The M Protein Is Dispensable for Maturation of Streptococcal Cysteine Protease SpeB

The streptococcal pyrogenic exotoxin B (SpeB) is an importantvirulence factor of group A streptococci (GAS) with cysteineprotease activity. Maturation of SpeB to a proteolytically activeform was suggested to be dependent on cell-wall-anchored M1protein, the major surface protein of GAS (M. Collin and A.Olsén, Mol. Microbiol. 36:1306-1318, 2000). Collin andOlsén showed that mutant GAS strains expressing truncatedM protein secrete a conformationally different form of unprocessedSpeB with no proteolytic activity. Alternatively, we hypothesizedthat a truncated M protein may interfere with processing ofthis secreted protease, and therefore we tested cysteine proteaseactivity in genetically defined mutant strains that expresseither no M protein or membrane-anchored M protein with an in-framedeletion of the AB repeat region. Measurements of SpeB activityby cleavage of a substrate n-benzoyl-Pro-Phe-Arg-p-nitroanilidehydrochloride showed that the proteolytic activities in culturesupernatants of both mutants were similar to those from thewild-type strain. In addition, Western blot analysis of culturesupernatants showed that SpeB expression and processing to amature form was unaffected by either deletion mutation. Therefore,we conclude that M protein is not required for maturation ofthe streptococcal cysteine protease SpeB.

INTRODUCTION

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Introduction

The gram-positive Streptococcus pyogenes (group A streptococci[GAS]) is widely considered an important human pathogen whichcauses a broad variety of diseases. It is commonly associatedwith mild infections, like tonsillopharyngitis and impetigo,and severe complications, such as necrotizing fasciitis andstreptococcal toxic shock syndrome. The latter have increasedsince the mid 1980s (8, 32, 49). The spectrum of nonsuppurativesequelae includes rheumatic fever, an acute poststreptococcalglomerulonephritis, and, more recently, neuropsychiatric syndromes(39, 48).

GAS produces a variety of surface-bound and secreted virulencefactors which are known to contribute to the severity of theirinfections. Among these, M protein which is anchored to thecell wall by an LPXTG motif (23) plays key roles in bacterialresistance to phagocytosis (28), adherence, invasion of humanepithelial cells (13), and microcolony formation in tonsillartissue (6). The M protein exhibits extensive sequence variationsbetween strains, accounting for over 120 different serotypesof GAS (21). The hypervariable NH2-terminal region of M1 protein,containing unique A and B repeat domains, has been shown tobe important for both adherence and entry into epithelial cells(13). GAS that lack emm genes are cleared faster in animal models(1).

The gene encoding the streptococcal cysteine protease (streptococcalpyrogenic exotoxin B), speB, is present in most isolates ofGAS and is highly conserved among them (52). Expression of SpeBis controlled by multiple transcriptional regulators, includingmga (multiple gene regulator in group A streptococci), ropB(regulation of proteinase), and pel (pleiotropic effect locus)(35, 38, 46). In addition, RopB regulation of SpeB expressionis known to be growth phase dependent (40). The oligopeptideand dipeptide permeases, Opp and Dpp, respectively, were suggestedto influence speB expression, whereas influence of the two-componentsystem, CsrR-CsrS, is still under discussion (22, 25, 43, 44).Trigger factor, a molecular chaperone and peptidyl-prolyl cis-transisomerase, is essential for secretion of the protease and itsmaturation (37, 38). SpeB, like many proteases, is secretedas an inactive precursor of the 40-kDa zymogen form, which isautocatalytically cleaved into an active enzyme of 28 kDa (9).At least six intermediates are produced during this process(18). Recently, analysis of the crystal structure of the zymogenshowed that the catalytic domain of SpeB is structurally similarto the papain superfamily of cysteine proteases but differsin the propeptide region (30). In vitro studies have shown thatSpeB is able to cleave immunoglobulin G (IgG) (11) and to degradeIgA, IgE, IgM, and IgD (10). In addition, SpeB is able to releasebradykinin from its precursor and to activate interleukin-1ß(26, 33). SpeB affects proteins displayed on the streptococcalsurface as well. For example, SpeB cleaves M protein and thestreptococcal C5a peptidase off the bacterial surface (3, 19,47). However, the contribution of SpeB to human infections isstill controversial (12, 15, 24, 27, 31, 50).

