Virulence Of Group Research Articles

CodY Is a Global Transcriptional Regulator Required for Virulence in Group B Streptococcus.

Group B Streptococcus (GBS) is a Gram-positive bacterium able to switch from a harmless commensal of healthy adults to a pathogen responsible for invasive infections in neonates. The signals and regulatory mechanisms governing this transition are still largely unknown. CodY is a highly conserved global transcriptional regulator that links nutrient availability to the regulation of major metabolic and virulence pathways in low-G+C Gram-positive bacteria. In this work, we investigated the role of CodY in BM110, a GBS strain representative of a hypervirulent lineage associated with the majority of neonatal meningitis. Deletion of codY resulted in a reduced ability of the mutant strain to cause infections in neonatal and adult animal models. The observed decreased in vivo lethality was associated with an impaired ability of the mutant to persist in the blood, spread to distant organs, and cross the blood-brain barrier. Notably, the codY null mutant showed reduced adhesion to monolayers of human epithelial cells in vitro and an increased ability to form biofilms, a phenotype associated with strains able to asymptomatically colonize the host. RNA-seq analysis showed that CodY controls about 13% of the genome of GBS, acting mainly as a repressor of genes involved in amino acid transport and metabolism and encoding surface anchored proteins, including the virulence factor Srr2. CodY activity was shown to be dependent on the availability of branched-chain amino acids, which are the universal cofactors of this regulator. These results highlight a key role for CodY in the control of GBS virulence.

Virulence of Group A Streptococci Is Enhanced by Human Complement Inhibitors

Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is an important human bacterial pathogen that can cause invasive infections. Once it colonizes its exclusively human host, GAS needs to surmount numerous innate immune defense mechanisms, including opsonization by complement and consequent phagocytosis. Several strains of GAS bind to human-specific complement inhibitors, C4b-binding protein (C4BP) and/or Factor H (FH), to curtail complement C3 (a critical opsonin) deposition. This results in diminished activation of phagocytes and clearance of GAS that may lead to the host being unable to limit the infection. Herein we describe the course of GAS infection in three human complement inhibitor transgenic (tg) mouse models that examined each inhibitor (human C4BP or FH) alone, or the two inhibitors together (C4BPxFH or ‘double’ tg). GAS infection with strains that bound C4BP and FH resulted in enhanced mortality in each of the three transgenic mouse models compared to infection in wild type mice. In addition, GAS manifested increased virulence in C4BPxFH mice: higher organism burdens and greater elevations of pro-inflammatory cytokines and they died earlier than single transgenic or wt controls. The effects of hu-C4BP and hu-FH were specific for GAS strains that bound these inhibitors because strains that did not bind the inhibitors showed reduced virulence in the ‘double’ tg mice compared to strains that did bind; mortality was also similar in wild-type and C4BPxFH mice infected by non-binding GAS. Our findings emphasize the importance of binding of complement inhibitors to GAS that results in impaired opsonization and phagocytic killing, which translates to enhanced virulence in a humanized whole animal model. This novel hu-C4BPxFH tg model may prove invaluable in studies of GAS pathogenesis and for developing vaccines and therapeutics that rely on human complement activation for efficacy.

Characteristics of major virulence of Group A β-hemolytic streptococcus isolated from children

