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Phenotypically distinct clinical isolates of Mycobacterium tuberculosis are capable of altering the balance that exists between the pathogen and human host and ultimately the outcome of infection. This study has identified two M. tuberculosis strains (i.e. HN885 and HN1554) among a bank of clinical isolates with a striking defect in phagocytosis by primary human macrophages when compared with strain Erdman, a commonly used laboratory strain for studies of pathogenesis. Mass spectrometry in conjunction with NMR studies unequivocally confirmed that both HN885 and HN1554 contain truncated and more branched forms of mannose-capped lipoarabinomannan (ManLAM) with a marked reduction of their linear arabinan (corresponding mainly to the inner Araf-α(1→5)-Araf unit) and mannan (with fewer 6-Manp residues and more substitutions in the linear Manp-α(1→6)-Manp unit) domains. The truncation in the ManLAM molecules produced by strains HN885 and HN1554 led to a significant reduction in their surface availability. In addition, there was a marked reduction of higher order phosphatidyl-myo-inositol mannosides and the presence of dimycocerosates, triglycerides, and phenolic glycolipid in their cell envelope. Less exposed ManLAM and reduced higher order phosphatidyl-myo-inositol mannosides in strains HN885 and HN1554 resulted in their low association with the macrophage mannose receptor. Despite reduced phagocytosis, ingested bacilli replicated at a fast rate following serum opsonization. Our results provide evidence that the clinical spectrum of tuberculosis may be dictated not only by the host but also by the amounts and ratios of surface exposed mycobacterial adherence factors defined by strain genotype.

Poststreptococcal glomerulonephritis is one of the oldest recognized renal diseases. In the past three decades, significant changes have occurred in its epidemiology, in new insight gained in the nephritogenic characteristics of streptococcal antigens, and in the natural history of the disease. The disease is now rare in industrialized nations, but in the underprivileged world, the burden of poststreptococcal glomerulonephritis ranges between 9.5 and 28.5 new cases per 100,000 individuals per year. Prophylactic antibiotic treatment is advisable in epidemic conditions and to household contacts of index cases in communities where the prevalence of the disease is high. The long-term prognosis is variable; in general, prognosis is excellent in children but significantly worse when it occurs in elderly individuals and in populations that present other risk factors of chronic kidney disease. Contemporary large-scale research strategies such as genome-wide sequencing may uncover new information about pathogenic factors contributing to disease.

Group A Streptococcus (GAS) infections may trigger autoimmune sequelae thought to involve streptococcal antibodies cross-reactive to human antigens. Here, the antigenicity of the streptococcal collagen-like (CL) proteins, Scl1 and Scl2, that exhibit structural similarity to human collagen, was analyzed. Antibodies to Scl1.1 protein were detected in human sera from healthy individuals previously infected with M1 GAS and from patients with various GAS infections, as well as in sera from mice infected with M1 GAS. Linear B-cell epitope mapping identified immunoreactive peptides corresponding to the CL region of Scl1.1. Humoral responses to Scl1.28 and Scl2.28 were also detected in pediatric patients and mice infected with M28 GAS.

Group A Streptococcus (GAS) genes that encode proteins putatively involved in polysaccharide utilization show growth phase-dependent expression in human saliva. We sought to determine whether the putative polysaccharide transcriptional regulator MalR influences the expression of such genes and whether MalR helps GAS infect the oropharynx. Analysis of 32 strains of 17 distinct M protein serotypes revealed that MalR is highly conserved across GAS strains. malR transcripts were detectable in patients with GAS pharyngitis, and the levels increased significantly during growth in human saliva compared to the levels during growth in glucose-containing or nutrient-rich media. To determine if MalR influenced the expression of polysaccharide utilization genes, we compared the transcript levels of eight genes encoding putative polysaccharide utilization proteins in the parental serotype M1 strain MGAS5005 and its Δ malR isogenic mutant derivative. The transcript levels of all eight genes were significantly increased in the Δ malR strain compared to the parental strain, especially during growth in human saliva. Following experimental infection, the Δ malR strain persistently colonized the oropharynx in significantly fewer mice than the parental strain colonized, and the numbers of Δ malR strain CFU recovered were significantly lower than the numbers of the parental strain CFU recovered. These data led us to conclude that MalR influences the expression of genes putatively involved in polysaccharide utilization and that MalR contributes to the persistence of GAS in the oropharynx.

