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Streptococcus pyogenes malate degradationpathway links pH regulation and virulenceElyse PaluscioWashington University School of Medicine in St. Louis

Michael G. CaparonWashington University School of Medicine in St. Louis

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Recommended CitationPaluscio, Elyse and Caparon, Michael G., ,"Streptococcus pyogenes malate degradation pathway links pH regulation and virulence."Infection and Immunity.83,3. 1162-1171. (2015).http://digitalcommons.wustl.edu/open_access_pubs/3725

Streptococcus pyogenes Malate Degradation Pathway Links pHRegulation and Virulence

Elyse Paluscio, Michael G. Caparon

Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri, USA

The ability of Streptococcus pyogenes to infect different niches within its human host most likely relies on its ability to utilizealternative carbon sources. In examining this question, we discovered that all sequenced S. pyogenes strains possess the genes forthe malic enzyme (ME) pathway, which allows malate to be used as a supplemental carbon source for growth. ME is comprised offour genes in two adjacent operons, with the regulatory two-component MaeKR required for expression of genes encoding amalate permease (maeP) and malic enzyme (maeE). Analysis of transcription indicated that expression of maeP and maeE is in-duced by both malate and low pH, and induction in response to both cues is dependent on the MaeK sensor kinase. Further-more, both maePE and maeKR are repressed by glucose, which occurs via a CcpA-independent mechanism. Additionally, malateutilization requires the PTS transporter EI enzyme (PtsI), as a PtsI– mutant fails to express the ME genes and is unable to utilizemalate. Virulence of selected ME mutants was assessed in a murine model of soft tissue infection. MaeP–, MaeK–, and MaeR– mu-tants were attenuated for virulence, whereas a MaeE– mutant showed enhanced virulence compared to that of the wild type.Taken together, these data show that ME contributes to S. pyogenes’ carbon source repertory, that malate utilization is a highlyregulated process, and that a single regulator controls ME expression in response to diverse signals. Furthermore, malate uptakeand utilization contribute to the adaptive pH response, and ME can influence the outcome of infection.

Although it has a relatively small genome (approximately 1.8Mbp), the pathogenic Gram-positive bacterium Streptococcus

pyogenes has a remarkable ability to adapt to a variety of humantissues. This trait allows it to cause numerous diseases, rangingfrom superficial and self-limiting infections in soft tissues likethe skin (impetigo) and pharynx (pharyngitis) to more prob-lematic infections at a number of diverse anatomical sites (1).Understanding the complex regulatory interactions that allowit to adapt to these diverse environments provides a uniqueopportunity to gain insight into how a pathogen can efficientlyemploy a relatively limited genetic repertory to maximize itsability to cause disease.

An important question is how S. pyogenes uses its limited met-abolic potential to grow efficiently in diverse tissues. Considerableevidence has accrued to suggest that the patterns by which S. pyo-genes exploits available growth substrates are intimately associatedwith both temporal and compartment-specific patterns of viru-lence gene expression (2–4). As a lactic acid bacterium, S. pyogenesrelies exclusively on fermentation via the homolactic and mixed-acid pathways to generate energy (5–7). However, the specific car-bon sources it preferentially utilizes in different tissues, the tem-poral patterns with which these are consumed, and how thesepatterns impact regulation of virulence gene expression are notwell understood.

One approach to gain insight into conditions encounteredduring infection has involved comparison of the S. pyogenes tran-scriptome between organisms recovered from various models ofinfection to organisms cultured under different in vitro condi-tions. In general, these studies have revealed that at the latter timepoints of infection, patterns of gene expression most closely re-semble those observed in vitro in environments of low pH (pH 6.0to 6.5) and low concentrations of glucose (8–10). These two con-ditions are likely related, as the fermentation of glucose by lacticacid bacteria results in the highest rates of production of acidic endproducts, including lactate, acetate, and formate (6, 7). This sug-

gests that S. pyogenes’ choice of carbon source results in a signifi-cant remodeling of its local tissue environment. It also indicatesthat over the course of infection, it must both adapt to its self-inflicted acid stress as well as exploit alternative carbon sources. Inthis regard, transcriptome profiling revealed that one of the mosthighly differentially activated gene clusters under conditions ofacid stress and glucose starvation and in murine soft tissue en-codes a putative operon of two genes predicted to function inthe catabolism of malate (10), annotated as the malic permease(maeP) and the malic enzyme (maeE) (Fig. 1A).

The dicarboxylic organic acid malate is found in abundance intissue and in the environment, so it is not surprising that numer-ous malate degradation pathways have been identified amongboth prokaryotic and eukaryotic organisms (11–17). In lactic acidbacteria, two distinct pathways have been identified that makevery different contributions to physiology. The most common ofthese is malolactic fermentation (MLF), which allows for the con-version of malate into lactate through the function of the malo-lactic enzyme (MLE). Typically, MLF does not contribute togrowth yields but does play an important role in maintenance of

Received 21 October 2014 Returned for modification 12 November 2014Accepted 28 December 2014

Accepted manuscript posted online 12 January 2015

Citation Paluscio E, Caparon MG. 2015. Streptococcus pyogenes malatedegradation pathway links pH regulation and virulence. Infect Immun83:1162–1171. doi:10.1128/IAI.02814-14.

