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Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110-1093
Received 25 August 2003/ Returned for modification 10 October 2003/ Accepted 18 November 2003
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ABSTRACT |
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INTRODUCTION |
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Orthologs of HtrA have been found in many gram-positive bacteria, and several have been implicated in virulence (30, 50). However, the specific contribution that HtrA makes to virulence is much less clear. These bacteria lack the outer membrane of the gram-negative bacteria, and as a consequence, lack a periplasmic space. Thus, the folding of secretory proteins does not take place in the confined compartment of the periplasmic space but rather likely occurs at the membrane-cell wall interface following translocation across the cytoplasmic membrane (54, 56). It is known that the peripheral membrane contains some accessory proteins to promote folding, including chaperones and disulfide oxidoreductases (54, 56). However, this peripheral membrane compartment is exposed to the environment and it is not clear that misfolded secreted proteins accumulate at this site, although it is likely that misfolded surface-anchored proteins would accumulate. The fact that the gram-positive orthologs of HtrA are predicted to be peripheral membrane proteins anchored to the membrane by a single transmembrane domain located near their N termini (44, 47) suggests that HtrA may function in protein quality control at this site. In addition, its dual role as a chaperone to promote folding of certain exported proteins in gram-negative bacteria (2, 52) may indicate a more central role in the biogenesis of secreted proteins in gram-positive bacteria. Consistent with this, HtrA has been implicated as the sole extracellular protease responsible for degradation of abnormal exported proteins, for processing secreted proproteins, and for maturation of native proteins in Lactococcus lactis (47).
Secreted proteins play critical roles in the pathogenesis of diseases caused by gram-positive bacteria. For example, culture supernatants of the group A streptococcus (Streptococcus pyogenes) contain at least 16 polypeptides with an identifiable export sequence (33). This bacterium is the causative agent of numerous suppurative infections of the pharynx (e.g., "strep throat") and soft tissues (impetigo, cellulitis, and necrotizing fasciitis), as well as several systemic diseases that can result from toxigenic (scarlet fever and toxic shock syndrome) or immunopathological (rheumatic fever) processes (11). For the most part, the contribution of any secreted factor to the pathogenesis of any disease caused by S. pyogenes is poorly understood. However, it has been reported that depletion of HtrA was shown to diminish the virulence of S. pyogenes in a mouse model of systemic infection (30), suggesting a possible role for HtrA in the biogenesis of secreted virulence factors.
Two important secreted virulence factors of S. pyogenes are the cysteine protease SpeB and the hemolysin streptolysin S (SLS) (18, 24). Both of these factors require extensive processing for the generation of their biologically active forms via pathways that are not well understood (37, 42). The SpeB protease is secreted across the cytoplasmic membrane and folds into an enzymatically inactive zymogen (37), whose subsequent maturation to a proteolytic active form may require at least six intermediate structures generated by sequential cleavages within the zymogen's prodomain (13). While the protease is autocatalytic under certain conditions (37), efficient activation is an intermolecular event (13). The activation pathway is influenced both by environmental factors (37) and by other streptococcal gene products (9, 38, 39). The SLS hemolysin is a predominantly cell-associated small peptide of 
30 amino acids that is posttranslationally processed and likely further modified by a poorly understood pathway that is encoded by a cluster of nine genes in the sag (for streptolysin-associated gene) locus (42). The sequence of the toxin precursor is highly enriched in amino acid residues that are the substrates for thio-ether bond modification found in other cyclic peptide toxins, suggesting that the biogenesis of SLS is similar to that of bacteriocins (42). Consistent with this, several of the genes in the sag locus have similarity to genes required for the synthesis of peptide bacteriocins (42). The roles of extracellular processing factors in the biogenesis of the SpeB protease or SLS have not been well defined.