Study of the maltose/maltodextrin binding protein MalE in Escherichia coli has resulted in fundamental insights into the molecular mechanisms of microbial transport. Whether gram-positive bacteria employ a similar pathway for maltodextrin transport is unclear. The maltodextrin binding protein MalE has previously been shown to be key to the ability of group A Streptococcus (GAS) to colonize the oropharynx, the major site of GAS infection in humans. Here we used a multifaceted approach to elucidate the function and binding characteristics of GAS MalE. We found that GAS MalE is a central part of a highly efficient maltodextrin transport system capable of transporting linear maltodextrins that are up to at least seven glucose molecules long. Of the carbohydrates tested, GAS MalE had the highest affinity for maltotriose, a major breakdown product of starch in the human oropharynx. The thermodynamics and fluorescence changes induced by GAS MalE-maltodextrin binding were essentially opposite those reported for E. coli MalE. Moreover, unlike E. coli MalE, GAS MalE exhibited no specific binding of maltose or cyclic maltodextrins. Our data show that GAS developed a transport system optimized for linear maltodextrins longer than two glucose molecules that has several key differences from its well-studied E. coli counterpart.

Phospholipases are ubiquitous and diverse enzymes that induce changes in membrane composition, activate the inflammatory cascade and alter cell signaling pathways. Recent evidence suggests that certain bacterial pathogens have acquired genes encoding secreted phospholipase A 2 enzymes through lateral gene transfer events. The two best-studied members of this class of enzyme are ExoU and SlaA, which are produced by Pseudomonas aeruginosa and group A Streptococcus , respectively. These enzymes modulate the host inflammatory response, increase the severity of disease and otherwise alter host–pathogen interactions. We propose that a key function of ExoU and SlaA is to increase the fitness of the subclones expressing these enzymes, thereby increasing the population size of the PLA 2 -positive strains and enhancing the likelihood of encountering an at-risk host.

Background Alcohol mediates detrimental alterations in the immune response to Mycobacterium tuberculosis. The association between quantity and frequency of alcohol use and the prevalence of cavitary disease in tuberculosis (TB) has not been analyzed. To investigate the relationship of alcohol use and the prevalence of cavitary disease in a 6-year population-based data set of individuals with TB. Methods We assessed quantity and frequency of alcohol use (daily alcohol use, years of alcohol use, and lifetime alcohol use) with a standardized questionnaire. The study group consisted of 1,250 patients analyzed for cavitary disease (HIV sero-negative subjects that were 18 years or older). Significant covariates for cavitary disease were entered into multiple logistic regression models. Results Although daily alcohol use, years of alcohol use, and alcohol use 30 days or 6 months before symptom onset were significant predictors of cavitary disease in univariate analysis, no independent associations were found between alcohol use and cavitary disease in the multivariate analysis. Only diabetes mellitus was independently associated with cavitary disease at any level or frequency of alcohol use. Conclusion Alcohol use is not independently associated with increased prevalence of cavitary disease in adult patients with TB.