Editor: A. Camilli

Address correspondence to Michael G. Caparon, caparon@wusm.wustl.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.02814-14.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.02814-14

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ATP pools during starvation and in protection from acid killing(18–21). Since malate is a stronger acid than lactate, its decarbox-ylation by MLF results in alkalinization of the cytoplasm, and theresulting pH gradient drives the malate/lactate antiporter coupledto ATP synthesis (7, 18–21).

Less commonly found is an alternative degradation pathwaythat converts malate to pyruvate and carbon dioxide (18) that isknown as the malic enzyme (ME) pathway (Fig. 1B). A uniquefeature of ME is that, unlike MLF, it enables cells to utilize malateas a carbon source for growth (16, 18). However, while the MLFsystem has been extensively studied, the regulation and physiolog-ical significance of the ME pathway are not as well understood.Studies in several lactic acid species, including Enterococcus faeca-lis, Streptococcus bovis, and Lactobacillus casei (13, 15, 17, 22), haveindicated that ME requires 4 genes organized into two adjacentoperons (Fig. 1A). These include the maePE operon that encodesthe transmembrane permease (maeP) and cytosolic malic enzyme(maeE). Expression of these genes is dependent on the adjacenttwo-component system (TCS), which includes a sensor histidinekinase (maeK) and response regulator (maeR) (15, 17, 18). Thissimilar organization is observed in the S. pyogenes chromosome(Fig. 1A), and as noted above, maePE is upregulated by acid stressand infection in S. pyogenes. In addition, examination of the S.

pyogenes profiling data shows that both the maePE operon and theadjacent TCS had similar patterns of regulation, suggesting thatthese two systems function together (10).

Interestingly, while other ME operons are activated by malate(15, 17, 18) and repressed by glucose (15, 17), regulation by pHhas been described only for Lactococcus casei (18). Whether thissystem is regulated by pH in other bacterial species that contain afunctional ME pathway and the physiological role of this regula-tion are not known. Rather, pH regulation is more commonlyassociated with the MLF pathway, where it is associated with acidresistance (20, 21). Examination of the S. pyogenes genome has notrevealed the presence of MLF genes (23), so the significance of pHregulation of the ME pathway and whether it compensates forMLF in acid tolerance are not clear. In this study, we examined thecontribution of malate catabolism and its unique pattern of regu-lation to S. pyogenes physiology and virulence. This analysis re-vealed that S. pyogenes has a functional ME pathway, that catabo-lism of malate contributes to growth, and that its regulation sharessome similarities with other lactic acid bacteria but also has severalunique features. Finally, we show that the presence or absence ofME genes can influence virulence in a murine model of soft tissueinfection.

FIG 1 The malic enzyme (ME) pathway in S. pyogenes. (A) The arrangement of the open reading frames that comprise the ME locus of S. pyogenes is shown bylarge arrows. Gene names are shown below, and the genomic loci listed within the open reading frames are based on the genome of S. pyogenes HSC5 (23). Known(black font) and predicted (gray font) regulatory elements of the intergenic region of S. pyogenes and Enterococcus faecalis JH2-2 (Ef JH2-2 [17]) and Lactobacilluscasei BL23 (Lc BL23 [15]) are shown below. Arrows indicate sites in DNA bound by MaeR, while sites bound by CcpA are boxes labeled “cre” (cataboliteregulatory element). Numbers below indicate intergenic distances in numbers of base pairs. (B) Schematic of the ME pathway. The subcellular localization,function, and reactions catalyzed by the various components of the ME pathway that are listed in the figure are shown.

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MATERIALS AND METHODSBacterial strains, media, and growth conditions. The Escherichia colistrain TOP10 (Invitrogen) was used for cloning using standard molecularbiology techniques. The Streptococcus pyogenes strain HSC5 (23) and mu-tant derivatives were utilized in this study. Strains were grown in Todd-Hewitt broth with 0.2% yeast extract (THY; Difco) or C medium (24). Cmedium was adjusted to pH 7.5 as described previously (24). Routinegrowth conditions utilized sealed culture tubes incubated at 37°C understatic conditions. Streptococcal strains grown on solid medium contain-ing 1.4% Bacto agar (Difco) were cultured in a sealed jar with a commer-cial gas generator (GasPak catalogue no. 70304; BBL). For experimentsutilizing malate supplementation, filter-sterilized 5% (wt/vol) stock solu-tion buffered to pH 7.0 with NaOH (Sigma) was used to add malate(Sigma) to a final concentration of 0.5% to the medium. For experimentsutilizing glucose or maltose supplementation, filter-sterilized 20% (wt/vol) stock solution was used to add glucose or maltose (Sigma) to a finalconcentration of 0.2% to the medium. For experiments utilizing bufferedmedium, 1 M stock solutions of HEPES (pH 7.5) or morpholineethane-sulfonic acid (MES) (pH 6.0) (both obtained from Sigma) were added toa final concentration of 0.1 M to the medium. All media used were steril-ized in an autoclave prior to supplementation. When appropriate, antibi-otics were added at the following concentrations: erythromycin at 1 �g/ml, kanamycin at 250 �g/ml, and chloramphenicol at 3 �g/ml.

