1 Department of Medical Microbiology, University Hospital of North Norway; 2 Department of Microbiology, University of Tromsø, Norway
Received 12 November 2001; returned 21 January 2002; revised 14 June 2002; accepted 21 June 2002
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Most cationic antimicrobial peptides require electrostatic interactions with the outer and/or cytoplasmic membrane to exert their effect. Alterations in lipopolysaccharide (LPS) structure and composition, and thus blocking of the initial interaction between peptide and Gram-negative bacteria, have been extensively studied in several Gram-negative species.36 In staphylococci, alterations in the composition of teichoic acid (TA) and membrane lipids have been reported to play a role in antimicrobial peptide susceptibility.78 Furthermore, antibodies targeted against TA abolish the antimicrobial effect of the antimicrobial peptide lactoferricin B.9 Modifications to other surface molecules, e.g. outer membrane protein C (OmpC) in Escherichia coli,10 and Yersinia adhesin protein (YadA) in Yersinia enterocolitica,11 have also been shown to increase the survival of bacteria exposed to antimicrobial peptides.
Other mechanisms, such as energy-dependent efflux systems, may also confer reduced susceptibility to antimicrobial peptides. They include the mtr system (multiple transferable resistance), conferring decreased susceptibility to protegrin 1 and LL-37 in gonococci,12 and the plasmid-encoded staphylococcal gene qacA, mediating resistance to thrombin-induced platelet microbicidal protein (tPMP-1).13
Proteases are of vital importance to all bacteria. Juretic et al.14 have suggested that one of the bacterial resistance mechanisms against magainins is mediated by proteases present on the microbial surface. Groisman et al.15 report a Salmonella mutant harbouring a mutation in the pepQ locus (encoding an X-Pro peptidase), showing increased susceptibility to magainin 2. Furthermore, outer membrane protein T (OmpT) in E. coli16 and the Salmonella homologue PgtE,17 have been identified as proteases responsible for resistance to antimicrobial peptides.
Lactoferricin B is a 25-amino-acid cationic peptide, derived from the N-terminal part of bovine lactoferrin.18 Lactoferricin B has an antimicrobial effect against fungi,19 protozoa,20,21 viruses,22 and Gram-positive and Gram-negative bacteria.23 Natural resistance to lactoferricin B has been reported for some bacterial species.24,25 Shortened derivatives of lactoferricin B have been shown to have reduced activity compared with the native peptide.26,27 Hence, proteolytic cleavage of the native lactoferricin B may result in reduced activity, i.e resistance. This study was aimed at revealing the possible effect of bacterial proteases on the antibacterial activity of lactoferricin B. The effect of protease inhibitors on the MIC of lactoferricin B was investigated using a reference strain, clinical isolates and protease-deficient strains of E. coli. The susceptibility of a reference strain and clinical isolates of Staphylococcus aureus was also studied.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Lactoferricin B (FKCRRWQWRMKKLGAPSITCVRRAF)28 was prepared by pepsin digestion of bovine lactoferrin by the Centre for Food Technology (Queensland, Australia). The peptide was dissolved in double-distilled sterile water and stored at 20°C.
Determination of MIC
The MIC of the peptide for all strains was determined as described previously24 under the growth conditions indicated below. All strains were tested in parallel and at least three times. The MIC was defined as the lowest peptide concentration at which no visible growth occurred.
Bacterial strains
E. coli ATCC 25922 and S. aureus ATCC 25923 were chosen as reference strains for the study. In addition, 20 clinical isolates of E. coli were collected from urinary samples, and 20 clinical isolates of S. aureus were collected from samples of pus, and used as test strains. All strains were stored at 70°C, and further grown in 2% Bacto Peptone Water (BPW) (Difco, Detroit, MI, USA), pH 6.8 at 37°C. All MIC tests were performed with bacteria in exponential growth, and the bacterial suspensions were adjusted in 2% BPW to give the desired final density.
Protease-deficient E. coli K-12 strains were kindly supplied by Dr Evert Bakker.16 The strains were stored at 70°C, and further grown in brainheart infusion (BHI) medium (Oxoid, Hampshire, UK), pH 7.4 at 37°C. All MIC tests were performed with bacteria in exponential growth, and the bacterial suspensions were adjusted in growth medium to give the desired final density.