The aim of the present study was to further investigate the role of HtrA in the pathogenesis of S. pyogenes disease. In particular, the contributions of HtrA to the biogenesis of the highly processed SpeB protease and SLS were examined. As previously reported (30), insertion of a polar element into htrA, the gene encoding HtrA, resulted in a mutant that grew very poorly under normal culture conditions. However, this phenotype was not observed for a nonpolar mutation of htrA, suggesting that the growth phenotype was the result of a polar effect on expression of an adjacent gene. Mutants of HtrA did display altered expression of proteolytic and hemolytic activities to imply a role for HtrA in the activation of the SpeB protease and the SLS hemolysin. Finally, examination of nonpolar HtrA mutants in a murine subcutaneous-infection model revealed that they displayed no significant defect in the ability to cause disease in the subcutaneous tissues.
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MATERIALS AND METHODS |
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(Life Technologies), and experiments with S. pyogenes used strain HSC5 (22). Routine culture of S. pyogenes employed Todd-Hewitt medium (BBL) supplemented with 0.2% yeast extract (Difco) (THY medium) in sealed tubes without agitation. Analyses of protease and hemolytic phenotypes were conducted on cultures grown in C medium (38). To produce solid media, Bacto Agar (Difco) was added to THY medium to a final concentration of 1.4%, and unless otherwise indicated, all solid cultures were incubated under anaerobic conditions produced using a commercial gas generator (GasPack, catalog no. 70304; BBL). When appropriate, antibiotics were added to the medium at the following concentrations: kanamycin, 50 µg/ml for E. coli and 500 µg/ml for S. pyogenes; erythromycin 750 µg/ml for E. coli and 1 µg/ml for S. pyogenes. DNA techniques. Plasmid DNA was isolated by standard techniques and was used to transform E. coli by the method of Kushner (32) and to transform S. pyogenes by electroporation as previously described (5). Restriction endonucleases, ligases, kinases, and polymerases were used according to the manufacturers' recommendations. Chromosomal DNA was purified from S. pyogenes as previously described (5). Fluorescently labeled dideoxynucleotides (Big Dye terminators; PE Applied Biosystems) were used in DNA-sequencing reactions according to the recommendations of the manufacturer for the confirmation of the DNA sequences generated by PCR.
Growth rate comparison. The growth rates of various strains described in this study were determined through the change in optical density at 600 nm (OD600) over time. Cultures were initiated from overnight growth in THY media that had been washed once with an equal volume of phosphate-buffered saline (PBS) (pH 7.4). The initial OD600 of the cultures was adjusted to 0.01, and absorbance values were determined at routine time intervals during incubation.
Insertional inactivation of htrA. For the construction of a polar insertion, a region internal to htrA (open reading frame Spy2Z16 [15]) was amplified using the primers HtrAinternal1 (CACAA CGAAT TCTAC TAAAG CTGTC AAAGC) and HtrAinternal2 (CTGAA TAGCA TCTGC AGAGA CAGTC TCACC). Subsequent insertion of the fragment between the EcoRI and PstI sites of the integrational plasmid pCIV2 using the sites embedded in the primers (underlined) generated pHTR1. For construction of a polar insertion immediately downstream of htrA, a fragment containing the 3' end of htrA and the adjacent chromosome was amplified using the primers HtrAinternal1 and HtrAafterstop (CATAA AACAG TCCTG CAGAC TGTTTT ATGT). This fragment was then inserted into pCIV2 as described above to generate pHTR2. Integration of pHTR1 and pHTR2 into the HSC5 chromosome via homologous recombination produced strains HTR1 and HTR2, respectively. The former contains a polar insertion in htrA, while the latter contains a polar insertion immediately downstream of an unaltered copy of htrA. The chromosomal structures of these mutants were verified through PCR and sequence analyses.
Construction of an in-frame deletion in htrA.