Objective To study the prevalence of superantigens, deoxy ribonuclease(DNase) genes and fibronectin-binding protein encoding (FBP) genes of Group A Streptococci(GAS) isolated from children, and to analyze its relationship with clinical infections and predominant emm type. Methods A total of 112 strains of GAS were collected from three Children's Hospitals during the period from 2008 to 2012, including 72 strains from patients and 40 strains from asymptomatic carriers.Emm, superantigens(speA, speC, speH, speI, speG, speJ, ssa, smeZ, speL, speM, speK and speF), DNase genes (sda, sdn, spd1, spd3 and spd4), phospholipase A2 gene and FBP genes (prtFl、prtF2、fba) were amplified by PCR, and emm was further typed by DNA sequencing. Results A total 7 types were identified, including emm1, emm12, emm22, emm4, emm77, emm86, and stG485.The most common emm type was emm12, which accounted for 68.8% of all isolates in this study.The emm type distribution of the pathogens was accorded with the carrier strains.The prevalence of 12 superantigens, including speA, speC, speF, speG, speH, speI, speJ, speK, speL, speM, ssa, smeZ were 13.4%, 95.5%, 85.7%, 74.1%, 63.4%, 63.4%, 14.3%, 0.9%, 0.9%, 0.9%, 74.1% and 93.8%, respectively.The prevalence of 5 DNase genes, including sda, sdn, spd1, spd3, spd4, was 66.96%, 12.5%, 94.64%, 27.68%, 0.89%, respectively.No sla gene was detected.The prevalence of fibronectin-binding protein, including prtF1, prtF2, fba, was 69.6%, 79.5%, 30.4%, respectively.The carriage rate of speJ, spd3 from invasive or noninvasive infections was higher than that from asymptomatic carriers, however, the positive rate of ssa, speF, speG, prtF1, prtF2 and prtF1/prtF2 was lower than that of asymptomatic carriers.The prevalence of speH, speI, speG, sda, spd1, prtF1, prtF2, prtF1/prtF2 from the emm12 isolates was higher than that from non-emm12 isolates, whereas speA, speJ, sdn, spd3 and fba were more common in non-emm12 isolates. Conclusions Emm12 is the prevalent genotype among S. pyogenes strains isolated from children, followed by emm1 and emm12, which is irrelevant to resources; Strains isolated from infected children and from asymptomatic carriers have different distribution characteristics of virulence genes; The prevalence of emm12 may be associated with the high carrier rate of the certain virulence gene. Key words: Streptococcus pyogenes; Virulence factors; Emm typing

Crystal Structure of Peroxide Stress Regulator from Streptococcus pyogenes Provides Functional Insights into the Mechanism of Oxidative Stress Sensing

Regulation of oxidative stress responses by the peroxide stress regulator (PerR) is critical for the in vivo fitness and virulence of group A Streptococcus. To elucidate the molecular mechanism of DNA binding, peroxide sensing, and gene regulation by PerR, we performed biochemical and structural characterization of PerR. Sequence-specific DNA binding by PerR does not require regulatory metal occupancy. However, metal binding promotes higher affinity PerR-DNA interactions. PerR metallated with iron directly senses peroxide stress and dissociates from operator sequences. The crystal structure revealed that PerR exists as a homodimer with two metal-binding sites per subunit as follows: a structural zinc site and a regulatory metal site that is occupied in the crystals by nickel. The regulatory metal-binding site in PerR involves a previously unobserved HXH motif located in its unique N-terminal extension. Mutational analysis of the regulatory site showed that the PerR metal ligands are involved in regulatory metal binding, and integrity of this site is critical for group A Streptococcus virulence. Interestingly, the metal-binding HXH motif is not present in the structurally characterized members of ferric uptake regulator (Fur) family but is fully conserved among PerR from the genus Streptococcus. Thus, it is likely that the PerR orthologs from streptococci share a common mechanism of metal binding, peroxide sensing, and gene regulation that is different from that of well characterized PerR from Bacillus subtilis. Together, our findings provide key insights into the peroxide sensing and regulation of the oxidative stress-adaptive responses by the streptococcal subfamily of PerR.

The two-component response regulator LiaR regulates cell wall stress responses, pili expression and virulence in group B Streptococcus

Group B Streptococcus (GBS) remains the leading cause of early onset sepsis among term infants. Evasion of innate immune defences is critical to neonatal GBS disease pathogenesis. Effectors of innate immunity, as well as numerous antibiotics, frequently target the peptidoglycan layer of the Gram-positive bacterial cell wall. The intramembrane-sensing histidine kinase (IM-HK) class of two-component regulatory systems has been identified as important to the Gram-positive response to cell wall stress. We have characterized the GBS homologue of LiaR, the response regulator component of the Lia system, to determine its role in GBS pathogenesis. LiaR is expressed as part of a three-gene operon (liaFSR) with a promoter located upstream of liaF. A LiaR deletion mutant is more susceptible to cell wall-active antibiotics (vancomycin and bacitracin) as well as antimicrobial peptides (polymixin B, colistin, and nisin) compared to isogenic wild-type GBS. LiaR mutant GBS are significantly attenuated in mouse models of both GBS sepsis and pneumonia. Transcriptional profiling with DNA microarray and Northern blot demonstrated that LiaR regulates expression of genes involved in microbial defence against host antimicrobial systems including genes functioning in cell wall synthesis, pili formation and cell membrane modification. We conclude that the LiaFSR system, the first member of the IM-HK regulatory systems to be studied in GBS, is involved in sensing perturbations in the integrity of the cell wall and activates a transcriptional response that is important to the pathogenesis of GBS infection.