Construction of mutants. All references to genomic loci are based onthe genome of HSC5 (23). In-frame deletion mutations in the genes en-coding MaeP (L897_04180), MaeE (L897_04185), MaeR (L897_04170),and MaeK (L897_04175), as well as the modified allele for HPr (L897_05590)(see Table S1 in the supplemental material), were generated using allelicreplacement and the PCR primers listed in Table S2 in the supplementalmaterial. The deletion alleles were transferred to the HSC5 chromosomeby using the allelic replacement vector pGCP213 (25) as described previ-ously (26) and listed in Table S3 in the supplemental material. Each dele-tion allele was obtained through overlap extension PCR (27) using theprimers listed in Table S2. All molecular constructs and chromosomalstructures of all mutants were verified using PCR and DNA sequencing(Genewiz, South Plainfield, NJ) by using oligonucleotide primers (IDT,Coralville, IA) of the appropriate sequences.

Complementation of ME mutants. To complement the maeE in-frame deletions, DNA fragments containing maeE from HSC5 in the ab-sence of its promoter were amplified using the primers listed in Table S2 inthe supplemental material and inserted under the control of the rofApromoter in pABG5 as previously described (28). The resulting plasmid,pEP66, was then used for ectopic expression of MaeE (see Table S3). Forcomplementation of the maeK in-frame deletion, a reversion strategy wasused to restore the wild-type (WT) locus in the MaeK– mutant back-ground. A DNA fragment containing the maeK open reading frame andflanking regions was amplified from HSC5 using primers listed in TableS2 and inserted into the plasmid pCRK (29). The resulting plasmid,pEP74, was then used to create the strain MaeKr as described previously(30) (see Table S1).

Metabolic assays. Malate, lactate, and formate concentrations weremeasured using commercially available kits (Sigma). Briefly, cultures ofeach individual strain tested were grown overnight in C medium with theappropriate supplement added (see the text). Cultures were then sub-jected to centrifugation, filtered through 0.22-�m-pore-size filters (Mil-lipore), and then assessed per the manufacturer’s protocol. Data shownare the means and standard deviations from duplicate determination ofthree separate biological samples prepared from at least 2 independentexperiments.

Isolation of RNA and transcript analysis. Transcript abundance ofselected genes was analyzed as previously described (31). Briefly, over-night cultures were diluted 1:25 into fresh C medium with the appropriatesupplement added (see the text) and harvested at mid-log phase (opticaldensity at 600 nm [OD600] of 0.2). Total RNA was isolated using QiagenRNeasy minikit per the manufacturer’s protocol. RNA was subjected to

reverse transcription (RT) using iScript (Bio-Rad) per the manufacturer’sprotocol. RT-PCR analysis of cDNA samples were performed using iQSYBR green supermix (Bio-Rad) and the primers listed in Table S2. Rel-ative transcript abundance was determined using the ��CT method usingrecA transcript as a standard and are presented in comparison to unmod-ified C medium or in comparison to the wild type. The data shown are themeans and the standard deviations from triplicate determinations of atleast two separate biological samples prepared from at least two indepen-dent experiments.

Infection of mice. As previously described (32, 33), 5- to 6-week-oldfemale SKH1 hairless mice (Charles River Labs) were injected subcutane-ously with approximately 107 CFU of the S. pyogenes strains indicated inthe text. Following infection, the resulting ulcers formed were docu-mented over a period of several days by digital photography, and lesionareas were measured as previously described (32). Data presented arepooled from at least two independent experiments with at least 10 miceper experimental group.

Growth rate calculations. Indicated bacterial strains were back-di-luted 1:50 into 1 ml of fresh C medium (unmodified or altered as indicatedin the text), and their growth was monitored at 37°C using a Tecan InfiniteM200 Pro plate reader. During growth, the plate was shaken every 10 minfor 30 s, followed by a 5-s wait period and measurement of the OD600.Data were normalized relative to uninoculated medium, and growth rateswere calculated as described previously (34). Growth rates are reported asdoubling time (t1/2) and were determined from a series of 7 time pointscollected over a 60-min period that defines the peak rate of growth, whichtypically occurred prior to the culture reaching 15 to 30% of the maxOD600. Growth yields were calculated from the maximum OD600 reachedby the culture and are expressed as a percentage relative to that of thewild-type strain under identical conditions. The average doubling timeand percent growth yield were calculated from each replicate from at leastthree independent experiments.