Primers htrA5720PstI (GGTAG GTCTG CAGAT AATTC TTTTG TC) and htrA7100BamHI (AAGGT ATAAG GATCC AAAGT TCTAT AAGC) were used to amplify a fragment containing the entire htrA open reading frame, which was inserted into pTOPO2.1 (Invitrogen) through a TA cloning procedure. The resulting plasmid (pHTR3) was used as a template in an "inside-out" PCR with the primers htrAIFDdown (GACCT GCTCT TGGAA TACAT ATGGT C) and htrAIFDup (GCTTT GACAG CTTTA GTCAT ATGGG TTGTG). Cleavage of the resulting product with NdeI (the sites are underlined), followed by subsequent religation, resulted in an in-frame deletion of the region of htrA that encodes A153-I299. The deletion allele (htrA
153-293) was then inserted between the PstI and BamHI sites of the streptococcal-E. coli shuttle vector pJRS233 using the sites embedded in the original primers (underlined). The resulting plasmid (pHTR10) was then used to replace the wild-type allele of htrA using the method of Ji et al. (28). This method produces a partial duplication with both wild-type and mutant alleles in the chromosome, and the duplication is then resolved to either the wild-type or mutant allele (28). Further analyses were conducted using one isolate that resolved to the mutant allele (HTR10) and a sibling that resolved to the wild-type allele (HTR11). Chromosomal structures were verified by PCR and sequence analysis using primers with the appropriate sequences.
Measurement of protease activity. Expression of the SpeB protease was analyzed in culture supernatants as follows. Cultures in C medium were initiated using cells from overnight growth in C medium, which were washed in PBS (pH 7.4) to remove any residual protease. The initial OD600 of the cultures was adjusted to 0.01; samples were removed at various time points during incubation at 37°C, and cells were removed by filtration (0.45-µm-pore-size Sterile Acrodisc; Gelman Sciences). The resulting supernatant fluids were diluted in fresh C medium to normalize for any differences in growth between samples based upon the OD600 of the culture at the time of harvest. The presence of the proprotein and processed forms of SpeB was determined through Western blot analysis as described previously (38). The proteolytic activities of supernatants were quantified by the method of Hauser et al. (23), which measures the increase in relative fluorescence generated by the proteolytic cleavage of fluorescein isothiocyanate-casein (Sigma). The activity of uninoculated culture medium was used to derive background values that were typically undetectable under the conditions of this assay. To ensure that all proteolytic activity was specifically the result of SpeB, the cysteine protease-specific inhibitor E-64 (final concentration, 10 mM; Sigma) was routinely added to selected samples. This treatment typically reduced activity by >95%.
Measurement of SLS activity. The production of SLS-specific hemolytic activity was determined as follows. An overnight culture of the strain under analysis was diluted to an OD600 of 0.01 in C medium and incubated at 37°C. At the times indicated in the text, aliquots were harvested from the culture, washed once in PBS (pH 7.4), and resuspended in PBS to an OD600 of 0.5. The cell-associated SLS activity was then determined by the method of Ofek et al. (45). Hemolytic activity was represented as the reciprocal of the minimum dilution that contained unlysed erythrocytes.
Murine subcutaneous-infection model. The method of Bunce et al. (4) as modified by others (36, 49) was used to establish an infection of S. pyogenes in the subcutaneous tissues of mice as described in detail elsewhere (3). Mock-infected animals received a subcutaneous injection of saline at a volume equivalent to the volume of the dose of streptococci injected. Ulcer formation was documented by digital photography, and the precise area contained by each ulcer was calculated from the digital record using MetaMorph image analysis software (version 4.6; Universal Imaging Corp.). The difference between the numbers of mice developing an ulcer following subcutaneous challenge with wild-type or mutant bacteria was tested for significance by the chi-square test with Yates' correction (19), and differences in the areas of the resulting ulcers were tested by the Mann-Whitney U test (19). For all test statistics, the null hypothesis was rejected when P was <0.05. The data presented were derived from two independent experiments.
