title: Salmonella enterica Serovar Typhimurium HtrA: regulation of expression and role of the chaperone and protease activities during infection.
abstract: HtrA is a bifunctional stress protein required by many bacterial pathogens to successfully cause infection. Salmonella enterica serovar Typhimurium (S. Typhimurium) htrA mutants are defective in intramacrophage survival and are highly attenuated in mice. Transcription of htrA in Escherichia coli is governed by a single promoter that is dependent on sigma(E) (RpoE). S. Typhimurium htrA also possesses a sigma(E)-dependent promoter; however, we found that the absence of sigma(E) had little effect on production of HtrA by S. Typhimurium. This suggests that additional promoters control expression of htrA in S. Typhimurium. We identified three S. Typhimurium htrA promoters. Only the most proximal promoter, htrAp3, was sigma(E) dependent. The other promoters, htrAp1 and htrAp2, are probably recognized by the principal sigma factor sigma(70). These two promoters were constitutively expressed but were also slightly induced by heat shock. Thus expression of htrA is different in S. Typhimurium and E. coli. The role of HtrA is to deal with misfolded/damaged proteins in the periplasm. It can do this either by degrading (protease activity) or folding/capturing (chaperone/sequestering, C/S, activity) the aberrant protein. We investigated which of these functions are important to S. Typhimurium in vitro and in vivo. Point or deletion mutants of htrA that encode variant HtrA molecules have been used in previous studies to investigate the role of different regions of HtrA in C/S and protease activity. These htrA variants were placed under the control of the S. Typhimurium htrAP123 promoters and expressed in a S. Typhimurium htrA mutant, GVB1343. Both wild-type HtrA and HtrA (HtrA S210A) lacking protease activity enabled GVB1343 to grow at high temperature (46 degrees C). Both molecules also significantly enhanced the growth/survival of GVB1343 in the liver and spleen of mice during infection. However, expression of wild-type HtrA enabled GVB1343 to grow to much higher levels than expression of HtrA S210A. Thus both the protease and C/S functions of HtrA operate in vivo during infection but the protease function is probably more important. Absence of either PDZ domain completely abolished the ability of HtrA to complement the growth defects of GVB1343 in vitro or in vivo.
authors.affiliation: Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK.
authors.forename: Claire
authors.initials: C
authors.lastname: Lewis
authors.affiliation: Institute of Molecular Biology, Slovak Academy of Science, Dubravska cesta 21, 845 51 Bratislava, Slovak Republik.
authors.forename: Henrieta
authors.initials: H
authors.lastname: Skovierova
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
authors.forename: Gary
authors.initials: G
authors.lastname: Rowley
authors.affiliation: Institute of Molecular Biology, Slovak Academy of Science, Dubravska cesta 21, 845 51 Bratislava, Slovak Republik.
authors.forename: Bronislava
authors.initials: B
authors.lastname: Rezuchova
authors.affiliation: Institute of Molecular Biology, Slovak Academy of Science, Dubravska cesta 21, 845 51 Bratislava, Slovak Republik.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Homerova
authors.affiliation: Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK.
authors.forename: Andrew
authors.initials: A
authors.lastname: Stevenson
authors.affiliation: Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK.
authors.forename: Janice
authors.initials: J
authors.lastname: Spencer
authors.affiliation: Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK.
authors.forename: Jacinta
authors.initials: J
authors.lastname: Farn
authors.affiliation: Institute of Molecular Biology, Slovak Academy of Science, Dubravska cesta 21, 845 51 Bratislava, Slovak Republik.
authors.forename: Jan
authors.initials: J
authors.lastname: Kormanec
authors.affiliation: Institute of Comparative Medicine, Faculty of Veterinary Medicine, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK.
authors.forename: Mark
authors.initials: M
authors.lastname: Roberts
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D051379:Mice; D008807:Mice, Inbred BALB C; D018832:Molecular Chaperones / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000235:genetics; D012480:Salmonella Infections / Q000382:microbiology; D012486:Salmonella typhimurium / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D012808:Sigma Factor / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D012329:RNA, Bacterial; D012808:Sigma Factor; C063202:sporulation-specific sigma factors; D010447:Peptide Hydrolases