Statistical analyses. Differences between mean values obtained forwild-type and mutant strains in in vitro assays were tested for significanceusing the Student t test. For infection of mice, differences in lesion areabetween the wild type and individual mutants were tested for significanceusing the Mann-Whitney U test. Computation of test statistics utilizedInstat (version 3.1) and Prism (version 6.0) from the GraphPad software(San Diego, CA). For all tests, the null hypothesis was rejected for P valuesof �0.05.

RESULTSME is necessary for S. pyogenes malate-enhanced growth. It isunclear why the ME pathway in S. pyogenes is regulated by pH, asMLF and not ME is typically associated with acid stress resistancein other lactic acid species (20, 21). However, S. pyogenes lacks thegenes necessary for MLF (23), so the contribution of the ME path-way to streptococcal physiology was investigated. The malic en-zyme uses NAD� to oxidize malate to produce CO2, NADH, andpyruvate (Fig. 1B). Since pyruvate can be further metabolized toproduce ATP, the signature function of ME is to allow cells toutilize malate as a carbon source for growth (16). To test thisgrowth phenotype, S. pyogenes HSC5 was cultured overnight in acarbohydrate-reduced medium (C medium) in the presence orabsence of 0.5% malate. Although cultures had comparablegrowth rates in both conditions (t1/2 � 56 min or 63 min, respec-tively), 0.5% malate enhanced growth yields by approximately50% (Fig. 2A). Additionally, pH measurements of cell-free super-natants taken throughout growth indicate that malate utilizationdoes not alter the pH of the medium compared to that of unmod-ified C medium (see Fig. S2A in the supplemental material). Thisis due to the fact that, unlike when grown in medium supple-mented with glucose, when grown on malate, the bacteria utilize

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mixed-acid fermentation, producing large amounts of formate,which has a much higher pKa than the lactate commonly pro-duced (see Fig. S2B).

In-frame deletion mutants in maeP (malate permease), maeE(malic enzyme), maeK (malate sensor kinase), and maeR (malateresponse regulator) were constructed and were found to havegrowth characteristics identical to those of the wild type in un-modified C medium. However, all mutants failed to show an in-creased growth yield in the presence of malate (Fig. 2B). Malateconcentrations were measured from cell-free supernatants ofovernight cultures grown in 0.5% malate to determine malateconsumption by the wild type and the four ME mutants. WTcultures exhibited an approximately 80% reduction from the ini-

tial concentration of 37.3 mM, while malate concentrations wereunchanged by growth of the mutants (Fig. 2C). With the excep-tion of maeE, it was not possible to express the ME genes from aplasmid for complementation. As an alternative, allelic replace-ment was used to restore the full-length maeK gene in a �MaeKmutant background to make the reversion strain MaeKr. In thisway, we were able to complement at least one gene from eachoperon. Complementation of maeE and reversion of maeK re-stored both enhanced growth yields in the presence of malate (Fig.2D) and consumption of malate (Fig. 2E).

Expression of ME genes is dependent on malate and requiresMaeK. In other lactic acid bacteria, expression of ME requiresboth the presence of malate and the ME TCS (15, 17, 18). To

FIG 2 ME mutants are deficient in malate catabolism. (A) WT bacteria were tested for malate utilization by measuring growth over the course of 16 h of culturesgrown in either unmodified C medium or C medium supplemented with 0.5% malate. Data are presented as means and standard deviations from 3 independentexperiments. (B and D) WT and ME mutants were grown in unmodified C medium or C medium supplemented with 0.5% malate. Following 16 h of incubation,growth yields were measured by OD600. Data are presented as the means and standard deviations from 3 independent experiments. (C and E) Malate concen-trations from cell-free culture supernatants from overnight cultures of WT or ME mutants grown in C medium supplemented with 0.5% malate were measured(see Materials and Methods). Data are presented as percent remaining (compared to initial concentration) and are presented as means and standard deviationsfrom 3 biological samples analyzed in duplicate. Asterisks indicate significant differences (***, P � 0.001) compared to WT in C medium.

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determine if this common regulatory mechanism is also utilized inS. pyogenes, an analysis of transcript levels of ME genes using real-time RT-PCR was performed. The results indicated that duringthe exponential phase of growth (OD600 � 0.2), maeP and maeEwere highly upregulated in the presence of malate by 100- and200-fold, respectively (Fig. 3A). This response was dependent onMaeK, as transcript levels were equivalent in the presence or ab-sence of malate in an MaeK� mutant. Restoration of the protein inan MaeKr reversion strain also restored malate-induced transcrip-tion (3A). Transcription of maeK and maeR was also increased inthe presence of malate in WT cells, with both genes showing anapproximately 3-fold increase compared to that in the unmodi-fied medium (Fig. 3B).