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RESULTS |
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8 days postinfection, the lesion begins to resolve; it is typically fully healed by day 14, and the animals rarely develop systemic infection (3). Weight loss, time to formation of the ulcer, ulcer size, and time to heal the ulcer are the quantitative parameters typically used to evaluate the severity of disease. When the wild-type and deletion mutant were compared, there was no significant difference in (i) the pattern of weight loss (Fig. 6A), (ii) the time to ulcer formation (data not shown), (iii) ulcer size at the time of maximal ulceration caused by the wild-type strain (day 3 [Fig. 6B]) or at any other time point (data not shown), or (iv) the time to heal the ulcer (data not shown). Thus, deletion of htrA is not associated with a significant reduction in virulence in this model of infection.
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DISCUSSION |
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Interestingly, while insertional mutagenesis of htrA in S. pyogenes has been associated with a reduced capacity to cause disease in an animal model of systemic infection (30), the in-frame deletion mutation analyzed in the present study indicated that deletion of htrA did not result in attenuation in the murine model of subcutaneous infection. One possible explanation for this difference is the observation that the deletion mutant did not demonstrate the growth defect at 37°C that was exhibited in the mutants derived by insertional inactivation of htrA. In addition, the growth defect of the insertion mutant did not appear to be due to the loss of htrA itself but to a polar effect. Immediately downstream of htrA is a gene with similarity to spo0J, whose product is associated with chromosome partitioning in Bacillus subtilis (27), and dnaA, whose product is the essential initiator of chromosome replication. Similar to what has been reported for Streptococcus pneumoniae, the htrA locus contains several regions with clusters of binding sites for DnaA (17) (Fig. 2) and likely represents the origin of chromosme replication (17). Thus, it is not surprising that any large insertion into this region may alter the efficiency of cell division. It appears that smaller changes can be tolerated, since in S. pneumoniae, allele replacement of either htrA or spo0J has no noticeable effect on growth, although htrA mutants are less fit in competition with the wild type for nasopharyngeal colonization (50).
While a temperature-sensitive phenotype was not observed for the deletion mutant, it may be premature to conclude that htrA does not contribute to thermal tolerance. Most evidence suggests that htrA acts as a housekeeping protease to degrade unfolded polypeptides during heat shock (46). The wild-type strain used in this study is typical of many strains of S. pyogenes and does not grow at temperatures of >40°C. It is not known whether there is significant accumulation of unfolded polypeptides for the organism at this temperature. In B. subtilis, the contribution of htrA to thermal stress is most clearly observed upon exposure to temperatures that are lethal. In addition, B. subtilis contains two htrA-like genes, and single mutation of either gene leads to a dramatic increase in resistance to stress as a result of compensating upregulation of the other gene (43). While the S. pyogenes genome contains a single copy of htrA, it is possible that mutation results in compensating upregulation of other stress resistance factors. Regulation of htrA expression is not understood in S. pyogenes. In B. subtilis and S. pneumoniae, htrA is regulated by two-component regulatory systems (26, 50), but clear orthologs of these regulators are not apparent in the S. pyogenes genome (W. Lyon and M. Caparon, unpublished data).
Upregulation of a compensating gene may also account for the observation that the htrA deletion mutant was not attenuated. The SLS hemolysin has been identified as an important virulence factor that contributes to ulcer formation in the subcutaneous-infection model (for a review, see reference 41). It is possible that any increase in sensitivity to stress in the mutant is compensated for by the observed hyperproduction of SLS, with the result that the courses of infection in the wild type and the mutant appear to be the same. On the other hand, it is also interesting that a large increase in production of the highly cytolytic SLS does not result in any increased severity of disease. There are multiple steps in the complex biogenesis pathway of SLS that could be influenced by HtrA. The SLS operon is subject to several transcriptional regulatory pathways (for a review, see reference 41), and the activity of these regulatory pathways may be influenced by HtrA. Alternatively, HtrA may degrade components of the SLS biogenesis machinery or even the SLS propeptide itself. It is also possible that HtrA alters the receptor that tethers SLS to the cell surface, making it less accessible to the carrier molecules that are required to solubilize SLS for the determination of hemolytic titers. Regardless of the mechanism, these data suggest that SLS production and stress responses may be linked via HtrA.