Effect of inhibition of bacterial proteases on antibacterial peptide activity
Protease Inhibitor Cocktail Set II (Calbiochem, La Jolla, CA, USA), containing 85 mM Na2-EDTA, 20 mM 4-(2-aminoethyl)-benzenesulphonylfluoride (AEBSF), 1.7 mM bestatin, 200 µM E-64 and 2 mM pepstatin A, was dissolved in 1 mL dimethylsulphoxide (DMSO) and further diluted in 4 mL distilled water as instructed by Calbiochem. Aliquots of 1 mL were transferred to cryo tubes and kept at 20°C until use.
The bacteria were grown to mid-logarithmic phase in 2% BPW or BHI and adjusted to give a density of ~3 x 106 cfu/mL. Aliquots of 50 µL bacteria, 50 µL protease inhibitor cocktail and 50 µL peptide (serial dilutions in sterile water) were added to a 96-well microtitre tray (Nunc, Roskilde, Denmark) and incubated at 37°C for 1824 h. In addition, the protease inhibitors from the cocktail kit were tested individually and in all possible combinations against the reference strains. The most efficient combinations of protease inhibitors were further tested against all the clinical isolates. The MIC was defined as described above.
As a control, a regular MIC determination without protease inhibitors was carried out with each experiment. Also, to exclude any direct effect of protease inhibitors on the bacteria, equal amounts of bacteria in exponential growth and protease inhibitor cocktail in 10-fold dilutions were added to the wells of a microtitre tray, incubated for 1824 h at 37°C, and the inhibitory effect of the protease inhibitors themselves determined. Based on this, the protease cocktail was diluted 1:10 000 in sterile water for use with the staphylococci, and 1:1000 with the E. coli strains. In all experiments involving protease inhibitors, the bacteria were also grown in the presence of protease inhibitors in the appropriate dilution, but in the absence of peptide.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The activity of lactoferricin B was tested against the reference strains of S. aureus and E. coli in addition to 20 clinical isolates of each bacterium. Lactoferricin B showed similar activity against both reference strains with a mean MIC of 19 mg/L for S. aureus and 20 mg/L for E. coli. The clinical isolates of S. aureus were more susceptible than clinical isolates of E. coli overall (Table 1).
|
The MIC of lactoferricin B for the clinical isolates of both E. coli and S. aureus in the presence of the protease inhibitor cocktail was determined. The MIC decreased significantly for all the clinical isolates of both E. coli and S. aureus when the protease inhibitors were present during MIC testing (Figure 1 and Table 1).
|
|
In S. aureus, sodium-EDTA combined with AEBSF gave an MIC of 3 mg/L, improving the MIC six-fold compared with the control without inhibitor (Figure 2). Sodium-EDTA and pepstatin in combination resulted in a MIC of 9.5 mg/L, the same MIC as observed for sodium-EDTA alone. The combination of sodium-EDTA with bestatin or E-64 did not cause a significant reduction in the MIC for S. aureus; nor did any of the other possible combinations of inhibitors (data not shown).
Based on the findings reported above, all sodium-EDTA combinations were tested against the clinical isolates of E. coli (n = 20), and the combination of sodium-EDTA and pepstatin or AEBSF against the clinical isolates of S. aureus (n = 20). All combinations caused the MIC of lactoferricin B to improve significantly for all the clinical isolates (Figure 3). For E. coli, there was no significant difference in the MIC between the combinations irrespective of the inhibitors present. Against S. aureus, there were small but significant differences between the two combinations in some strains, with sodium-EDTA and AEBSF being the most efficient combination in all but one strain.
|
The activity of lactoferricin B was determined against six protease-deficient E. coli K-12 strains.16 The MIC of lactoferricin B in the presence or absence of protease inhibitors was also investigated against the K-12 strains KS 272 and KS 474. The MIC for the native strain decreased significantly in the presence of protease inhibitors (Figure 4). Furthermore, deletions in the degP gene resulted in MICs comparable to that for the native strain in the presence of protease inhibitors. Additional deletions in the ompT and ptr genes did not increase the susceptibility further than deletions in the degP gene alone.