Strains of emm89 Streptococcus pyogenes have become one of the major causes of invasive infections worldwide in the last 10 years. We recently sequenced the genome of 1,125 emm89 strains and identified three major phylogenetic groups, designated clade 1, clade 2, and the epidemic clade 3. Epidemic clade 3 strains, which now cause the great majority of infections, have two distinct genetic features compared to clade 1 and clade 2 strains. First, all clade 3 organisms have a variant 3 nga promoter region pattern, which is associated with increased production of secreted cytolytic toxins SPN (S. pyogenes NADase) and SLO (streptolysin O). Second, all clade 3 strains lack the hasABC locus mediating hyaluronic acid capsule synthesis, whereas this locus is intact in clade 1 and clade 2 strains. We constructed isogenic mutant strains that produce different levels of SPN and SLO toxins and capsule (none, low, or high). Here we report that emm89 strains with elevated toxin production are significantly more virulent than low-toxin producers. Importantly, we also show that capsule production is dispensable for virulence in strains that already produce high levels of SPN and SLO. Our results provide new understanding about the molecular mechanisms contributing to the rapid emergence and molecular pathogenesis of epidemic clade 3 emm89 S. pyogenes.S. pyogenes (group A streptococcus [GAS]) causes pharyngitis ("strep throat"), necrotizing fasciitis, and other human infections. Serious infections caused by emm89 S. pyogenes strains have recently increased in frequency in many countries. Based on whole-genome sequence analysis of 1,125 strains recovered from patients on two continents, we discovered that a new emm89 clone, termed clade 3, has two distinct genetic features compared to its predecessors: (i) absence of the genes encoding antiphagocytic hyaluronic acid capsule virulence factor and (ii) increased production of the secreted cytolytic toxins SPN and SLO. emm89 S. pyogenes strains with the clade 3 phenotype (absence of capsule and high expression of SPN and SLO) are highly virulent in mice. These findings provide new understanding of how new virulent clones emerge and cause severe infections worldwide. This newfound knowledge of S. pyogenes virulence can be used to help understand future epidemics and conduct new translational research.

Strains of emm89 Streptococcus pyogenes have become one of the major causes of invasive infections worldwide in the last 10 years. We recently sequenced the genome of 1,125 emm89 strains and identified three major phylogenetic groups, designated clade 1, clade 2, and the epidemic clade 3. Epidemic clade 3 strains, which now cause the great majority of infections, have two distinct genetic features compared to clade 1 and clade 2 strains. First, all clade 3 organisms have a variant 3 nga promoter region pattern, which is associated with increased production of secreted cytolytic toxins SPN (S. pyogenes NADase) and SLO (streptolysin O). Second, all clade 3 strains lack the hasABC locus mediating hyaluronic acid capsule synthesis, whereas this locus is intact in clade 1 and clade 2 strains. We constructed isogenic mutant strains that produce different levels of SPN and SLO toxins and capsule (none, low, or high). Here we report that emm89 strains with elevated toxin production are significantly more virulent than low-toxin producers. Importantly, we also show that capsule production is dispensable for virulence in strains that already produce high levels of SPN and SLO. Our results provide new understanding about the molecular mechanisms contributing to the rapid emergence and molecular pathogenesis of epidemic clade 3 emm89 S. pyogenes., IMPORTANCE S. pyogenes (group A streptococcus [GAS]) causes pharyngitis (“strep throat”), necrotizing fasciitis, and other human infections. Serious infections caused by emm89 S. pyogenes strains have recently increased in frequency in many countries. Based on whole-genome sequence analysis of 1,125 strains recovered from patients on two continents, we discovered that a new emm89 clone, termed clade 3, has two distinct genetic features compared to its predecessors: (i) absence of the genes encoding antiphagocytic hyaluronic acid capsule virulence factor and (ii) increased production of the secreted cytolytic toxins SPN and SLO. emm89 S. pyogenes strains with the clade 3 phenotype (absence of capsule and high expression of SPN and SLO) are highly virulent in mice. These findings provide new understanding of how new virulent clones emerge and cause severe infections worldwide. This newfound knowledge of S. pyogenes virulence can be used to help understand future epidemics and conduct new translational research.