Collin and Olsén reported that M1 protein influencesfolding and maturation of SpeB (9). They and we constructedstrains with insertion mutations in emm1 (9, 17). These mutationscaused expression of a truncated M1 protein without a cell wallanchor that is in part secreted into the growth media. Bothstrains had significantly reduced proteolytic activity in culturesupernatants and were unable to generate mature 28-kDa enzymes.They suggested that without M protein the protease assumes anaberrant conformation that blocks the active site and preventsmaturation into an active protease. This potentially importantand novel observation, however, left several unanswered questionswith regard to the role of M protein in protease maturation.

Experiments described below sought to confirm dependence onM1 protein for SpeB maturation and to define M protein segmentsrequired for processing. Genetically defined emm1 mutants thatexpress either no M protein or cell-wall-anchored M1 proteinwith a deletion of the AB repeat region were constructed andinvestigated for their capacity to generate mature and activecysteine protease. We also analyzed the effect of a deletionof emm49 as well as emm-like genes mrp and enn on protease activity.Collin and Olsén's results (9) were replicated usingthe same emm1 insertion mutant, but deletion of the emm1 genehad no effect on protease maturation and activity. Therefore,their interpretation was incorrect: the M protein is not requiredfor maturation of SpeB.

MATERIALS AND METHODS

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Materials and Methods

Bacteria. GAS M1 strain 90-226 was obtained from the World Health OrganizationCenter for Reference and Research on Streptococci at the Universityof Minnesota. It was originally isolated from the blood of apatient with sepsis (14). Strain CS101 is a spontaneous streptomycin-resistantderivative of an opacity-factor-positive M49 strain (29). AllGAS strains and mutants are summarized in Table 1. In addition,Fig. 1 shows the emm1 gene locus and corresponding M proteinsof wild-type and mutant strains. Streptococci were grown at37°C under 5% CO₂ and 20% O₂ atmosphere. For most experiments,Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) supplementedwith 1% yeast extract (THY) was used as the culture medium.For solid media, 1.5% granulated agar (Difco) was added to THY.After transformation, GAS were grown on blood agar plates (BAP)containing 5% sterile sheep blood. Escherichia coli was grownin Luria-Bertani broth (Difco). Antibiotics were used at thefollowing concentrations: erythromycin, 5 µg/ml (streptococci)and 300 µg/ml (E. coli); spectinomycin, 60 µg/ml(streptococci); and kanamycin, 500 µg/ml (streptococci).Strain 90-226 emm1::Km (17), previously shown to produce inactiveSpeB, was used in all experiments as a control to rule out technicaldifferences.

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TABLE 1. Strains used in this study

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FIG. 1. The top graphic shows the emm1 gene locus with corresponding M proteins of 90-226, 90-226 emm1::Km, and 90-226 emm1( ${Delta}$ AB). The bottom graphic shows the mga49 regulon of CS101 and MJY1-3.