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The metalloprotease inhibitor sodium-EDTA was the only protease inhibitor that, when used alone, significantly increased the activity of lactoferricin B against the reference strains of both S. aureus and E. coli. This suggests that a metalloprotease, or metalloproteases, may be involved in decreased susceptibility to lactoferricin B. Against E. coli, the aminopeptidase inhibitor bestatin and the aspartic protease inhibitor pepstatin caused a minor decrease in MIC when used alone. In combination, these inhibitors acted in a synergic manner and caused a further increase in susceptibility towards lactoferricin B. Thus, metalloproteases may not be the only proteases protecting E. coli against the peptide, but aminopeptidases and aspartic proteases may be involved as well.
Also acting in a synergic manner against E. coli, combinations of sodium-EDTA and bestatin, pepstatin, AEBSF or E-64, resulted in lower MICs of lactoferricin B than the MICs obtained when the inhibitor cocktail or sodium-EDTA alone was used. The sodium-EDTA combinations also significantly improved the MICs for all the clinical isolates and KS 272, indicating that the phenomenon is common to E. coli. In addition to being a metalloprotease inhibitor, sodium-EDTA is known to increase the permeability of the outer membrane in Gram-negative bacteria by replacing divalent cations in the LPS. Hence, the observed effect of sodium-EDTA alone, and the synergy between sodium-EDTA and the other inhibitors tested, may have at least two explanations. First, several different proteases are involved in the inactivation of lactoferricin B, and sodium-EDTA acts as a true metalloprotease alone and in synergy with other proteases. Or secondly, sodium-EDTA increases the permeability of the outer membrane, thereby enhancing the effect of lactoferricin B and/or protease inhibitors by increasing the concentration of the peptide and/or protease inhibitor at its target.
In the LPS-free bacterium S. aureus, sodium-EDTA also improved the MIC of lactoferricin B significantly (Figure 2). Here, it is likely that sodium-EDTA acts as a true metalloprotease inhibitor, inhibiting proteases involved in the inactivation of lactoferricin B. Further, a serine protease (inhibited by AEBSF in combination with sodium-EDTA) and an aspartic protease (inhibited by a combination of sodium-EDTA and pepstatin) may be responsible for reduced susceptibility to lactoferricin B in S. aureus. This is valid for both the reference strain and the clinical isolates (Figures 2 and 3).
Stumpe et al.16 suggest that one of the major functions of extracytoplasmic proteases is to protect the cells against the effects of toxic peptides. Using protease-deficient mutants of E. coli K-12, they have identified the outer membrane-bound protease OmpT as being responsible for decreased susceptibility to the antimicrobial peptide protamine. The OmpT deletion had only minor effects on the MIC of lactoferricin B. However, deletions in the gene encoding the periplasmic, heat shock-induced protease DegP, resulted in increased susceptibility to lactoferricin B.
DegP, also known as protease Do or HtrA, is essential for E. coli to survive at elevated temperatures, and degrades abnormal and misfolded periplasmic proteins.31,32 In vitro, it is known to cleave substrates such as casein, the DNA replication inhibitor IciA and the DNA repair protein Ada.33 DegP is an ATP-dependent endopeptidase in the serine protease family.32 Kolmar et al.34 report the cleavage sites to be ValXaa and IleXaa peptide bonds. Lactoferricin B contains one isoleucine residue at position 18 and a valine residue at position 21, making lactoferricin B a possible substrate for DegP.
However, AEBSF and pepstatin, an in vitro inhibitor and partial inhibitor of DegP, respectively,35 only increased the susceptibility of E. coli to lactoferricin B when used in combination with sodium-EDTA. Sodium-EDTA also enhanced the effect of E-64 and bestatin. Given the two possible actions of sodium-EDTA noted above, sodium-EDTA may permeabilize the outer membrane in E. coli sufficiently to allow the inhibitors and/or the peptide to have easier access to its target. Alternatively, sodium-EDTA, being a metalloprotease inhibitor that does not inhibit DegP in vitro, may inhibit proteases other than DegP involved in the inactivation of lactoferricin B.