In contrast to that of SLS, the role of SpeB protease in the subcutaneous-infection model is much less clear (1), although it is a major virulence factor in a humanized SCID mouse model of streptococcal impetigo (53). Major questions about the biogenesis pathway for the protease are how the nascent protease folds into its zymogen form following its secretion from the cell and how the zymogen's complex activation pathway is initiated (8). The contribution of HtrA may be to make an initial cleavage in the zymogen to begin the activation cascade. However, structural studies of HtrA from E. coli have shown that the protease exists as a hexamer derived from two interlocking trimers. The protease domains are sequestered in a central cavity that is only accessible laterally and likely can act only on polypeptides that are highly unfolded (31). Unlike E. coli, the streptococcal HtrA has only one instead of two adjacent PDZ domains and may have a structure more similar to that of mitochondrial HtrA2. This protease also has a single PDZ domain and forms a trimeric structure in which the protease domains are kept inactive by the PDZ domains, which then are displaced upon binding to the target polypeptide (35). This allows HtrA2 to act on more highly folded substrates, including an ability to activate apoptotic proteases (35). It is also possible that the role of HtrA is to act primarily as a chaperone to promote the folding of the zymogen into an activation-competent conformation, and this may or may not involve the protease activity of HtrA. Further analysis of the contribution of HtrA to the biogenesis of SLS and SpeB will be useful for understanding the production of virulence factors by S. pyogenes and pathways of protein secretion in gram-positive bacteria.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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| 1. | Ashbaugh, C. D., and M. R. Wessels. 2001. Absence of a cysteine protease effect on bacterial virulence in two murine models of human invasive group A streptococcal infection. Infect. Immun. 69:6683-6688. |
| 2. | Bass, S., Q. Gu, and A. Christen. 1996. Multicopy suppressors of prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated R1pA. J. Bacteriol. 178:1154-1161. |
| 3. | Brenot, A., K. Y. King, B. Janowiak, O. Griffith, and M. Caparon. 2004. Contribution of glutathione peroxidase to the virulence of Streptococcus pyogenes. Infect. Immun. 72:408-413. |
| 4. | Bunce, C., L. Wheeler, G. Reed, J. Musser, and N. Barg. 1992. Murine model of cutaneous infection with gram-positive cocci. Infect. Immun. 60:2636-2640. |
| 5. | Caparon, M. G., D. S. Stephens, A. Olsén, and J. R. Scott. 1991. Role of M protein in adherence of group A streptococci. Infect. Immun. 59:1811-1817. |
| 6. | Chaussee, M. S., E. R. Phillips, and J. J. Ferretti. 1997. Temporal production of streptococcal erythrogenic toxin B (streptococcal cysteine proteinase) in response to nutrient depletion. Infect. Immun. 65:1956-1959.[Abstract] |
| 7. | Clausen, T., C. Southan, and M. Ehrmann. 2002. The HtrA family of proteases: implications for protein composition and cell fate. Mol. Cell. 10:443-455.[CrossRef][Medline] |
| 8. | Collin, M., and A. Olsén. 2003. Extracellular enzymes with immunomodulating activities: variations on a theme in Streptococcus pyogenes. Infect. Immun. 71:2983-2992. |