Serine/Threonine Phosphatase Stp1 Mediates Post-transcriptional Regulation of Hemolysin, Autolysis, and Virulence of Group B Streptococcus

Elucidating how serine/threonine phosphatases regulate kinase function and bacterial virulence is critical for our ability to combat these infections. Group B streptococci (GBS) are β-hemolytic Gram-positive bacteria that cause invasive infections in humans. To adapt to environmental changes, GBS encodes signaling mechanisms comprising two component systems and eukaryotic-like enzymes. We have previously described the importance of the serine/threonine kinase Stk1 to GBS pathogenesis. However, how the presence or absence of the cognate serine/threonine phosphatase Stp1 affects Stk1 function and GBS virulence is not known. Here, we show that GBS deficient only in Stp1 expression are markedly reduced for their ability to cause systemic infections, exhibit decreased β-hemolysin/cytolysin activity, and show increased sensitivity to autolysis. Although transcription of genes important for β-hemolysin/cytolysin expression and export is similar to the wild type (WT), 294 genes (excluding stp1) showed altered expression in the stp1 mutant and included autolysin genes. Furthermore, phosphopeptide enrichment analysis identified that 35 serine/threonine phosphopeptides, corresponding to 27 proteins, were unique to the stp1 mutant. This included phosphorylation of ATP synthase, DNA and RNA helicases, and proteins important for cell division and protein synthesis. Collectively, our results indicate that Stp1 is important for appropriate regulation of Stk1 function, hemolysin activity, autolysis, and GBS virulence.

Role of Serine/Threonine Phosphatase (SP-STP) in Streptococcus pyogenes Physiology and Virulence

Reversible phosphorylation is the key mechanism regulating several cellular events in prokaryotes and eukaryotes. In prokaryotes, signal transduction is perceived to occur primarily via the two-component signaling system involving histidine kinases and cognate response regulators. Although an alternative regulatory pathway controlled by the eukaryote-type serine/threonine kinase (Streptococcus pyogenes serine/threonine kinase; SP-STK) has been shown to modulate bacterial growth, division, adherence, invasion, and virulence in group A Streptococcus (GAS; S. pyogenes), the precise role of the co-transcribing serine/threonine phosphatase (SP-STP) has remained enigmatic. In this context, this is the first report describing the construction and characterization of non-polar SP-STP mutants in two different strains of Type M1 GAS. The STP knock-out mutants displayed increased bacterial chain lengths in conjunction with thickened cell walls, significantly reduced capsule and hemolysin production, and restoration of the phenotypes postcomplementation. The present study also reveals important contribution of cognately regulated-reversible phosphorylation by SP-STK/SP-STP on two major response regulators of two-component systems, WalRK and CovRS. We also demonstrate a distinct role of SP-STP in terms of expression of surface proteins and SpeB in a strain-specific manner. Further, the attenuation of virulence in the absence of STP and its restoration only in the complemented strains that were generated by the use of a low copy plasmid and not by a high copy one emphasize not only the essential role of STP in virulence but also highlight the tightly regulated SP-STP/SP-STK-mediated cognate functions. SP-STP thus is an important regulator of GAS virulence and plays a critical role in GAS pathogenesis.

Listeriolysin S, a Novel Peptide Haemolysin Associated with a Subset of Lineage I Listeria monocytogenes

Streptolysin S (SLS) is a bacteriocin-like haemolytic and cytotoxic virulence factor that plays a key role in the virulence of Group A Streptococcus (GAS), the causative agent of pharyngitis, impetigo, necrotizing fasciitis and streptococcal toxic shock syndrome. Although it has long been thought that SLS and related peptides are produced by GAS and related streptococci only, there is evidence to suggest that a number of the most notorious Gram-positive pathogenic bacteria, including Listeria monocytogenes, Clostridium botulinum and Staphylococcus aureus, produce related peptides. The distribution of the L. monocytogenes cluster is particularly noteworthy in that it is found exclusively among a subset of lineage I strains; i.e., those responsible for the majority of outbreaks of listeriosis. Expression of these genes results in the production of a haemolytic and cytotoxic factor, designated Listeriolysin S, which contributes to virulence of the pathogen as assessed by murine- and human polymorphonuclear neutrophil–based studies. Thus, in the process of establishing the existence of an extended family of SLS-like modified virulence peptides (MVPs), the genetic basis for the enhanced virulence of a proportion of lineage I L. monocytogenes may have been revealed.