As expected, the known DegP inhibitors AEBSF and pepstatin gave no significant reduction in susceptibility to lactoferricin B when tested against the DegP+ strain E. coli KS 272. Further, sodium-EDTA, alone or in the combinations given above, only gave a minor decrease in the MIC of lactoferricin B. This minor increase in susceptibility may be attributable to the permeabilizing effect of sodium-EDTA, allowing the peptide molecules easier access to the antibacterial target. Hence, in DegP+ E. coli strains, sodium-EDTA may exert a permeabilizing effect, act as a true protease inhibitor or a combination of both.
Other non-protease functions of DegP have been reported, including non-destructive protein processing and modulation of signalling pathways.32 Spiess et al.36 have shown that DegP, controlled by a temperature-dependent switch, changes from chaperone to protease activity. It has also been shown that DegP is involved in the virulence of several bacterial species.3740 The increased susceptibility of degP mutants of E. coli to lactoferricin B may thus be a result of non-protease mechanisms rather than the lack of proteolytic inactivation of the peptide. Shifting the temperature to 48°C for up to 60 min does not affect the MIC of lactoferricin B for E. coli KS 272 (H. Ulvatne, Ø. Samuelsen, H. H. Haukland, M. Krämer & L. H. Vorland, unpublished results), further arguing a non-protease mechanism. Mutants lacking this multifunctional protein may be more susceptible to the stress lactoferricin B induces to the cell, resulting in increased susceptibility to the peptide.
The results presented here imply that proteases are responsible for an overall reduction in the activity of lactoferricin B in both E. coli and S. aureus. In E. coli, the periplasmic protein DegP plays a hereto unidentified role in the susceptibility to lactoferricin B. At the present time we have not been able to identify specific proteases responsible for the inactivation of lactoferricin B observed in S. aureus. S. aureus does possess two genes homologous to the degP gene identified in E. coli,41 and DegP may therefore contribute to the decreased susceptibility to lactoferricin B observed here.
![]() |
Acknowledgements |
---|
![]() |
Footnotes |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2 . Groisman, E. A. & Aspedon, A. (1997). The genetic basis of microbial resistance to antimicrobial peptides. Methods in Molecular Biology 78, 20515.[Medline]
3 . Loeffelholz, M. J. & Modrzakowski, M. C. (1986). Plasmid RP1-mediated susceptibility of Acinetobacter calcoaceticus to rat polymorphonuclear leukocyte granule contents. Infection and Immunity 54, 7059.[ISI][Medline]
4
.
Banemann, A., Deppisch, H. & Gross R. (1998). The lipopolysaccharide of Bordetella bronchiseptica acts as a protective shield against antimicrobial peptides. Infection and Immunity 66, 560712.
5
.
McCoy, A. J., Liu, H., Falla, T. J. & Gunn, J. S. (2001). Identification of Proteus mirabilis mutants with increased sensitivity to antimicrobial peptides. Antimicrobial Agents and Chemotherapy 45, 20307.
6
.
Gunn, J. S., Ryan, S. S., van Velkinburgh, J. C., Ernst, R. K. & Miller, S. I. (2000). Genetic and functional analysis of a PmrA-PmrB-regulated locus necessary for lipopolysaccharide modification, antimicrobial peptide resistance, and oral virulence of Salmonella enterica serovar typhimurium. Infection and Immunity 68, 613946.
7
.
Peschel, A., Otto, M., Jack, R. W., Kalbacher, H., Jung, G. & Götz, F. (1999). Inactivation of the dlt operon in Staphylococcus aureus confers sensitivity to defensins, protegrins, and other antimicrobial peptides. Journal of Biological Chemistry 274, 840510.
8
.
Peschel, A., Jack, R. W., Otto, M., Collins, L. V., Staubitz, P., Nicholson, G. et al. (2001). Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on the modification of membrane lipids with L-lysine. Journal of Experimental Medicine 193, 106776.
9 . Vorland, L. H., Ulvatne, H., Rekdal, Ø. & Svendsen, J. S. (1999). Initial binding sites of antimicrobial peptides in Staphylococcus aureus and Escherichia coli. Scandinavian Journal of Infectious Diseases 31, 46773.[ISI][Medline]
10 . Siden, I. & Boman, H. G. (1983). Escherichia coli mutants with an altered sensitivity to cecropin D. Journal of Bacteriology 154, 1706.[ISI][Medline]
11 . Visser, L. G., Hiemstra, P. S., van den Barselaar, M. T., Ballieux, P. A. & van Furth, R. (1996). Role of YadA in resistance to killing of Yersinia enterocolitica by antimicrobial polypeptides of human granulocytes. Infection and Immunity 64, 16538.[Abstract]
12
.