| 9. | Collin, M., and A. Olsén. 2000. Generation of a mature streptococcal cysteine proteinase is dependent on cell wall-anchored M1 protein. Mol. Microbiol. 36:1306-1318.[CrossRef][Medline] |
| 10. | Cortes, G., B. de Astorza, V. J. Benedi, and S. Alberti. 2002. Role of the htrA gene in Klebsiella pneumoniae virulence. Infect. Immun. 70:4772-4776. |
| 11. | Cunningham, M. W. 2000. Pathogenesis of group A streptococcal infections. Clin. Microbiol. Rev. 13:470-511. |
| 12. | Diaz-Torres, M. L., and R. R. Russell. 2001. HtrA protease and processing of extracellular proteins of Streptococcus mutans. FEMS Microbiol. Lett. 204:23-28.[CrossRef][Medline] |
| 13. | Doran, J. D., M. Nomizu, S. Takebe, R. Menard, D. Griffith, and E. Ziomek. 1999. Auotcatalytic processing of the streptococcal cysteine protease zymogen: processing mechanism and characterization of the autoproteolytic cleavage sites. Eur. J. Biochem. 263:145-151.[Medline] |
| 14. | Elzer, P. H., R. W. Phillips, G. T. Robertson, and R. M. Roop II. 1996. The HtrA stress response protease contributes to resistance of Brucella abortus to killing by murine phagocytes. Infect. Immun. 64:4838-4841.[Abstract] |
| 15. | Ferretti, J. J., W. M. McShan, D. Adjic, D. Savic, G. Savic, K. Lyon, C. Primeaux, S. S. Sezate, A. N. Surorov, S. Kenton, H. Lai, S. Lin, Y. Qian, H. G. Jia, F. Z. Najar, Q. Ren, H. Zhu, L. Song, J. White, X. Yuan, S. W. Clifton, B. A. Roe, and R. E. McLaughlin. 2001. Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc. Natl. Acad. Sci. USA 98:4658-4663. |
| 16. | Gajic, O., G. B. Buist, M. Kojic, L. Topisirovic, O. P. Kuipers, and J. Kok. 2003. Novel mechanism of bacteriocin secretion and immunity carried out by lactococcal MDR proteins. J. Biol. Chem. 278:34291-34298. |
| 17. | Gasc, A.-M., P. Biammarinaro, S. Richter, and M. Sicard. 1998. Organization around the dnaA gene of Streptococcus pneumoniae. Microbiology 144:433-439.[Abstract] |
| 18. | Gerlach, D., H. Knoll, W. Kohler, J. H. Ozegowski, and V. Hribalova. 1983. Isolation and characterization of erythrogenic toxins. V. Communication: identity of erythrogenic toxin type B and streptococcal proteinase precursor. Zentbl. Bakteriol. Mikrobiol. Hyg. A 255:221-233. |
| 19. | Glantz, S. 2002. Primer of biostatistics, 5th ed. McGraw-Hill, New York, N.Y. |
| 20. | Gottesman, S. 1996. Proteases and their targets in Escherichia coli. Annu. Rev. Genet. 30:465-506.[CrossRef][Medline] |
| 21. | Gottesman, S., S. Wickner, and M. R. Maurizi. 1997. Protein quality control: triage by chaperones and proteases. Genes Dev. 11:815-823. |
| 22. | Hanski, E., P. A. Horwitz, and M. G. Caparon. 1992. Expression of protein F, the fibronectin-binding protein of Streptococcus pyogenes JRS4, in heterologous streptococcal and enterococcal strains promotes their adherence to respiratory epithelial cells. Infect. Immun. 60:5119-5125. |
| 23. | Hauser, A. R., and P. M. Schlievert. 1990. Nucleotide sequence of the streptococcal pyrogenic exotoxin type B gene and relationship between the toxin and the streptococcal proteinase precursor. J. Bacteriol. 172:4536-4542. |
| 24. | Herbert, D., and E. W. Todd. 1944. The oxygen-stable haemolysin of group A haemolytic streptococci (streptolysin S). Br. J. Exp. Pathol. 25:242-254. |