Construction of strain 90-226 ${Delta}$ emm1. Plasmid pG⁺host5::mga-sic was constructed by ligation of theDNA fragment mga-sic into pG⁺host5 digested with NotI and XhoI.The DNA fragment mga-sic, containing sequences correspondingto the C-terminal half of the mga gene contiguous to the N-terminalhalf of the sic gene with a deletion of the entire emm1 sequence,was generated by overlap PCR amplification of chromosomal DNAfrom the strain 90-226. Briefly, the mga and sic fragments wereamplified by PCR using oligonucleotide primer pairs MGAF (5'-CCCCGC GGC CGC CTA CGC CTT TTT CAT CAC-3') and MGAR (5'-GTA AGGGCT GCC GCT GTT TTC AGG GTT TAA CTC TAA-3') as well as SICF(5'-TTA GAG TTA AAC CCT GAA AAC AGC GGC AGC CCT TAC-3') andSICR (5'-CCC CCC CTC GAG TCT TCA GGC CAA TCT TCA-3') for mgaand sic, respectively. The amplified DNA fragments, which overlapeach other at one end, were then used as templates for subsequentPCR to generate DNA fragment mga-sic by using primers MGAF andSICR. The resulting DNA fragment was digested with NotI andXhoI (restriction sites are underlined in primer sequences above)and ligated to pG⁺host5. Plasmid pG⁺host5::mga-sic was usedto create the deletion mutant 90-226 ${Delta}$ emm1 by gene replacementas described previously (4). PCR-amplified DNA from one recombinant,including 1.2 kb of mga-sic and the flanking region, was sequencedto confirm deletion of the emm1 gene.

Construction of strain 90-226 emm1( ${Delta}$ AB). Plasmid pG⁺host5::emm1 ${Delta}$ AB, carrying a 486-bp in-frame deletionwithin emm1, was constructed by subcloning a BamHI/HindIII fragmentfrom the plasmid pPE6 (42) into pG⁺host5 after deleting a BglII/BclIfragment. In addition to the deletion, emm1 ${Delta}$ AB has an insertionof a 17-residue ovalbumin epitope. This insertion has no effecton known M-protein functions (42). The deletion recombinant,90-226 emm1( ${Delta}$ AB), was created using this construct by gene replacement(4). The chromosomal AB deletion in the emm1 gene was confirmedby sequencing PCR-amplified DNA from one recombinant. The oligonucleotideprimers used for PCR were complementary to flanking sequencesof the deleted region of emm1. The expression of a truncatedM protein in the mutant strain was confirmed by immunoblottingof cell wall proteins extracted from streptococci (Fig. 2).

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FIG. 2. (a) Expression of an M protein with deletion of the AB repeat on the surface of 90-226 emm1( ${Delta}$ AB). Bacterial surface proteins were extracted as described in Materials and Methods, separated on SDS-12% PAGE, transferred to a polyvinylidene difluoride membrane, and probed with antibody 10B6. Antibody 10B6 is a monoclonal antibody raised against a 15-amino-acid peptide within the C repeats of M protein. Lanes: 1, 90-226; 2, 90-226 emm1( ${Delta}$ AB). (b) M1 protein associated with the surface of 90-226 emm1::Km. Bacterial surface proteins were extracted and electrophoresed through 8% polyacrylamide-SDS gels and were transferred to a nitrocellulose membrane. The membrane was incubated with an anti-M antibody and developed with horseradish peroxidase-conjugated secondary antibody and chromogenic substrate. Anti-M1 serum was raised by immunizing rabbits with an M1 peptide corresponding to the N-terminal amino acids 1 to 23 of the mature M1 protein. Lanes: 1, 90-226; 2, 90-226 emm1( ${Delta}$ AB); 3, 90-226 emm1::Km.

Construction of a 90-226 speB mutant. Plasmid pUCErm1, carrying a speB knockout construct (2, 36),was introduced into 90-226 by transformation. Transformantswere selected on BAP supplemented with erythromycin (5 µg/ml).The plasmid integration into the chromosome was verified byPCR. Culture supernatants of the mutant showed a truncated proteinreactive with anti-SpeB serum in immunoblots, which has no cysteineprotease activity (data not shown).

Phagocytosis assays. Human blood phagocytosis assays were performed as describedpreviously (34). Briefly, streptococcal log-phase cultures werediluted in THY to 5 x 10³ CFU/ml. One-hundred microliters ofbacterial suspension was mixed with 0.9 ml of fresh human bloodor plasma and rotated at 37°C for 3 h. Viable counts weredetermined by plating diluted samples onto BAP.