Shafer, W. M., Qu, X. D., Waring, A. J. & Lehrer, R. I. (1998). Modulation of Neisseria gonorrhoeae susceptibility to vertebrate antibacterial peptides due to a member of the resistance/nodulation/division efflux pump family. Proceedings of the National Academy of Sciences, USA 95, 182933.
13
.
Kupferwasser, L. I., Skurray, R. A., Brown, M. H., Firth, N., Yeaman, M. R. & Bayer, A. S. (1999). Plasmid-mediated resistance to thrombin-induced platelet microbicidal protein in staphylococci: role of the qacA locus. Antimicrobial Agents and Chemotherapy 43, 23959.
14 . Juretic, D., Chen, C. H., Brown, J. H., Morell, J. L., Hendler, R. W. & Westerhoff, H. V. (1989). Magainin 2 amide and analogues. Antimicrobial activity, membrane depolarisation and susceptibility to proteolysis. FEBS Letters 249, 21923.[ISI][Medline]
15 . Groisman, E. A., Parra-Lopez, C., Salcedo, M., Lipps, C. J. & Hefron, F. (1992). Resistance to host antimicrobial peptides is necessary for Salmonella virulence. Proceedings of the National Academy of Sciences, USA 89, 1193943.[Abstract]
16
.
Stumpe, S., Scmid, R., Stephens, D. L., Georgiou, G. & Bakker, E. P. (1998). Identification of OmpT as the protease that hydrolyzes the antimicrobial peptide protamine before it enters growing cells of Escherichia coli. Journal of Bacteriology 180, 40026.
17
.
Guina, T., Yi, E. C., Wang, H., Hackett, M. & Miller, S. I. (2000). A PhoP-regulated outer membrane protease of Salmonella enterica serovar typhimurium promotes resistance to -helical antimicrobial peptides. Journal of Bacteriology 182, 407786.
18
.
Tomita, M., Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H. & Kawase, K. (1991). Potent antibacterial peptides generated by pepsin digestion of bovine lactoferrin. Journal of Dairy Science 74, 413742.
19 . Bellamy, W., Wakabayashi, H., Takase, M., Kawase, K., Shimamura, S. & Tomita, M. (1993). Killing of Candida albicans by lactoferricin B, a potent antimicrobial peptide derived from the N-terminal region of bovine lactoferrin. Medical Microbiology and Immunology 182, 97105.[ISI][Medline]
20 . Turchany, J. M., Aley, S. B. & Gillin, F. B. (1995). Giardicidal activity of lactoferrin and N-terminal peptides. Infection and Immunity 63, 45502.[Abstract]
21 . Isamida, T., Tanaka, T., Omata, Y., Yamauchi, K., Shimazaki, K. & Saito, A. (1998). Protective effect of lactoferricin against Toxoplasma gondii infection in mice. Journal of Veterinary Medical Science 60, 2414.[ISI][Medline]
22 . Andersen, J. H., Osbakk, S. A., Vorland, L. H., Traavik, T. & Gutteberg, T. J. (2001). Lactoferrin and cyclic lactoferricin inhibit the entry of human cytomegalovirus into human fibroblasts. Antiviral Research 51, 1419.[ISI][Medline]
23 . Bellamy, W., Takase, M., Wakabayashi, H., Kawase, K. & Tomita, M. (1992). Antibacterial spectrum of lactoferricin B, a potent bactericidal peptide derived from the N-terminal region of bovine lactoferrin. Journal of Applied Bacteriology 73, 4729.[ISI][Medline]
24 . Vorland, L. H., Ulvatne, H., Andersen, J., Haukland, H. H., Rekdal, Ø., Svendsen, J. S. & Gutteberg, T. J. (1998). Lactoferricin of bovine origin is more effective than lactoferricins of human, murine and caprine origin. Scandinavian Journal of Infectious Diseases 30, 5137.[ISI][Medline]
25 . Martinez de Tejada, G., Pizarro-Cerda, J., Moreno, E. & Moriyon, I. (1995). The outer membranes of Brucella spp. are resistant to bactericidal cationic peptides. Infection and Immunity 63, 305461.[Abstract]
