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The disulfide bond in the Aeromonas hydrophila lipase/acyltransferase stabilizes the structure but is not required for secretion or activity.

ABSTRACT: Vibrio and Aeromonas spp. secrete an unusual 35-kDa lipase that shares several properties with mammalian lecithin-cholesterol acyltransferase. The Aeromonas hydrophila lipase contains two cysteine residues that form an intramolecular disulfide bridge. Here we show that changing either of the cysteines to serine does not reduce enzymatic activity, indicating that the disulfide bond is not required for correct folding. However, when either of the cysteines is replaced, the enzyme is more readily denatured by urea and more sensitive to degradation by trypsin than is the wild-type enzyme, evidence that the bridge has an important role in stabilizing the protein's structure. The two mutant proteins with serine-for-cysteine replacements were secreted by Aeromonas salmonicida containing the cloned genes, although the levels of both in the culture supernatants were lower than the level of the wild-type enzyme. When the general secretory pathway was blocked with carbonyl cyanide chlorophenylhydrazone, the cell-associated pools of the mutant enzymes appeared to be degraded, whereas the wild-type pool remained stable. We conclude that reduced extracellular levels of the mutant proteins are the result of their increased sensitivities to proteases encountered inside the cell during export.

SUBMITTER: Brumlik MJ

PROVIDER: S-EPMC179086 | biostudies-other | 1997 May

REPOSITORIES: biostudies-other

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The disulfide bond in the Aeromonas hydrophila lipase/acyltransferase stabilizes the structure but is not required for secretion or activity.

Brumlik M J MJ van der Goot F G FG Wong K R KR Buckley J T JT

Journal of bacteriology 19970501 10

Vibrio and Aeromonas spp. secrete an unusual 35-kDa lipase that shares several properties with mammalian lecithin-cholesterol acyltransferase. The Aeromonas hydrophila lipase contains two cysteine residues that form an intramolecular disulfide bridge. Here we show that changing either of the cysteines to serine does not reduce enzymatic activity, indicating that the disulfide bond is not required for correct folding. However, when either of the cysteines is replaced, the enzyme is more readily d ...[more]

PMID: 9150203

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Project description:Aeromonas hydrophila is an important pathogen of fish, and its high-virulence strains display a two-dimensional paracrystalline layer (S-layer) on their outermost surfaces. The nucleotide sequence of a 4.1-kb region located 700 bp upstream of the A. hydrophila TF7 S-layer protein gene (ahsA) has been determined. A sequence analysis of the region revealed the presence of three complete open reading frames ending in a gene encoding a 79.8-kDa polypeptide that shows high homology to the PulD family of secretion proteins. The sequenced region displays both organizational and sequence homology to the Xanthomonas campestris pv. campestris Xps secretory system. Insertional inactivation of the spsD (S-protein secretion D) gene showed that the loss of expression of the PulD homolog coincided with the localization of the S-protein in the periplasm and the loss of the S-layer from the surface of the bacterium. However, the secretion of the enzymes hemolysin, amylase, and protease was unaffected in the mutant with the nonfunctional spsD gene, as was the export of flagella and fimbrial proteins. Southern blot analysis showed that the spsD gene was not conserved among all strains of S-protein-producing A. hydrophila or Aeromonas veronii biotype sobria. Use of the promoterless chloramphenicol acetyltransferase gene showed that unlike pulD and its homologs, spsD contains its own promoter. A. hydrophila has been shown to contain the exe operon, which is responsible for the secretion of a number of extracellular enzymes in this bacterium. A fragment of DNA was generated from the exeD gene of A. hydrophilia Ah65 by PCR and was subsequently used in hybridization studies to probe the chromosome of A. hydrophila TF7. The presence of an exeD homolog in A. hydrophila TF7 was found; therefore, the spsD gene encodes a second pulD homolog that displays a high specificity for the secretion of the S-protein. This gene appears to be part of a second terminal branch of the general secretory pathway in A. hydrophila.

Project description:Many gram-negative bacteria use a type III secretion system (TTSS) to deliver effector proteins into host cells. Here we report the characterization of a TTSS chromosomal operon from the diarrheal isolate SSU of Aeromonas hydrophila. We deleted the gene encoding Aeromonas outer membrane protein B (AopB), which is predicted to be involved in the formation of the TTSS translocon, from wild-type (WT) A. hydrophila as well as from a previously characterized cytotoxic enterotoxin gene (act)-minus strain of A. hydrophila, thus generating aopB and act/aopB isogenic mutants. The act gene encodes a type II-secreted cytotoxic enterotoxin (Act) that has hemolytic, cytotoxic, and enterotoxic activities and induces lethality in a mouse model. These isogenic mutants (aopB, act, and act/aopB) were highly attenuated in their ability to induce cytotoxicity in RAW 264.7 murine macrophages and HT-29 human colonic epithelial cells. The act/aopB mutant demonstrated the greatest reduction in cytotoxicity to cultured cells after 4 h of infection, as measured by the release of lactate dehydrogenase enzyme, and was avirulent in mice, with a 90% survival rate compared to that of animals infected with Act and AopB mutants, which caused 50 to 60% of the animals to die at a dose of three 50% lethal doses. In contrast, WT A. hydrophila killed 100% of the mice within 48 h. The effects of these mutations on cytotoxicity could be complemented with the native genes. Our studies further revealed that the production of lactones, which are involved in quorum sensing (QS), was decreased in the act (32%) and aopB (64%) mutants and was minimal (only 8%) in the act/aopB mutant, compared to that of WT A. hydrophila SSU. The effects of act and aopB gene deletions on lactone production could also be complemented with the native genes, indicating specific effects of Act and the TTSS on lactone production. Although recent studies with other bacteria have indicated TTSS regulation by QS, this is the first report describing a correlation between the TTSS and Act of A. hydrophila and the production of lactones.

Dataset Information

The disulfide bond in the Aeromonas hydrophila lipase/acyltransferase stabilizes the structure but is not required for secretion or activity.

Publications

The disulfide bond in the Aeromonas hydrophila lipase/acyltransferase stabilizes the structure but is not required for secretion or activity.

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