Protein preparations and antibodies. Surface proteins were extracted using a method described byJi et al. (29). Anti-M1(1-23) serum (13) was raised againsta synthetic peptide, corresponding to the N-terminal amino acidresidues 1 to 23 of M1 protein, and was a gift from J. Dale,University of Tennessee, Memphis. Antibody 10B6 reacts witha 15-amino-acid sequence within the C repeats of M protein andwas kindly provided by V. A. Fischetti, Rockefeller University,New York, N.Y. (23). To prepare supernatant proteins of M1 strains,GAS were grown in THY for 15 h and supernatant was collected.Proteins in supernatants were concentrated fivefold by filtrationusing Centricon YM-10 filters (Millipore, Billerica, Mass.).M49 strains were grown in THY for 15 h, and bacterial cellswere harvested by centrifugation at 4,000 x g for 10 min at4°C. The supernatant was filtered through a 0.22-µm-pore-sizemembrane and concentrated by precipitation with 80% ammoniumsulfate at room temperature. After dialysis, protein was concentratedtwofold using Centriprep YM-10 filters (Millipore). Anti-SpeBserum, as well as purified recombinant SpeB, were kindly providedby P. M. Schlievert, Minneapolis, Minn. (5).

Cysteine protease activity assay. Cysteine protease activity was measured using the assay describedby North (41). Briefly, 50-µl culture supernatants fromM1 strains were incubated with 2 mM dithiothreitol (DTT) for30 min at 37°C to activate the enzyme. Sixty microlitersof 2.5 M n-benzoyl-proline-phenylalanine-arginine-p-nitroanilidehydrochloride (pH 4) (Sigma, St. Louis, Mo.) in distilled waterwas mixed with 90 µl of 0.1 M phosphate buffer (pH 6)and then added to activated culture supernatant. The changein absorbance was determined at an optical density of 405 nm(OD₄₀₅). Assays were performed in triplicate; protein preparationswere tested at least three times. E64 cysteine protease inhibitorwas tested in parallel assays to confirm specificity. To measurecysteine protease activity of M49 strains, 7 µg of concentratedsupernatant protein was activated with 10 mM DTT for 2 h at37°C. Protein concentrations were determined by the bicinchoninicacid method (Pierce Chemical Co., Rockford, Ill.), and densitometricanalysis of the amount of SpeB protein specifically evaluatedband intensities on Western blot membranes by using Scion Image1.62. Cleavage of n-Bz-Pro-Phe-Arg-p-nitroanilide was measuredas described above.

SDS-polyacrylamide gel electrophoresis (PAGE) and Western blot analysis. Sodium dodecyl sulfate (SDS)—8 or 12% polyacrylamide gelswere run using a Bio-Rad Protein Gel kit (Hercules, Calif.).Proteins were either stained with Coomassie brilliant blue ortransferred to a nitrocellulose membrane (Bio-Rad). Membraneswere blocked with 0.5% gelatin for 1 h, washed three times inTris-buffered saline, and incubated for 2 h with the antibodiesdescribed above. After the membranes were washed, horseradishperoxidase-conjugated goat anti-rabbit IgG or rabbit anti-mouseIgG antibodies (Sigma) were added for 2 h. Blots were developedwith 4-chloro-1-naphthol (Bio-Rad).

RESULTS

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Results

Characterization of strains 90-226 ${Delta}$ emm1 and 90-226 emm1( ${Delta}$ AB). To evaluate the effect of complete and partial deletions ofthe emm1 gene on resistance to phagocytosis, survival of bacteriain human blood was measured. After 3 h of incubation in freshhuman blood, viable counts of both mutants 90-226 ${Delta}$ emm1 and 90-226emm1( ${Delta}$ AB) were significantly reduced ( $~$ 50-fold), whereas wild-typestreptococci increased in numbers by 10- to 30-fold. Duringthe same period, growth rates of wild-type and mutant strainsin human plasma were similar. This result confirmed that M proteinplays an important role in resistance to phagocytosis and alsosuggested that this function is in part dependent on the ABsequence of M protein.