Glucose regulation of ME is CcpA independent. Malate ca-tabolism in other lactic acid bacteria is repressed by glucose, indi-cating this pathway is regulated through a mechanism of carboncatabolite repression (CCR) (15, 17). CCR allows the bacteria tometabolize preferable carbon sources in the environment, usuallythrough transcriptional repression of genes involved in the pro-cessing of alternative, and less favorable, carbon sources (reviewedin references 35–37). A key transcriptional regulator of globalCCR in Gram-positive bacteria is CcpA (35), which has beenshown to regulate ME in response to glucose in both Lactobacilluscasei and Enterococcus faecalis (15, 17). To test for CCR regulationof the ME pathway in S. pyogenes, transcription of the four MEgenes was analyzed in the absence or presence of glucose (0.2%) byreal-time RT-PCR. Results showed a significant repression of 4- to6-fold (log2 scale) for all four ME genes (Fig. 4), consistent withobservations in other lactic acid species (15, 17). However, in con-trast to these other species, repression occurred independently ofCcpA, as the addition of glucose still repressed expression of allME genes in a CcpA– mutant (Fig. 4) (38). Repression does havethe characteristics of CCR, as glucose was repressive even in thepresence of malate (Fig. 4), indicating that S. pyogenes has adopteda CcpA-independent CCR mechanism for regulation of malatecatabolism.

Malate catabolism is regulated by PTS-mediated phosphor-ylation. An alternative mechanism of CCR in bacteria is known as

induction prevention, which is dependent on the sugar phospho-transferase (PTS) system and the phosphorylation state of a con-served histidine residue of the phosphocarrier protein HPr (36),which in the case of S. pyogenes is His15 (39). If the ME loci arecontrolled by a mechanism similar to induction prevention, thencells unable to produce phosphorylated His-HPr (P�His-HPr)should be unable to utilize malate. To test this hypothesis, twomutants were constructed. The first is an allelic exchange mutantwith a swap of a chloramphenicol cassette with ptsI, which en-codes EI, the enzyme responsible for phosphorylation of the His15site within the HPr protein. The second mutant contains a singleamino acid substitution in HPr, replacing His15 with alanine,which has been shown to maintain HPr capability to be phosphor-ylated at Ser46 and is functional for sugar transport but lacking inthe ability to participate in regulation (HPrH15A) (36, 40, 41).

When grown in the presence of malate, both the PtsI– mutantand the HPrH15A mutant have a significant growth defect com-pared to WT growth, resulting in a reduced growth rate and lowerfinal culture density (Fig. 5A and B). To verify that this growthdefect was specific for malate utilization and not a general defectunder all conditions, strains were also grown in unmodified Cmedium, as well as in C medium supplemented with 0.2% maltose(a non-PTS sugar) (34). Comparisons of final yield from over-night cultures demonstrate that growth of both the PtsI– andHPrH15A mutants is similar to the WT in unmodified medium(Fig. 5B). In addition, upon supplementation of maltose, all threestrains showed an identical increase in growth (Fig. 5B). Thus,mutations that block formation of P�His-HPr are deficient inmalate utilization but are still able to utilize non-PTS carbonsources.

Expression of the ME genes was then examined in the presenceof malate, and it was discovered that compared to the WT, theHPrH15A mutant had a substantial reduction in transcript levelsfor all four ME genes (Fig. 5C). Taken together, these data dem-onstrate that the ME pathway in S. pyogenes is repressed by glucosethrough a mechanism similar to induction prevention.

pH regulation of ME is independent of malate but dependenton maeK. Prior transcriptional profiling revealed that maeP and

FIG 3 MaeK regulates malate-dependent expression of maePE. (A) WT,MaeK–, and MaeKr strains were grown in C medium supplemented with 0.5%malate until exponential phase (OD600 of 0.2). Total RNA was isolated andused for real-time RT-PCR analysis of maeP and maeE transcripts. (B) WTbacteria were grown in C medium supplemented with 0.5% malate as de-scribed before. Total RNA was isolated and used for real-time RT-PCR analysisof maeK and maeR transcripts. Data presented for all genes are the ratios oftranscript abundance in modified medium to that of unmodified C mediumand represent the means and standard deviations from 3 biological samples,each analyzed in triplicate.

FIG 4 Carbon catabolite repression of ME genes is CcpA independent. WTand CcpA– bacteria were grown in C medium supplemented with 0.2%glucose until exponential phase (OD600 of 0.2). Total RNA was isolated andused for real-time RT-PCR analysis of the individual mae transcripts. Data arepresented as the ratios of transcript abundance in modified medium to that inunmodified C medium and represent the means and standard deviations de-rived from 4 biological samples, each analyzed in triplicate.