Despite the high frequency of asymptomatic carriage of bacterial pathogens, we understand little about the bacterial molecular genetic underpinnings of this phenomenon. To obtain new information about the molecular genetic mechanisms underlying carriage of group A Streptococcus (GAS), we performed whole-genome sequencing of GAS strains recovered from a single individual during acute pharyngitis and subsequent asymptomatic carriage. We discovered that compared to the initial infection isolate, the strain recovered during asymptomatic carriage contained three single nucleotide polymorphisms, one of which was in a highly conserved region of a gene encoding a sensor kinase, liaS , resulting in an arginine-to-glycine amino acid replacement at position 135 of LiaS (LiaS R135G ). Using gene replacement, we demonstrate that introduction of the carrier allele ( liaS R135G ) into a serotype-matched invasive strain increased mouse nasopharyngeal colonization and adherence to cultured human epithelial cells. The carrier mutation also resulted in a reduced ability to grow in human blood and reduced virulence in a mouse model of necrotizing fasciitis. Repair of the mutation in the GAS carrier strain restored virulence and decreased adherence to cultured human epithelial cells. We also provide evidence that the carrier mutation alters the GAS transcriptome, including altered transcription of GAS virulence genes, providing a potential mechanism for the pleiotropic phenotypic effects. Our data obtained using isogenic strains suggest that the liaS R135G mutation in the carrier strain contributes to the transition from disease to asymptomatic carriage and provides new information about this poorly described regulatory system in GAS.

Group A Streptococcus (GAS) predominantly exists as a colonizer of the human oropharynx that occasionally breaches epithelial barriers to cause invasive diseases. Despite the frequency of GAS carriage, few investigations into the contributory molecular mechanisms exist. To this end, we identified a naturally occurring polymorphism in the gene encoding the streptococcal collagen-like protein A (SclA) in GAS carrier strains. All previously sequenced invasive serotype M3 GAS possess a premature stop codon in the sclA gene truncating the protein. The carrier polymorphism is predicted to restore SclA function and was infrequently identified by targeted DNA sequencing in invasive strains of the same serotype. We demonstrate that a strain with the carrier sclA allele expressed a full-length SclA protein, while the strain with the invasive sclA allele expressed a truncated variant. An isoallelic mutant invasive strain with the carrier sclA allele exhibited decreased virulence in a mouse model of invasive disease and decreased multiplication in human blood. Further, the isoallelic invasive strain with the carrier sclA allele persisted in the mouse nasopharynx and had increased adherence to cultured epithelial cells. Repair of the premature stop codon in the invasive sclA allele restored the ability to bind the extracellular matrix proteins laminin and cellular fibronectin. These data demonstrate that a mutation in GAS carrier strains increases adherence and decreases virulence and suggest selection against increased adherence in GAS invasive isolates.

Analysis of multiple group A Streptococcus (GAS) genomes shows that genes encoding proteins involved in carbohydrate utilization comprise some 15% of the core GAS genome. Yet there is a limited understanding of how carbohydrate utilization contributes to GAS pathogenesis. Previous genome-wide GAS studies led us to a focused investigation of MalE, a putative maltodextrin-binding protein. Analysis of 28 strains of 22 distinct M protein serotypes showed that MalE is highly conserved among diverse GAS strains. malE transcript levels were significantly increased during growth in human saliva compared to growth in a chemically defined glucose-containing medium or a nutrient-rich medium. MalE was accessible to antibody binding, indicating that it is expressed on the GAS cell surface. Moreover, growth in human saliva appeared to increase MalE surface expression compared to growth in a nutrient-rich medium. Analysis of a Δ malE isogenic mutant strain revealed decreased growth in human saliva compared to wild-type GAS. Radiolabeled carbohydrate binding assays showed that MalE was required for the binding of maltose but not glucose. The Δ malE isogenic mutant strain colonized a lower percentage of GAS-challenged mice compared to wild-type and genetically complemented strains. Furthermore, decreased numbers of CFU were recovered from mice infected with the Δ malE strain compared to those infected with wild-type GAS. These data demonstrate that maltodextrin acquisition is likely to be a key factor in the ability of GAS to successfully infect the oropharynx. Further investigation into carbohydrate transport and metabolism pathways may yield novel insights into GAS pathogenesis.

