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Infection and Immunity, December 2004, p. 7357-7359, Vol. 72, No. 12
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.12.7357-7359.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Effect of Inactivation of the HtrA-Like Serine Protease DegQ on the Virulence of Salmonella enterica Serovar Typhimurium in Mice

Jacinta Farn{dagger} and Mark Roberts*

Molecular Bacteriology Group, Institute of Comparative Medicine, Department of Veterinary Pathology, Glasgow University Veterinary School, Glasgow, United Kingdom

Received 10 June 2004/ Returned for modification 13 August 2004/ Accepted 19 August 2004


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ABSTRACT
 
DegQ is a serine protease that is highly homologous to HtrA, an important virulence determinant of Salmonella enterica serovar Typhimurium. We examined if DegQ is involved in serovar Typhimurium pathogenesis. A serovar Typhimurium degQ mutant was as virulent as the wild-type strain in mice. However, a serovar Typhimurium htrA degQ mutant survived less well in murine organs, particularly in the liver, than a serovar Typhimurium htrA mutant. DegQ is not essential for serovar Typhimurium pathogenesis but may play a small role during salmonella growth at systemic sites.


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TEXT
 
The serine protease HtrA is involved in the survival of Salmonella enterica serovar Typhimurium within macrophages in vitro and host tissues in vivo (6, 7, 9). HtrA seems to be particularly important for survival of serovar Typhimurium at systemic sites in organs, such as the liver and spleen (4, 6). HtrA is a peripheral membrane protein that is active in the periplasm, and it possesses both protease and chaperone activities (9, 11). The main function of HtrA is believed to be degradation or refolding of damaged proteins that accumulate in the periplasm due to various insults (extracytoplasmic or envelope stress) (9). Expression of the htrA gene is positively controlled by two regulators of the extracytoplasmic stress response, the alternative sigma factor {sigma}E and the two-component regulator CpxAR (2, 3, and unpublished data). The importance of HtrA to serovar Typhimurium in vivo is somewhat surprising, because Salmonella spp. (and many other gram-negative bacteria) possess a second highly homologous protease, DegQ (HhoA), that is also active in the periplasm (9). The main domains of HtrA, the serine protease domain and the two PDZ domains, are highly conserved between HtrA and DegQ (9). Where they has been compared, the substrate specificities of HtrA and DegQ were identical (8).

We wished to see if DegQ plays a role in S. enterica serovar Typhimurium pathogenesis and also if DegQ was important in the absence of HtrA. To this end we inactivated the degQ gene of the wild-type (WT) mouse virulent strain S. enterica serovar Typhimurium SL1344 and an isogenic htrA mutant, BRD915 (1). The complete degQ open reading frame was amplified by PCR. The cloned gene was inactivated by digestion with EcoRV, which removed an internal 420-bp fragment from the center of the gene. A 1,252-bp HincII fragment from pUC4K containing the kanamycin resistance gene (aphI) was blunt-end ligated with EcoRV-digested degQ. The degQ::Kmr construct was cloned into the suicide plasmid pRDH10 and was used to inactivate the gene of strains SL1344 and BRD915 as previously described (6). The genotype of the resulting degQ and htrA degQ strains was confirmed by PCR and Southern blotting, and the phenotype was confirmed by Western blotting with anti-HtrA and anti-DegQ antisera (data not shown).

The colony morphology of both the degQ and htrA degQ mutants appeared normal on solid media, and both mutants grew normally at 37 and 42°C in Luria-Bertani broth (data not shown).

The virulence of the serovar Typhimurium degQ mutant was compared to that of its WT parent. Initially the strains were compared by competition assay. An inoculum containing ~103 CFU of each strain was inoculated intraperitoneally into groups of mice, and 3 days later animals were euthanized. Liver and spleens were removed and homogenized, and the number of CFU of each strain present determined. The competitive index for degQ versus that of WT was ~1.0, indicating that there was no difference in the ability of the two strains to compete in vivo following parenteral challenge. To examine if the degQ mutation impaired the ability of serovar Typhimurium to infect mice via the natural route of infection, groups of mice were inoculated orally with either the WT or degQ strain. There was no difference in the growth kinetics of the two strains in the Peyer's patches (PPs), mesenteric lymph nodes (MLN), liver, and spleen (Fig. 1).



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  		FIG. 1. Comparison of the ability of S. enterica serovar Typhimurium WT and degQ strains to infect mice via the oral route. Groups of adult female BALB/c mice were inoculated orally with either 1.1 x 107 CFU of the WT S. enterica serovar Typhimurium strain or 9.2 x 106 CFU of the serovar Typhimurium degQ mutant. The number of bacteria present in different organs was determined by viable count at time points postchallenge. Each data point represents the mean of three or four mice, and the error bar indicates the standard error.