The Importance of the Group AStreptococcusCapsule in the Pathogenesis of Human Infections: A Historical Perspective

The virulence of group A streptococci (GAS) correlates closely with expression of its surface antigen, M protein, and its hyaluronic acid capsule. In studies of human GAS infection, the former has received considerable attention. For several decades, however, systematic identification of encapsulated virulent strains by the mucoid colonies they produce has been neglected in clinical studies. In part, this may be due to the capsule's evanescent expression on artificial media, its repression during convalescent carriage, lack of expertise in recognizing its colonial morphology, and the growing tendency for clinical laboratories to eschew throat cultures in favor of rapid laboratory tests for group A polysaccharide. Older and more recent studies are reviewed here that emphasize the capsule's basic role in infection. We believe that it is time to refocus newer clinical studies and techniques on achieving early recognition of potentially dangerous, heavily encapsulated strains of GAS for which spread may be prevented.

The Maintenance of High Affinity Plasminogen Binding by Group A Streptococcal Plasminogen-binding M-like Protein Is Mediated by Arginine and Histidine Residues within the a1 and a2 Repeat Domains

Subversion of the plasminogen activation system is implicated in the virulence of group A streptococci (GAS). GAS displays receptors for the human zymogen plasminogen on the cell surface, one of which is the plasminogen-binding group A streptococcal M-like protein (PAM). The plasminogen binding domain of PAM is highly variable, and this variation has been linked to host selective immune pressure. Site-directed mutagenesis of full-length PAM protein from an invasive GAS isolate was undertaken to assess the contribution of residues in the a1 and a2 repeat domains to plasminogen binding function. Mutagenesis to alanine of key plasminogen binding lysine residues in the a1 and a2 repeats (Lys98 and Lys111) did not abrogate plasminogen binding by PAM nor did additional mutagenesis of Arg101 and His102 and Glu104, which have previously been implicated in plasminogen binding. Plasminogen binding was only abolished with the additional mutagenesis of Arg114 and His115 to alanine. Furthermore, mutagenesis of both arginine (Arg101 and Arg114) and histidine (His102 and His115) residues abolished interaction with plasminogen despite the presence of Lys98 and Lys111 in the binding repeats. This study shows for the first time that residues Arg101, Arg114, His102, and His115 in both the a1 and a2 repeat domains of PAM can mediate high affinity plasminogen binding. These data suggest that highly conserved arginine and histidine residues may compensate for variation elsewhere in the a1 and a2 plasminogen binding repeats, and may explain the maintenance of high affinity plasminogen binding by naturally occurring variants of PAM.

Human immune response to streptococcal C5a peptidase in Indian children and adults

Streptococcal C5a peptidase (SCPA) is a cell bound protease associated with virulence of group A streptococci (GAS). In India, as the incidence of GAS infections and their late sequelae like rheumatic fever (RF), rheumatic heart disease (RHD) and acute glomerulo nephritis (AGN) is very high, it is necessary to characterize the immune response to SCPA. SCPA is considered as a standard vaccine candidate, as anti-SCPA antibodies can neutralize the activity of this enzyme in vitro and in vivo. In the present study, the anti-SCPA IgG levels and their subtypes were determined in 199 serum samples collected from RF/RHD/AGN/NORMAL subjects and the results were correlated with the anti-streptolysin O (ASO) titres. The mean anti-SCPA IgG in normal healthy subjects was 7.35 μg/ml. The level of these antibodies in RF, RHD and AGN cases was found to be 17.58 μg/ml, 12.74 μg/ml and 11.1 μg/ml, respectively. The rise of anti-SCPA IgG observed in patients was irrespective of different emm types of GAS strains isolated from these patients. The findings are helpful for generating base line data of anti-SCPA IgG levels.

Oxygen regulates invasiveness and virulence of group B streptococcus.