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maeE are among the genes most highly regulated by pH in S.pyogenes (10). Additionally, growth of WT cells in acidified mediawas enhanced with the addition of malate, demonstrating thatmalate catabolism can occur in a low-pH environment (see Fig. S3in the supplemental material). To further characterize the role ofenvironmental pH on the ME pathway, WT S. pyogenes was grownin C medium buffered to either low (pH 6.0) or neutral (pH 7.5)pH, and transcription of the ME genes was analyzed by real-timeRT-PCR. Compared to unbuffered medium and in the absence ofthe addition of malate, growth at low pH, but not neutral pH,enhanced abundance of the maeP and maeE transcripts by ap-proximately 10- and 20-fold, respectively (Fig. 6A). Neither lownor high pH environments altered expression of maeK or maeRcompared with that in unmodified medium (Fig. 6B). However,MaeK itself was required for the enhanced expression of maeP andmaeE, as the abundance of these transcripts did not increase in theMaeK– mutant during growth at low pH, but regulation was re-stored in the MaeKr strain (Fig. 6C). Finally, to address the hier-archy of stimuli between malate and pH, quantitative RT-PCRwas performed on cells in the presence of both high malate (0.5%)concentrations and neutral pH and compared to malate alone.Results show that, for both maeP and maeE, transcription is dra-matically increased in the presence of 0.5% malate and that thisenhanced expression is unaffected by the pH of the medium (Fig.6D). Overall, these data show that mae gene expression is regu-

lated by environmental pH and that this regulation is mediatedthrough MaeK and is independent of malate regulation.

Loss of MaeE causes enhanced virulence in vivo. The in vitroexperiments presented in this work were done with bacterial cul-tures grown in C medium, which is characterized as having lowcarbohydrate concentrations and high salt and peptide levels (10).It has been demonstrated previously that these conditions arehighly analogous to the in vivo milieu of carbon sources within themurine soft tissue environment (10). Additionally, malate, beingone of the intermediate products of the citric acid cycle, is abun-dant in host tissue (42). Therefore, the fact that S. pyogenes is ableto utilize malate in vitro when added to C medium lends strongsupport that it can also be utilized in vivo during host tissue infec-tions.

Thus, it was of interest to determine if malate utilization andthe ME pathway play a role in virulence. To assess this, a murinesoft tissue model was used. Briefly, approximately 107 bacteriawere injected subcutaneously into the flank of immunocompetenthairless mice. Infection of WT S. pyogenes HSC5 produces a local-ized necrotic lesion and formation of an eschar within 24 h butdoes not cause a systemic infection (33). Lesion size increases overtime, peaking in size at day 3 postinfection (32). Measurement ofthe lesion area over time is therefore used as a marker for virulencein this model. For this analysis, mice were infected with the WT,MaeP–, MaeE–, MaeK–, or MaeR– strains, and lesion areas were

FIG 5 Carbon catabolite repression of ME genes controlled by P�His-HPr. WT, PtsI–, and HPrH15A strains were grown in unmodified C medium or C mediumplus 0.5% malate. (A) Growth of the WT and PTS mutants in malate-supplemented medium was measured by OD600 over the course of 16 h. Data presented arefrom a representative experiment. (B) The WT and PTS mutants were grown in unmodified C medium or C medium supplemented with 0.5% malate or 0.2%maltose. Following 16 h of incubation, growth yields were measured by OD600. Data are presented as the means and standard deviations from 3 independentexperiments. Asterisks indicate significant differences (**, P � 0.01) compared to the WT under each condition. (C) WT and HPrH15A strains were grown in Cmedium supplemented with 0.5% malate until exponential phase (OD600 of 0.2). Total RNA was isolated and used for real-time RT-PCR analysis of transcriptabundance of the individual mae transcripts. Data are presented as ratios of transcript abundance of HPrH15A to that of WT and represent the means and standarddeviations derived from 3 biological samples, analyzed in triplicate.

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compared 3 days postinfection. Mice infected with the MaeK–,MaeR–, and MaeP– strains all formed lesions that were signifi-cantly smaller than those in mice infected with the WT (Fig. 7).Conversely, mice infected with the MaeE– strain formed lesionsthat were significantly larger than those in mice infected with theWT (Fig. 7). These results demonstrate that malate catabolism is

an important factor during a soft tissue infection and that the lossof individual ME genes can have differential effects on the out-come of an infection.

DISCUSSION

In this study, we have shown that the ME genes of S. pyogenes allowthe bacterium to use malate as a carbon source for growth. Addi-tionally, we have shown that this pathway is subjected to regula-tion by both positive and negative signals, including glucose,malate, and pH. The former of these is via PTS-mediated phos-phorylation, while the latter two signals are recognized by theMaeKR regulatory system. Finally, these data show that loss of anyindividual mae gene can alter the outcome of a soft tissue infectionin mice, suggesting that the abilities to transport and utilize malateare both key processes in pathogenesis.

Regulation of ME gene expression in S. pyogenes was found toshare features in common with other bacterial species (15, 17, 18).Most notably is that they are induced by malate and that inductionrequires the MaeKR TCS. A prior analysis of the maePE andmaeKR promoter regions in L. casei identified the DNA-bindingsite for MaeR as a series of direct repeats, and a similar site isshared among other lactic acid bacteria, including S. pyogenes (seeFig. S1 in the supplemental material) (15, 17). Another commonfeature to ME pathway regulation is that all species repress MEgene expression in the presence of glucose, a regulatory mecha-nism known as carbon catabolite repression (CCR) (36, 37, 43).

