of cell-associated Hsp60 epitopes and increased relative levels of surface epitopes, suggesting that Hsp60 was released by stressed bacteria. Putative secretion of Hsp60 by L. pneumophila was further indicated by the accumulation of Hsp60 in the endosomal space, between replicating intracellular bacteria. These results are consistent with an extracytoplasmic location for Hsp60 in L. pneumophilaand further suggest both the existence of a novel secretion mechanism (not present in E. coli) and a potential role in pathogenesis.

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Purification, partial characterization, and seroreactivity of a genuswide 60-kilodalton Legionella protein antigen

Cited by 14 publications

References 12 publications

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

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

Surface-Associated Hsp60 Chaperonin of Legionella pneumophila Mediates Invasion in a HeLa Cell Model

Immunolocalization of Hsp60 in Legionella pneumophila

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