To determine if the absence of degQ further attenuated an S. enterica serovar Typhimurium htrA mutant, we inoculated the htrA and htrA degQ mutants individually into mice intravenously and monitored their persistence in the liver and spleen (Fig. 2). The htrA degQ double mutant survived less well in both the liver and spleen than the htrA mutant, but the difference in the numbers of the two strains was only statistically different (P < 0.05) in the liver at days 3 and 4. We examined if the addition of the degQ mutation affected the ability of a serovar Typhimurium htrA mutant to colonize murine tissues when administered orally to mice. There was no difference in the persistence of the two mutants in the PPs and MLN (data not shown), but it was not possible to discern any difference in the ability of the mutants to persist in the liver and spleen because there were very few, if any, organisms isolated from these sites at any time point (data not shown). We also found that the degQ mutation did not diminish the ability of a serovar Typhimurium htrA mutant to act as a single-dose live oral vaccine against serovar Typhimurium (data not shown)



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  		FIG. 2. Comparison of the ability of S. enterica serovar Typhimurium htrA and htrA degQ mutants to survive and persist in murine tissues. Groups of adult female BALB/c mice were inoculated intravenously with either 3.7 x 103 CFU of the S. enterica serovar Typhimurium htrA strain or 2.7 x 103 CFU of the serovar Typhimurium htrA degQ mutant. The number of bacteria present in different organs was determined by viable count at periods after challenge. Each data point represents the mean of three mice, and the error bar indicates the standard error.

The DegQ protein is not essential for S. enterica serovar Typhimurium to cause infection, at least in mice. HtrA appears to be more important for serovar Typhimurium to cause systemic infection than to grow in tissues associated with the gut (4, 6, and this study). At systemic sites, the spleen and specifically the liver, it would seem that DegQ can partially compensate for the absence of HtrA. As mentioned, it is not readily apparent why DegQ cannot more efficiently substitute for HtrA in vivo. It may be due to differences in the regulation of degQ and htrA; degQ is constitutively expressed, whereas htrA is positively controlled by regulators that respond to envelope stress and is upregulated within macrophages in vitro and in murine tissues in vivo (5, 9, 10). Alternatively, sequence or structural variation between HtrA and DegQ may determine differences in the function of HtrA and DegQ in vivo. We are presently investigating these different possibilities.


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ACKNOWLEDGMENTS
 
This work was supported by grant PRS12222 from the BBSRC.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Comparative Medicine, Department of Veterinary Pathology, Glasgow University Veterinary School, Bearsden Rd., Glasgow G61 1QH, United Kingdom. Phone: 141 330 5780. Fax: 141 330 5602. E-mail: m.roberts{at}vet.gla.ac.uk. Back

Editor: J. T. Barbieri

{dagger} Present address: Department of Microbiology and Immunology, The University of Melbourne, Parkville, Victoria, Australia 3052. Back


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REFERENCES
 
    1
  1. Chatfield, S. N., K. Strahan, D. Pickard, I. G. Charles, C. E. Hormaeche, and G. Dougan. 1992. Evaluation of Salmonella typhimurium strains harbouring defined mutations in htrA and aroA in the murine salmonellosis model. Microb. Pathog. 12:145-151.[CrossRef][Medline]
    2. 2
  2. Connolly, L., P. De Las, B. M. Alba, and C. A. Gross. 1997. The response to extracytoplasmic stress in Escherichia coli is controlled by partially overlapping pathways. Genes Dev. 11:2012-2021.[Abstract/Free Full Text]
    3. 3
  3. Danese, P. N., W. B. Snyder, C. L. Cosma, L. J. Davis, and T. J. Silhavy. 1995. The Cpx two-component signal transduction pathway of Escherichia coli regulates transcription of the gene specifying the stress-inducible periplasmic protease, DegP. Genes Dev. 9:387-398.[Abstract/Free Full Text]
    4. 4
  4. Dunstan, S. J., C. P. Simmons, and R. A. Strugnell. 1998. Comparison of the abilities of different attenuated Salmonella typhimurium strains to elicit humoral immune responses against a heterologous antigen. Infect. Immun. 66:732-740.[Abstract/Free Full Text]
    5. 5
  5. Everest, P., G. Frankel, J. Li, P. Lund, S. Chatfield, and G. Dougan. 1995. Expression of LacZ from the htrA, nirB and groE promoters in a Salmonella vaccine strain: influence of growth in mammalian cells. FEMS Microbiol. Lett. 126:97-101.[CrossRef][Medline]
    6. 6
  6. Humphreys, S., A. Stevenson, A. Bacon, A. B. Weinhardt, and M. Roberts. 1999. The alternative sigma factor, {sigma}E, is critically important for the virulence of Salmonella typhimurium. Infect. Immun. 67:1560-1568.[Abstract/Free Full Text]
    7. 7
  7. Johnson, K., I. Charles, G. Dougan, D. Pickard, P. O'Gaora, G. Costa, T. Ali, I. Miller, and C. Hormaeche. 1991. The role of a stress-response protein in Salmonella typhimurium virulence. Mol. Microbiol. 5:401-407.[Medline]
    8. 8
  8. Kolmar, H., P. R. Waller, and R. T. Sauer. 1996. The DegP and DegQ periplasmic endoproteases of Escherichia coli: specificity for cleavage sites and substrate conformation. J. Bacteriol. 178:5925-5929.[Abstract/Free Full Text]
    9. 9
  9. Pallen, M. J., and B. W. Wren. 1997. The HtrA family of serine proteases. Mol. Microbiol. 26:209-221.[CrossRef][Medline]
    10. 10
  10. Roberts, M., J. Li, A. Bacon, and S. Chatfield. 1998. Oral vaccination against tetanus: comparison of the immunogenicities of Salmonella strains expressing fragment C from the nirB and htrA promoters. Infect. Immun. 66:3080-3087.[Abstract/Free Full Text]
    11. 11
  11. Spiess, C., A. Beil, and M. Ehrmann. 1999. A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97:339-347.[CrossRef][Medline]

Infection and Immunity, December 2004, p. 7357-7359, Vol. 72, No. 12
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.12.7357-7359.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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