Expression of M protein by mutant streptococci was comparedto that of wild-type strains by Western blot analysis, usingthe antibody 10B6 that reacts with a 15-amino-acid sequencewithin the C repeats (23). 90-226 ${Delta}$ emm1 extracts did not bindthe antibody (data not shown), whereas a 31-kDa band was observedin extracts from strain 90-226 emm1( ${Delta}$ AB) (Fig. 2a, lane 2). Blotsof surface protein extracts from 90-226, 90-226 emm1( ${Delta}$ AB), and90-226 emm1::Km were also treated with anti-M1(1-23) serum (13).The extract of 90-226 emm1( ${Delta}$ AB) did not react with this antiserum,confirming the expected deletion (Fig. 2b, lane 2). Extractsfrom 90-226 emm1::Km, the same strain analyzed by Collin andOlsén (9), revealed variable amounts of M1 protein associatedwith the bacterial envelope, even though the truncated proteinwithout cell wall anchor and sorting sequences is expected tobe secreted into the media (Fig. 2b, lane 3). The molecularsize of the band was estimated to be 29 kDa, which is in accordancewith the location of transposon insertion.

SpeB expression in culture supernatants. The migration of SpeB in SDS gels was investigated to evaluatethe maturation of the cysteine protease produced by strainscarrying the deletions of the emm1 gene. Western blot analysisof culture supernatants from cultures of 90-226, 90-226 ${Delta}$ emm1,and 90-226 emm1( ${Delta}$ AB) showed mature enzymes that were primarily28 kDa in size (Fig. 3a). As reported by Collin and Olsén(9), however, extracts of strain 90-226 emm1::Km revealed adifferent pattern of bands; the 40-kDa band predominated, andno mature 28-kDa enzyme was detected in the supernatants ofthis strain (Fig. 3a). Protease folding and cleavage of thepropeptide is accelerated under reducing conditions (20). Therefore,supernatants were incubated with 10 mM DTT for 30 min, but therewas no effect on the size of protease produced by strain 90-226emm1::Km (Fig. 3a, lanes 5 and 6). Hence, Western blot analysisof strain 90-226 and these mutants suggested that cell-wall-anchoredM1 protein was not required for the cleavage process that resultsin an active protease.

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FIG. 3. (a) SpeB in culture supernatants of 90-226 and isogenic mutants. Culture supernatant from bacteria grown in THY overnight was collected and concentrated as described in Materials and Methods. Samples were then separated on SDS-12% PAGE, blotted to a membrane, and probed with antibody against mature cysteine proteinase. Lanes: 1, purified mature SpeB protein; 2, 90-226; 3, 90-226 emm1( ${Delta}$ AB); 4, 90-226 ${Delta}$ emm1; 5, 90-226 emm1::Km; 6, 90-226 emm1::Km (30 min DTT). (b) SpeB in culture supernatants of CS101 and MJY1-3. Culture supernatants from bacteria grown in THY for 15 h were collected and concentrated by precipitation with ammonium sulfate. Samples were then incubated at 37°C for 2 h in the presence or absence of 10 mM DTT, run on SDS-12% PAGE, and probed with antibody against mature cysteine proteinase. Lanes: 1, purified mature SpeB protein; 2, CS101 (no DTT); 3, CS101 (2 h DTT); 4, MJY1-3 (no DTT); 5, MJY1-3 (2 h DTT).

The above results were confirmed using another GAS serotype.Supernatant proteins from the M49 strain CS101 and its isogenicmutant MJY1-3 were analyzed. Strain MJY1-3 lacks the emm49,mrp, and enn genes, thus, no M or M-like proteins are expressedon its surface (29). In both strains the 40-kDa zymogen andseveral intermediates were detected when analyzed without activationby DTT (Fig. 3b, lane 2 and 4). After 2 h of incubation with10 mM DTT, however, CS101 and MJY1-3 extracts contained onlythe 28-kDa band, confirming that zymogen and intermediates wereprocessed to mature enzyme (Fig. 3b, lane 3 and 5). This experimentagain confirms that M protein is not required for maturationof the cysteine protease, SpeB.