FIG 6 pH regulation of ME is malate independent but requires MaeK. (A and B) WT bacteria were grown in C medium buffered to pH 6.0 or pH 7.5 untilexponential phase (OD600 of 0.2). Total RNA was isolated and used for real-time RT-PCR analysis of the individual mae transcripts. Data are presented as theratios of transcript abundance in buffered medium to that in unmodified C medium and represent the means and standard deviations derived from 3 biologicalsamples, each analyzed in triplicate. (C) WT, MaeK–, and MaeKr strains were grown in C medium (pH 6.0), and total RNA was isolated as described before andused for real-time RT-PCR analysis of maeP and maeE transcripts. Data are presented as ratios of transcript abundance in buffered medium to that in unmodifiedC medium and represent the means and standard deviations derived from 3 biological samples, each analyzed in triplicate. (D) WT bacteria were grown in Cmedium plus 0.5% malate or C medium plus 0.5% malate buffered to pH 7.5, and total RNA was isolated as described before and used for real-time RT-PCRanalysis of maeP and maeE transcripts. Data are presented as ratios of transcript abundance in modified medium to that in unmodified C medium and representthe means and standard deviations derived from 3 biological samples, each analyzed in triplicate.

FIG 7 Loss of MaeE causes enhanced virulence in vivo. Hairless SKH1 micewere infected subcutaneously with WT or individual ME mutant strains, andthe resulting lesions formed at day 3 postinfection were measured. Each sym-bol plotted represents the value derived from an individual animal. Datashown are pooled from at least 2 independent experiments, with the mean andstandard deviation indicated. Differences between groups were tested for sig-nificance using the Mann-Whitney U test (*, P � 0.05; **, P � 0.01).

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However, in S. pyogenes, glucose-mediated regulation of the MEloci functions independently of the major carbon catabolite pro-tein CcpA. In support of this finding is the fact that, unlike theother characterized lactic acid bacteria, the promoter region in S.pyogenes lacks any identifiable cre sites (Fig. 1; see also Fig. S1) (15,35, 43, 44).

Instead, an alternative method of CCR, induction prevention,is likely regulating ME genes in S. pyogenes. Evidence to supportthis idea includes the fact that multiple genetic strains that areunable to form P�His-HPr (either through loss of the EI enzymeor direct mutation of HPr) are likewise deficient in ME transcrip-tion and malate utilization. This method of regulation, therefore,enables the cell to activate ME genes only in the presence of a highconcentration of P�His-HPr. During normal growth utilizingpreferred carbohydrates, levels of intracellular P�His-HPr wouldlikely be low (36, 45). This is due to either rapid accumulation ofP�Ser-HPr or transfer of the phosphate group on P�His-HPr todownstream PTS transporters to allow uptake of PTS sugars. Inthis way, the bacterium is able to preferentially utilize a numberof available PTS sugars before turning on the alternative MEpathway.

In order for this form of regulation to control ME expression, itrequires a transfer of the phosphate from P�His-HPr to an MEregulatory protein. Phosphorylation of non-PTS protein byP�His-HPr is known to occur in a variety of species (for a review,see reference 36) and often involves a PTS regulatory domain(PRD)-containing protein. Currently, the only non-PTS proteinshown to act as a phosphate acceptor from P�His-HPr in S. pyo-genes is the transcriptional regulator Mga (46). This protein hasbeen characterized as containing several unique, but related, PRDdomains (PRD_Mga), and previous work has shown that P�His-HPr is able to phosphorylate specific residues within these do-mains in vitro (46, 47). Although Mga has been shown to regulatea large number of target genes (3, 4, 47, 48), the ME cluster has notyet been identified as part of its regulon. Alternatively, there aretwo additional transcription regulators in S. pyogenes HSC5 pre-dicted to include a PRD_Mga domain (E. Paluscio and M. G.Caparon, unpublished data), both within the RofA family of reg-ulators (46, 49–51). It remains possible that one of these proteinsmay be necessary for ME gene expression. One likely mechanismfor this regulation would be that the phosphate from the P�His-HPr gets transferred to one of the PRD transcriptional regulators,which then allows this protein to bind to the promoter region ofmaeKR and induce its expression. Further evidence to support thishypothesis is the presence of several putative regulatory elementswithin the mae promoter region of S. pyogenes (see Fig. S1), whichare absent from the promoters of the other lactic acid bacteria.These sequences may serve as binding sites for one of the PRD-containing regulatory proteins mentioned above.