| 25. | Huang, D. L., T. L. Raivio, C. H. Jones, T. J. Silhavy, and S. J. Hultgren. 2001. Cpx signaling pathway monitors biogenesis and affects assembly and expression of P pili. EMBO J. 20:1508-1518.[CrossRef][Medline] |
| 26. | Hyyryläinen, H.-L., A. Bolhuis, E. Darmon, L. Muukkonen, P. Koski, M. Vitikainen, M. Sarvas, Z. Prágai, S. Bron, J. M. van Dijl, and V. P. Kontinen. 2001. A novel two-component regulatory system in Bacillus subtilis for the survival of severe secretion stress. Mol. Microbiol. 41:1159-1172.[CrossRef][Medline] |
| 27. | Ireton, K., H. W. T. Gunther, and A. D. Grossman. 1994. spo0J is required for normal chromosome segregation as well as the initiation of sporulation in Bacillus subtilis. J. Bacteriol. 176:5320-5329. |
| 28. | Ji, Y., L. McLandsborough, A. Kondagunta, and P. P. Cleary. 1996. C5a peptidase alters clearance and trafficking of group A streptococci by infected mice. Infect. Immun. 64:503-510.[Abstract] |
| 29. | 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] |
| 30. | Jones, C. H., T. C. Bolken, K. F. Jones, G. O. Zeller, and D. E. Hruby. 2001. Conserved DegP protease in gram-positive bacteria is essential for thermal and oxidative tolerance and full virulence in Streptococcus pyogenes. Infect. Immun. 69:5538-5545. |
| 31. | Krojer, T., M. Garrido-Franco, R. Huber, M. Ehrmann, and T. Clausen. 2002. Crystal structure of DegP (HtrA) reveals a new protease-chaperone machine. Nature 416:455-459.[CrossRef][Medline] |
| 32. | Kushner, S. R. 1978. An improved method for transformation of Escherichia coli with ColE1-derived plasmids, p. 17-23. In H. W. Boyer, and S. Micosia (ed.), Genetic engineering. Elsevier-North Holland Biomedical Press, New York, N.Y. |
| 33. | Lei, B., S. Mackie, S. Lukomski, and J. M. Musser. 2000. Identification and immunogenicity of group A Streptococcus culture supernatant proteins. Infect. Immun. 68:6807-6818. |
| 34. | Li, S. R., N. Dorrell, P. H. Everest, G. Dougan, and B. W. Wren. 1996. Construction and characterization of a Yersinia enterocolitica O:8 high-temperature requirement (htrA) isogenic mutant. Infect. Immun. 64:2088-2094.[Abstract] |
| 35. | Li, W., S. M. Srinivasula, J. Chai, P. Li, J.-W. Wu, Z. Zhang, E. S. Alnemri, and Y. Shi. 2002. Structural insights into the pro-apoptotic function of mitochondrial serine protease HtrA2/Omi. Nat. Struct. Biol. 9:436-441.[CrossRef][Medline] |
| 36. | Limbago, B., V. Penumalli, B. Weinrick, and J. R. Scott. 2000. Role of streptolysin O in a mouse model of invasive group A streptococcal disease. Infect. Immun. 68:6384-6390. |
| 37. | Liu, T.-Y., and S. D. Elliot. 1965. Streptococcal protease: the zymogen to enzyme transformation. J. Biol. Chem. 240:1138-1142. |
| 38. | Lyon, W. R., C. M. Gibson, and M. G. Caparon. 1998. A role for Trigger Factor and an Rgg-like regulator in the transcription, secretion and processing of the cysteine proteinase of Streptococcus pyogenes. EMBO J. 17:6263-6275.[CrossRef][Medline] |
| 39. | Lyon, W. R., J. C. Madden, J. C. Levin, J. L. Stein, and M. G. Caparon. 2001. Mutation of luxS affects growth and virulence factor expression in Streptococcus pyogenes. Mol. Microbiol. 42:145-157.[CrossRef][Medline] |
| 40. | Miyoshi, S., and S. Shinoda. 2000. Microbial metalloproteases and pathogenesis. Microbes Infect. 2:91-98.[CrossRef][Medline] |
| 41. | Nizet, V. 2002. Streptococcal beta-hemolysins: genetics and role in disease pathogenesis. Trends Microbiol. 10:575-580.[CrossRef][Medline] |