The facultative anaerobe group B Streptococcus (GBS) is an opportunistic pathogen of pregnant women, newborns, and the elderly. Although several virulence factors have been identified, environmental factors that regulate the pathogenicity of GBS have not been well characterized. Using the dynamic in vitro attachment and invasion system (DIVAS), we examined the effect of oxygen on the ability of GBS to invade immortalized human epithelial cells. GBS type III strain M781 invaded human epithelial cells of primitive neurons, the cervix, the vagina, and the endometrium in 5- to 400-fold higher numbers when cultured at a cell mass doubling time (t(d)) of 1.8 h than at a slower t(d) of 11 h. Invasion was optimal when GBS was cultured at a t(d) of 1.8 h in the presence of >or=5% oxygen and was significantly reduced without oxygen. Moreover, GBS grown in a chemostat under highly invasive conditions (t(d) of 1.8 h, with oxygen) was more virulent in neonatal mice than was GBS grown under suboptimal invasion conditions (t(d) of 1.8 h, without oxygen), suggesting a positive association between in vitro invasiveness with DIVAS and virulence.

Spa contributes to the virulence of type 18 group A streptococci.

Streptococcal protective antigen (Spa) is a newly described surface protein of group A streptococci that was recently shown to evoke protective antibodies (J. B. Dale, E. Y. Chiang, S. Liu, H. S. Courtney, and D. L. Hasty, J. Clin. Investig. 103:1261--1268, 1999). In this study, we have determined the complete sequence of the spa gene from type 18 streptococci. Purified, recombinant Spa protein evoked antibodies that were bactericidal against type 18 streptococci, confirming the presence of protective epitopes. Sera from patients with acute rheumatic fever contained antibodies against recombinant Spa, indicating that the Spa protein is expressed in vivo and is immunogenic in humans. To determine the role of Spa in the virulence of group A streptococci, we created a series of insertional mutants that were (i) Spa negative and M18 positive, (ii) Spa positive and M18 negative, and (iii) Spa negative and M18 negative. The mutants and the parent M18 strain (18-282) were used in assays to determine resistance to phagocytosis, growth in human blood, and mouse virulence. The results show that Spa is a virulence determinant of group A streptococci and that expression of both Spa and M18 is required for optimal virulence of type 18 streptococci.

Is streptolysin S of group A streptococci a virulence factor?

The possible role played by streptolysin S (SLS) of group A streptococci in the pathophysiology of streptococcal infections and in post-streptococcal sequelae is discussed. The following properties of SLS justify its definition as a distinct virulence factor: 1) its presence on the streptococcus surface in a cell-bound form, 2) its continuous and prolonged synthesis by resting streptococci, 3) its non-immunogenicity, 4) its extractability by serum proteins (albumin, alpha lipoprotein), 5) its ability to become transferred directly to target cells while being protected from inhibitory agents in the milieu of inflammation, 6) its ability to bore holes in the membrane phospholipids in a large variety of mammalian cells, 7) its ability to synergize with oxidants, proteolytic enzymes, and with additional host-derived proinflammatory agonists, and 8) its absence in streptococcal mutants associated with a lower pathogenicity for animals. Because tissue damage in streptococcal and post-streptococcal sequelae might be the end result of a distinct synergism between streptococcal and host-derived proinflammatory agonists it is proposed that only cocktails of anti-inflammatory agents including distinct inhibitors of SLS (phospholipids), gamma globulin, inhibitors of reactive oxygen species, proteinases, cationic proteins cytokines etc., will be effective in inhibiting the multiple synergistic interactions which lead to fasciitis, myositis and the flesh-eating syndromes, and often develop into sepsis, septic shock and multiple organ failure. The creation of mutants deficient in SLS and in proteases will help shed light on the specific role played by SLS in the virulence of group A hemolytic streptococci.

Rheumatic Fever—A Half-Century Perspective

Rheumatic Fever—A Half-Century Perspective

The Role of ICAM-1 in Host Response to Group A Streptococcus ♦ 913

The virulence of Group A Streptococcus (GAS) is due to multiple factors including the production of superantigens such as Streptococcus Pyrogenic Exotoxin A (SPEA). Superantigens activate the T-lymphocyte through a non-specific MHC-antigen presenting cell complex of which intercellular adhesion molecule-1 (ICAM-1) is a necessary component. We sought to determine the contribution of ICAM-1 in the host response to SPEA-producing and non-SPEA producing GAS.

Role of emm and mrp genes in the virulence of group A streptococcal isolate 64/14 in a mouse model of skin infection.