SpeB activity in culture supernatants. The above immunoblot experiments suggested that M^– strains90-226 ${Delta}$ emm1 and 90-226 emm1( ${Delta}$ AB) and MJY1-3 should produce enzymaticallyactive SpeB protease activity at levels comparable to that ofthe wild-type culture. Strain 90-226 and its mutants were grownin THY for 15 h, the filtered supernatants were activated withDTT, and then they were incubated with the chromogenic substraten-Bz-Pro-Phe-Arg-p-nitroanilide, as described in Materials andMethods. After 2 and 6 h of incubation, relative amounts ofcleaved substrate were determined spectrophotometrically. Mutantstrains 90-226 ${Delta}$ emm1 and 90-226 emm1( ${Delta}$ AB) produced slightly increasedlevels of protease activity compared to that of the wild type(Fig. 4a, columns 2 and 3). Strain 90-226 emm1::Km, as previouslyreported, failed to cleave the substrate (column 4). Therefore,the implications of the Western blot analyses confirmed thatthe deletion of M1 did not decrease the proteolytic activityof supernatant proteins. This conclusion was also supportedby proteolytic activity assays of supernatant proteins fromstrains CS101 and MJY1-3. Proteolysis assays were performedas described above for M1 strains. After 24 h of incubationwith n-Bz-Pro-Phe-Arg-p-nitroanilide, supernatant proteins fromCS101 and MJY1-3 strains showed similar levels of proteolyticactivity (Fig. 4b). Overall, the amount of protease activityproduced by these strains was much lower than that of M1 strain90-226, requiring longer incubation times to produce measurablecleavage of substrate. Nevertheless, Western immunoblot studieswere again confirmed: deletion of emm49, mrp, and enn did notaffect the activity of SpeB. Taken together, these results showthat maturation of the streptococcal cysteine protease SpeBto an active 28-kDa enzyme does not require M protein.

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FIG. 4. (a) Relative proteolytic activity in culture supernatants of M1 strain 90-226 and isogenic mutants. Culture supernatants were activated with a reducing agent, and proteolytic cleavage of n-Bz-Pro-Phe-Arg-p-nitroanilide was measured as absorbancy at 405 nm after incubation at 37°C for 2 h (left) or 6 h (right). Bars: 1, 90-226; 2, 90-226 ${Delta}$ emm1; 3, 90-226 emm1( ${Delta}$ AB); 4, 90-226 emm1::Km. Column 4 is too small to be visible in the figure. (b) Proteolytic activity of culture supernatant proteins of M49 strain CS101 and isogenic mutant. Culture supernatants were concentrated by precipitation with ammonium sulfate. After dialysis, samples were further concentrated using Centriprep YM-10 and then activated with a reducing agent. Proteolytic cleavage of n-Bz-Pro-Phe-Arg-p-nitroanilide was measured as absorbancy at 405 nm after incubation at 37°C for 6 h (left) or 24 h (right). Bars: 1, CS101; 2, MJY1-3.

DISCUSSION

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Discussion

A variety of activities have been attributed to the streptococcalcysteine protease, or SpeB, which may influence the virulenceof GAS. These included activation of apoptosis, activation ofinterleukin-1 precursor, superantigenicity, and binding of fibronectin.Expression of SpeB is globally regulated and growth phase dependent.SpeB was found to be primarily produced after periods of growthlonger than 8 h (7, 18, 45). Although posttranslational activationof the enzyme has long been known to depend on sulfhydryl reductionof the protein, Collin and Olsén more recently reportedthat in vitro maturation of SpeB expressed by GAS was also dependenton cell-wall-anchored M1 protein (9). They suggested that thezymogen form of SpeB, produced by M^– mutant strains, wasfolded in an aberrant manner (9) which physically blocked theactive site of the protease. Their experiments showed that additionof M protein to growth medium did not influence cleavage ofthe zymogen, but they did not question whether mutation interferedwith processing or activation of other surface-bound or -secretedstreptococcal proteins. Their conclusion that maturation ofSpeB depended on M protein was primarily based on two M^–strains with plasmid insertion mutations in the emm1 gene. Onemutant, strain 90-226 emm1::Km, was constructed in our laboratoryand was, therefore, available to us for further study. We investigatedthe alternative hypothesis that truncated fragments of M1 proteinproduced by insertion mutants could interfere with folding and/orsecretion of the protease.