| 42. | Nizet, V., B. Beall, D. J. Bast, V. Datta, L. Kilburn, D. E. Low, and J. C. De Azavedo. 2000. Genetic locus for streptolysin S production by group A streptococcus. Infect. Immun. 68:4245-4254. |
| 43. | Noone, D., A. Howell, R. Collery, and K. M. Devine. 2001. YkdA and YvtA, HtrA-like serine proteases in Bacillus subtilis, engage in negative autoregulation and reciprocal cross-regulation of ykdA and yvtA gene expression. J. Bacteriol. 183:654-663. |
| 44. | Noone, D., A. Howell, and K. M. Devine. 2000. Expression of ykdA, encoding a Bacillus subtilis homologue of HtrA, is heat shock inducible and negatively autoregulated. J. Bacteriol. 182:1592-1599. |
| 45. | Ofek, I., D. Zafriri, J. Goldhar, and B. I. Eisenstein. 1990. Inability of toxin inhibitors to neutralize enhanced toxicity caused by bacteria adherent to tissue culture cells. Infect. Immun. 58:3737-3742. |
| 46. | Pallen, M. J., and B. W. Wren. 1997. The HtrA family of serine proteases. Mol. Microbiol. 26:209-221.[CrossRef][Medline] |
| 47. | Poquet, I., V. Saint, E. Seznec, N. Simoes, A. Bolotin, and A. Gruss. 2000. HtrA is the unique surface housekeeping protease in Lactococcus lactis and is required for natural protein processing. Mol. Microbiol. 35:1042-1051.[CrossRef][Medline] |
| 48. | Purdy, G. E., M. Hong, and S. M. Payne. 2002. Shigella flexneri DegP facilitates IcsA surface expression and is required for efficient intercellular spread. Infect. Immun. 70:6355-6364. |
| 49. | Schrager, H. M., J. G. Rheinwald, and M. R. Wessels. 1996. Hyaluronic acid capsule and the role of streptococcal entry into keratinocytes in invasive skin infection. J. Clin. Investig. 98:1954-1958.[Medline] |
| 50. | Sebert, M. E., L. M. Palmer, M. Fosenberg, and J. N. Weiser. 2002. Microarray-based identification of htrA, a Streptococcus pneumoniae gene that is regulated by the CiaRH two-component system and contributes to nasopharyngeal colonization. Infect. Immun. 70:4059-4067. |
| 51. | Skorko-Glonek, J., D. Zurawa, E. Kuxzwara, M. Wozniak, and Z. Wypych. 1999. The Escherichia coli heat shock protease HtrA participates in defense against oxidative stress. Mol. Gen. Genet. 262:342-350.[CrossRef][Medline] |
| 52. | 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] |
| 53. | Svensson, M. D., D. A. Scaramuzzino, U. Sjöbring, A. Olsén, C. Frank, and D. E. Bessen. 2000. Role for a secreted cysteine proteinase in the establishment of host tissue tropism by group A streptococci. Mol. Microbiol. 38:242-253.[CrossRef][Medline] |
| 54. | Tjalsma, H., A. Bolhuis, J. D. H. Jongbloed, S. Bron, and J. M. van Dijl. 2000. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome. Microbiol. Mol. Biol. Rev. 64:515-547. |
| 55. | Travis, J., and J. Potempa. 2000. Bacterial proteinases as targets for the development of second-generation antibiotics. Biochem. Biophys. Acta 1477:35-50.[CrossRef][Medline] |
| 56. | van Wely, K. H. M., J. Swaving, R. Fruedl, and A. J. M. Driessen. 2001. Translocation of proteins across the cell envelope of Gram-positive bacteria. FEMS Microbiol. Rev. 25:437-454.[Medline] |
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Km-2 antibiotic resistance determinant insertionally inactivated htrA and produced HTR1. (B) Integration of pHTR2 into the HSC5 chromosome by a single recombination event regenerated htrA and placed the polar 