This work also demonstrated that the maePE operon is regu-lated by a pH-dependent mechanism, whereby acidic pH inducestranscription of these genes and neutral pH is inhibitory. In S.pyogenes, it appears that, in addition, the MaeKR TCS was neces-sary for this regulation, as the loss of MaeK prevents the pH-dependent expression of maePE seen in wild-type cells. Interest-ingly, this work is the first to identify a signal other than malatethat is recognized by the MaeKR regulatory system. Though un-common, MaeK is not the first transcriptional regulator identifiedthat is able to respond to multiple extracellular signals. In Esche-richia coli, the cad operon is regulated by CadC, which recognizes

both acidic pH and lysine to induce transcription of the cad genes(52, 53). In Streptococcus mutans, the AguR protein, which con-trols the expression of the agmatine deiminase system (AgDS),recognizes acidic pH and agmatine (54, 55). An important distinc-tion between CadC, AguR, and MaeK is that the former two pro-teins require both signals to be present in order to allow for acti-vation and transcription of their target genes. This work hasshown, however, that MaeK functions in the presence of eithersignal and does not require both for transcriptional activation.Nonetheless, given that all three proteins respond to multiple sig-nals, one of which is low pH, they may all share some similarmechanisms of activation. It is hypothesized that for both CadCand AguR, the acidic pH environment induces a conformationalchange in the protein, and this change then allows for binding ofthe substrate (lysine and agmatine, respectively) (53, 54). Like-wise, in the presence of acidic pH, MaeK may undergo a confor-mational change that induces activation of the protein. However,unlike CadC and AguR, the MaeK protein likely has a separatemalate sensor domain that can bind malate in the presence orabsence of acidic pH. This mechanism would predict that MaeKhas two distinct regions required for signal recognition and thateither can control the activity of the protein.

The identification of a pH-dependent response for ME expres-sion is of particular interest due to the metabolism of the bacte-rium. S. pyogenes is a member of the lactic acid bacteria, a groupthat relies on a mix of homolactic and mixed-acid fermentation asa means of generating energy in the cell (6, 56). Over time, in thepresence of rapidly metabolized carbohydrates such as glucose,high concentrations of organic acid end products will accumulate(see Fig. S2 in the supplemental material) (5, 6, 56, 57). In this way,there is a direct link between carbohydrate availability and pH,with depletion of glucose leading to a corresponding reduction ofthe surrounding pH. In this respect, low pH could function as anearly warning signal for changes in carbohydrate availability.Thus, low pH may function as an inducer of expression for mul-tiple alternative catabolic operons and is likely not exclusive tomalate catabolism alone. Additionally, the MaeKR TCS may benecessary for controlling this pH-adaptive response for theseother catabolic operons.

Taken together, this work demonstrates that under conditionsof low glucose or acidic pH, malic acid catabolic genes are highlyexpressed. Given that these signals are among those that S. pyo-genes encounters at specific points in a soft tissue infection (8–10,57), the question of whether this alternative metabolic pathwaywas important for virulence was of particular interest. Althoughall of the ME mutants have similar phenotypes in vitro, a loss ofgrowth on malate, the mechanism to cause this deficiency is dif-ferent for each strain. All three attenuated strains are unable totransport extracellular malate into the cell (either through dele-tion of the malate transporter gene or loss of maeP expression inan MaeK– or MaeR– mutant). Alternatively, an MaeE– mutant isable to transport malate into the cell but cannot convert this mol-ecule into pyruvate. This differentiation may, in part, explain thein vivo phenotypes observed.

In this case, it remains possible that malate may serve a second-ary, as-yet-unknown benefit to the bacterial cell independent ofincreased pyruvate concentrations. Thus, the ability of an MaeE–

mutant to allow uptake of malate may allow for this unused malateto be shuttled into an alternative pathway, ultimately serving tobenefit S. pyogenes during infection. Alternatively, MaeP–, MaeK–,

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and MaeR– mutants would be depleted of any internal malateaccumulation, and this loss would ultimately decrease fitness forthe cells compared to a WT or an MaeE– mutant. Another possi-bility is that the accumulation of intracellular malate in the MaeE–

mutant may cause the misregulation of virulence factor expres-sion, leading to enhanced virulence. In preliminary studies, wehave found that while expression of the SpeB cysteine proteasedoes not differ between WT and the MaeE– and MaeP– mutants(see Fig. S4A in the supplemental material), the addition of malatealters the temporal pattern of SpeB expression in both mutantscompared to that in the WT (see Fig. S4B). Since this alteration inSpeB expression is similar between the two mutants, it cannotexplain the enhanced virulence of the MaeE– mutant. However, itdoes support the possibility that alterations to malate metabolismcan result in changes in patterns of virulence factor expression.Further analyses of virulence factor expression will be required inorder to determine if specific factors are specifically misregulatedin the MaeE– mutant and whether these factor are responsible forhypervirulence. This work does, however, provide novel insightsinto the unique regulatory mechanisms utilized by S. pyogenes formalate degradation, as well as demonstrate for the first time theimportance of this alternative metabolic pathway on influencingpathogenesis.

ACKNOWLEDGMENTS

We thank Y. Le Breton and K. McIver for providing the plasmid pCRK.This work was supported by Public Health Service Grant AI070759

from the National Institutes of Health. The work cited in this publicationwas performed in a facility supported by NCRR grant C06 RR015502.

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