The virulence of group A streptococcal isolate 64/14 and paired isogenic mutants in which either the emm or mrp gene had been insertionally inactivated was compared in mice. Loss of expression of the emm gene product resulted in a significant loss of virulence when the isolate was injected into the skin but had no significant difference when injected intraperitoneally. By contrast, inactivation of the mrp gene caused the organism to be more virulent in the skin, while having no significant effect intraperitoneally. An isogenic mutant, in which the mga gene was inactivated and neither the emm gene nor the mrp gene was expressed, demonstrated no significant difference in virulence from the wild type organism. Organisms recovered from the spleen of mice lethally infected with the mga mutant expressed all Mga-regulated IgG-binding gene products despite the presence of the spectinomycin-resistance cassette, which was used to inactivate the mga gene, in its original position.

M protein expression is not required for resistance to phagocytosis of type 18 group A streptococci.

The major determinant of virulence of group A streptococci is the ability to resist phagocytosis by polymorphonuclear leukocytes. It has long been held that the M protein of group A streptococci is the surface component that is primarily responsible for conferring this property upon the organisms. Many highly virulent group A streptococci also express a hyaluronic acid capsule, which could contribute to the ability of the organism to resist phagocytic killing by forming a physical barrier between complement deposited close to the cell wall and the phagocyte. Previous studies by Wessels and his colleagues used transposon mutagenesis to inactivate the hyaluronate synthase gene (has) of type 18 streptococci (3). The unencapsulated mutant grew less well in human blood than the parent strain, despite the expression of M protein on its surface (3). The present studies were performed to directly determine the role of M protein in resistance to phagocytosis of type 18 streptococci.

Streptococcal Toxic Shock—like Syndrome

After decades marked by a decrease in the incidence and severity of scarlet fever and streptococcal soft-tissue infections, invasive infections with group A streptococci have reemerged as a global public health problem. Sporadic outbreaks of a rapidly progressive disorder characterized by fever, shock, desquamating rash, and multiorgan system failure often associated with severe suppurative soft-tissue infection have recently been recognized in young, otherwise-healthy adults. Referred to as streptococcal toxic shock-like syndrome, this acute, progressive, often fatal illness appears to be related to an overall change in the virulence of group A streptococci and the reappearance of highly mucoid exotoxin-producing strains. However, the relationship between virulence factors, epidemiological features, and precise pathogenesis of these infections remains unclear. The 80% incidence of soft-tissue involvement in streptococcal toxic shock-like syndrome and the high frequency of progression to necrotizing fasciitis, myositis, or even death are unprecedented. The type and extent of tissue injury, systemic toxicity, and multiorgan system failure associated with this syndrome appear to be mediated by streptococcal pyrogenic exotoxins via the induction of biologically potent endogenous cytokines. The observed increase in the incidence and severity of invasive group A streptococcal infections coincides with the resurgence of genetically related, highly virulent strains of Staphylococcus pyogenes that express an invasive phenotype and carry a specific gene for pyrogenic exotoxin A. The changing spectrum of invasive group A streptococcal infections has prompted a reexamination of the microorganism and its diverse clinical manifestations. A clonal basis for increased virulence expression is supported epidemiologically. As the incidence of streptococcal toxic shock-like syndrome appears to be increasing, it is imperative that clinicians become familiar with the presentation of this syndrome so they can diagnose and treat it in a timely and effective manner.

Environmental regulation of virulence in group A streptococci: transcription of the gene encoding M protein is stimulated by carbon dioxide.

We have found that different atmospheres can have significant effects on the transcription of emm, the gene that encodes M protein, the major virulence factor of the group A streptococcus (Streptococcus pyogenes). Expression of emm was monitored by constructing a transcriptional fusion of the promoter for emm6.1 from S. pyogenes JRS4 to a promoterless chloramphenicol acetyltransferase gene. Transcription, as measured by determining chloramphenicol acetyltransferase specific activity, was stimulated by as much as 25-fold by increased carbon dioxide tension. Expression was greater in the latter stages of growth and was not affected by growth at 30 instead of 37 degrees C. Insertional inactivation of mry, a gene encoding a positive regulator of emm6.1, reduced chloramphenicol acetyltransferase activity below the detectable level. We conclude that expression of emm is influenced by environmental factors and that the level of carbon dioxide is one signal that may influence expression of M protein during infection.