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

ABSTRACT: 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.

SUBMITTER: Hoffman PS

PROVIDER: S-EPMC313664 | biostudies-other | 1990 Oct

REPOSITORIES: biostudies-other

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

Hoffman P S PS Houston L L Butler C A CA

Infection and immunity 19901001 10

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 acid ...[more]

PMID: 2205580

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Project description:BACKGROUND:Heat shock is a potential control strategy for Legionella pneumophila in hot water plumbing systems. However, it is not consistently effective, with little understanding of its influence on the broader plumbing microbiome. Here, we employed a lab-scale recirculating hot water plumbing rig to compare the pre- and post-"heat shock" (i.e., 40???60???40 °C) microbiota at distal taps. In addition, we used a second plumbing rig to represent a well-managed system at 60 °C and conducted a "control" sampling at 60 °C, subsequently reducing the temperature to 40 °C to observe the effects on Legionella and the microbiota under a simulated "thermal disruption" scenario. RESULTS:According to 16S rRNA gene amplicon sequencing, in the heat shock scenario, there was no significant difference or statistically significant, but small, difference in the microbial community composition at the distal taps pre- versus post-heat shock (both biofilm and water; weighted and unweighted UniFrac distance matrices). While heat shock did lead to decreased total bacteria numbers at distal taps, it did not measurably alter the richness or evenness of the microbiota. Quantitative PCR measurements demonstrated that L. pneumophila relative abundance at distal taps also was not significantly different at 2-month post-heat shock relative to the pre-heat shock condition, while relative abundance of Vermamoeba vermiformis, a known Legionella host, did increase. In the thermal disruption scenario, relative abundance of planktonic L. pneumophila (quantitative PCR data) increased to levels comparable to those observed in the heat shock scenario within 2 months of switching long-term operation at 60 to 40 °C. Overall, water use frequency and water heater temperature set point exhibited a stronger effect than one-time heat shock on the microbial composition and Legionella levels at distal taps. CONCLUSIONS:While heat shock may be effective for instantaneous Legionella control and reduction in total bacteria numbers, water heater temperature set point and water use frequency are more promising factors for long-term Legionella and microbial community control, illustrating the importance of maintaining consistent elevated temperatures in the system relative to short-term heat shock.

Project description:The eukaryotic protein synthesis inhibitor cycloheximid has been used by many investigators to selectively radiolabel intracellular bacteria. Although cycloheximide has no direct effect on bacterial gene expression, there are concerns that long-term inhibition of the host cell protein synthesis may have secondary effects on bacterial gene expression. Therefore, prior to further identification and cloning of the macrophage-induced (MI) genes of Legionella pneumophila, the effects of cycloheximide on L. pneumophila-infected U937 cells were evaluated by transmission electron microscopy. Inhibition of protein synthesis of the host cell for 6 h had no major effect on the ultrastructure of the host cell, on the formation of rough endoplasmic reticulum-surrounded replicative phagosome, or on initiation of intracellular bacterial replication. In contrast, by 15 h of cycloheximide treatment, there was profound deterioration in the host cell as well as in the phagosome. To examine protein synthesis by L. pneumophila during the intracellular infection, U937 macrophage-like cells were infected with L. pneumophila, and intracellular bacteria were radiolabeled during a 2-h cycloheximide treatment or following 12 h of cycloheximide treatment. Comparison by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the protein profile of radiolabeled in vitro-grown L. pneumophila to that of intracellularly radiolabeled bacteria showed that 23 proteins were induced in response to the intracellular environment during 2 h of inhibition of host cell protein biosynthesis. Twelve MI proteins of L. pneumophila were artifactually induced due to prolonged inhibition of the host cell protein synthesis. The gene encoding a 20-kDa MI protein was cloned by a reverse genetics technique. Sequence analysis showed that the cloned gene encoded a protein that was 80% similar to the enzyme inorganic pyrophosphatase. Studies of promoter fusion to a promoterless lacZ gene showed that compared to in vitro-grown bacteria, expression of the pyrophosphatase gene (ppa) was induced fourfold throughout the intracellular infection. There was no detectable induction in transcription of the ppa promoter during exposure to stress stimuli in vitro. The ppa gene of L. pneumophila is the first example of a regulated ppa gene which is selectively induced during intracellular infection and which may reflect enhanced capabilities of macromolecular biosynthesis by intracellular L. pneumophila. The data indicate caution in the long-term use of inhibition of host cell protein synthesis to selectively examine gene expression by intracellular bacteria.

Dataset Information

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

Publications

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

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