To test the above hypothesis, we constructed two strains thatcontain complete or partial deletion of emm1. Neither of thesestrains had decreased cysteine protease activity. In fact, theactivity had increased compared to that of the wild type. Westernblot analysis clearly revealed that despite the mutation ofM1 protein, both strains were able to generate a 28-kDa enzyme.We also evaluated the effect of a mutation in an M49 strainMJY1-3 (29) that deleted all M-like surface proteins encodedby emm49, mrp, and enn genes on secretion and maturation ofthe cysteine protease. These experiments confirmed studies withthe serotype M1 strain: deletion of M49 and M-like proteinsdid not affect the translocation or activation of the cysteineprotease. It is very unlikely, therefore, that cell-wall-anchoredM protein influences the maturation of SpeB. It seems plausiblethat truncated M1 protein expressed in insertional mutant strainsdramatically changes the transport and processing machineryof GAS. It could interfere with processes involved in proteinexport across the cell wall, for example, which ultimately inhibita correct secretion of the cysteine protease.

Translocation of secreted proteins across the cytoplasmic membraneand the cell wall for gram-positive bacteria has been studiedextensively in Bacillus subtilis (51). The genome of GAS revealsthe sequence of proteins with significant homologies to SecAand SecY proteins, two essential components of the cytoplasmicmembrane translocation apparatus, as well as the signal peptidaseSipS. Thus, it seems likely that secretion mechanisms in GASare similar to those in bacilli. We postulate that secretedmalformed M protein interacts with this apparatus to disruptappropriate folding of secreted proteins. We observed variousamounts of truncated M protein in cell surface extracts fromstrain 90-226 emm1::Km, one of the strains examined previously(9). The nature of this interaction is unknown, but it is likelythat the truncated M1 protein is trapped either in the membraneor the peptidoglycan cell wall. Dieye et al. (16) reported asimilar phenomenon for Lactococcus lactis. Approximately 50%of a mutated protein was found associated with the cell wall,rather than being secreted into the growth media as expected.The interaction between the secreted molecule and the cell wallwas suggested to involve electrostatic interactions (16). Collinand Olsén analyzed two different M^– strains withplasmid insertions in the emm1 gene (9). Both failed to produceproteolytically active SpeB. During translocation, M1 couldaccumulate in a similar way and, for that reason, interferewith the secretion of other proteins.

In conclusion, we reproduced Collin and Olsén's results(9) that insertion mutations in the emm1 gene impede maturationof SpeB into an active protease. However, complete or partialdeletion of M1 protein did not negatively affect protease activityor prevent conformational changes required for autocleavageof the 40-kDa zymogen into the smaller 28-kDa mature enzyme.Therefore, the results of Collin and Olsén were correct,but their interpretation failed to consider the potential negativeeffect of M-protein fragments produced by the mutants they studied.We conclude that M protein is not required for secretion, folding,or autocatalytic activation of the cysteine protease.

ACKNOWLEDGMENTS

This work was supported by a grant from the Cusanuswerk, Germany,and by National Institutes of Health grant AI34503.

We thank Myrna Rezcallah and Bernd Kreikemeyer for helpful discussionand Tim Leonard for preparing the figures.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology, University of Minnesota Medical School, 1460 Mayo Bldg., MMC196, 420 Delaware St. SE, Minneapolis, MN 55455. Phone: (612) 624-3932. Fax: (612) 626-0623. E-mail: cleary{at}mail.ahc.umn.edu.

Editor: V. J. DiRita

REFERENCES

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