26 . Kang, J. H., Lee, M. K., Kim, K. L. & Hahm, K. (1996). Structurebiological activity relationships of 11-residue highly basic peptide segment of bovine lactoferrin. International Journal of Peptide and Protein Research 48, 35763.[ISI][Medline]
27 . Rekdal, Ø., Andersen, J., Vorland, L. H. & Svendsen, J. S. (1999). Construction and synthesis of lactoferricin derivatives with enhanced antibacterial activity. Journal of Peptide Science 5, 3245.[ISI]
28 . Bellamy, W., Takase, M., Yamauchi, K., Wakabayashi, H., Kawase, K. & Tomita, M. (1992). Identification of the bactericidal domain of lactoferrin. Biochimica et Biophysica Acta 1121, 1306.[ISI][Medline]
29 . Meerman, H. J. & Georgiou, G. (1994). Construction and characterisation of a set of E. coli strains deficient in all known loci affecting the proteolytic stability of secreted recombinant proteins. Biotechnology 12, 110710.[ISI][Medline]
30 . Elish, M. E., Pierce, J. R. & Earhart, C. F. (1988). Biochemical analysis of spontaneous fepA mutants of Escherichia coli. Journal of General Microbiology 134, 135564.[ISI][Medline]
31 . Miller, C. G. (1996). Protein degradation and proteolytic modification. In Escherichia coli and Salmonella: Cellular and Molecular Biology, 2nd edn (Neidhardt, F. C. et al., Eds), pp. 93854, ASM Press, Washington, DC, USA.
32 . Pallen, M. J. & Wren, B. W. (1997). The HtrA family of serine proteases. Molecular Microbiology 26, 20921.[ISI][Medline]
33 . Chung, C. H. (1993). Proteases in Escherichia coli. Science 262, 3724.[ISI][Medline]
34 . Kolmar, H., Waller, P. R. H. & Sauer, R. T. (1996). The DegP and DegQ periplasmic endoproteases of Escherichia coli: specificity for cleavage sites and substrate conformation. Journal of Bacteriology 178, 59259.[Abstract]
35 . Lipinska, B., Zylicz, M. & Georgopoulos, C. (1990). The HtrA (DegP) protein, essential for Escherichia coli survival at high temperatures, is an endopeptidase. Journal of Bacteriology 172, 17917.[ISI][Medline]
36 . Spiess, C., Beil. A. & Ehrmann, M. (1999). A temperature-dependent switch from chaperone to protease in a widely conserved heat shock protein. Cell 97, 33947.[ISI][Medline]
37 . Sinha, K., Mastroeni, P., Harrison, J., de Hormaeche, R. D. & Hormaeche, C. E. (1997). Salmonella typhimurium, aroA, htrA, and aroD htrA mutants cause progressive infections in athymic (nu/nu) BALB/c mice. Infection and Immunity 65, 15669.[Abstract]
38 . Elzer, P. H., Phillips, R. W., Robertson, G. R. & Roop, R. M., III (1996). The HtrA stress response protease contributes to resistance of Brucella abortus to killing by murine phagocytes. Infection and Immunity 64, 483841.[Abstract]
39 . Li, S. R., Dorrell, N., Everest, P. H., Dougan, G. & Wren, B. W. (1996). Construction and characterization of a Yersinia enterocolitica O:8 high-temperature requirement (htrA) isogenic mutant. Infection and Immunity 64, 208894.[Abstract]
40 . Williams, K., Oyston, P. C., Dorrell, N., Li, S., Titball, R. W. & Wren, B. W. (2000). Investigation into the role of the serine protease HtrA in Yersinia pestis pathogenesis. FEMS Microbiology Letters 186, 2816.[ISI][Medline]
41
.
Jones, C. H., Bolken, T. C., Jones, K. F., Zeller, G. O. & Hruby, D. E. (2001). Conserved DegP protease in Gram-positive bacteria is essential for thermal and oxidative tolerance and full virulence in Streptococcus pyogenes. Infection and Immunity 69, 553845.