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Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH-AopB-AopD complexes in the type III secretion system of Aeromonas hydrophila.

ABSTRACT: In the type III secretion system (T3SS) of Aeromonas hydrophila, AcrH acts as a chaperone for translocators AopB and AopD. AcrH forms a stable 1:1 monomeric complex with AopD, whereas the 1:1 AcrH-AopB complex exists mainly as a metastable oligomeric form and only in minor amounts as a stable monomeric form. Limited protease digestion shows that these complexes contain highly exposed regions, thus allowing mapping of intact functional chaperone binding regions of AopB and AopD. AopD uses the transmembrane domain (DF1, residues 16-147) and the C-terminal amphipathic helical domain (DF2, residues 242-296) whereas AopB uses a discrete region containing the transmembrane domain and the putative N-terminal coiled coil domain (BF1, residues 33-264). Oligomerization of the AcrH-AopB complex is mainly through the C-terminal coiled coil domain of AopB, which is dispensable for chaperone binding. The three proteins, AcrH, AopB, and AopD, can be coexpressed to form an oligomeric and metastable complex. These three proteins are also oligomerized mainly through the C-terminal domain of AopB. Formation of such an oligomeric and metastable complex may be important for the proper formation of translocon of correct topology and stoichiometry on the host membrane.

SUBMITTER: Tan YW

PROVIDER: S-EPMC2776960 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH-AopB-AopD complexes in the type III secretion system of Aeromonas hydrophila.

Tan Yih Wan YW Yu Hong Bing HB Sivaraman J J Leung Ka Yin KY Mok Yu-Keung YK

Protein science : a publication of the Protein Society 20090801 8

In the type III secretion system (T3SS) of Aeromonas hydrophila, AcrH acts as a chaperone for translocators AopB and AopD. AcrH forms a stable 1:1 monomeric complex with AopD, whereas the 1:1 AcrH-AopB complex exists mainly as a metastable oligomeric form and only in minor amounts as a stable monomeric form. Limited protease digestion shows that these complexes contain highly exposed regions, thus allowing mapping of intact functional chaperone binding regions of AopB and AopD. AopD uses the tra ...[more]

PMID: 19530229

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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

Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH-AopB-AopD complexes in the type III secretion system of Aeromonas hydrophila.

Publications

Mapping of the chaperone AcrH binding regions of translocators AopB and AopD and characterization of oligomeric and metastable AcrH-AopB-AopD complexes in the type III secretion system of Aeromonas hydrophila.

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