Group AStreptococcus(GAS) is a gram-positive human bacterial pathogen that causes infections ranging in severity from pharyngitis to life-threatening invasive disease, such as necrotizing fasciitis. Serotype M28 strains are consistently isolated from invasive infections, particularly puerperal sepsis, a severe infection that occurs during or after childbirth. We recently sequenced the genome of a serotype M28 GAS strain and discovered a novel 37.4-kb foreign genetic element designated region of difference 2 (RD2). RD2 is similar in gene content and organization to genomic islands found in group B streptococci (GBS), the major cause of neonatal infections. RD2 encodes seven proteins with conventional gram-positive secretion signal sequences, six of which have not been characterized. Herein, we report that one of these six proteins (M28_Spy1325; Spy1325) is a member of the antigen I/II family of cell surface-anchored molecules produced by oral streptococci. PCR and DNA sequence analysis found that Spy1325 is very well conserved in GAS strains of distinct M protein serotypes. As assessed by real-time TaqMan quantitative PCR, the Spy1325 gene was expressed in vitro, and Spy1325 protein was present in culture supernatants and on the GAS cell surface. Western immunoblotting and enzyme-linked immunosorbent assays indicated that Spy1325 was produced by GAS in infected mice and humans. Importantly, the immunization of mice with recombinant Spy1325 fragments conferred protection against GAS-mediated mortality. Similar to other antigen I/II proteins, recombinant Spy1325 bound purified human salivary agglutinin glycoprotein. Spy1325 may represent a shared virulence factor among GAS, GBS, and oral streptococci.

In recent years we have studied the relationship between strain genotypes and patient phenotypes in group A Streptococcus (GAS), a model human bacterial pathogen that causes extensive morbidity and mortality worldwide. We have concentrated our efforts on serotype M3 organisms because these strains are common causes of pharyngeal and invasive infections, produce unusually severe invasive infections, and can exhibit epidemic behavior. Our studies have been hindered by the lack of genome-scale phylogenies of multiple GAS strains and whole-genome sequences of multiple serotype M3 strains recovered from individuals with defined clinical phenotypes. To remove some of these impediments, we sequenced to closure the genome of four additional GAS strains and conducted comparative genomic resequencing of 12 contemporary serotype M3 strains representing distinct genotypes and phenotypes. Serotype M3 strains are a single phylogenetic lineage. Strains from asymptomatic throat carriers were significantly less virulent for mice than sterile-site isolates and evolved to a less virulent phenotype by multiple genetic pathways. Strain persistence or extinction between epidemics was strongly associated with presence or absence, respectively, of the prophage encoding streptococcal pyrogenic exotoxin A. A serotype M3 clone significantly underrepresented among necrotizing fasciitis cases has a unique frameshift mutation that truncates MtsR, a transcriptional regulator controlling expression of genes encoding iron-acquisition proteins. Expression microarray analysis of this clone confirmed significant alteration in expression of genes encoding iron metabolism proteins. Our analysis provided unprecedented detail about the molecular anatomy of bacterial strain genotype-patient phenotype relationships.

SummaryGroup A Streptococcus secretes cysteine proteases named Mac-1 and Mac-2 that mediate host immune evasion by targeting both IgG and Fc receptors. Here, we report the crystal structures of Mac-1 and its catalytically inactive C94A mutant in two different crystal forms. Despite the lack of sequence homology, Mac-1 adopts the canonical papain fold. Alanine mutations at the active site confirmed the critical residues involved in a papain-like catalytic mechanism. Mac-1 forms a symmetric dimer in both crystal forms and displays the unique dimer interface among papain superfamily members. Mutations at the dimer interface resulted in a significant reduction in IgG binding and catalysis, suggesting that the dimer contributes to both IgG specificity and enzyme cooperativity. A tunnel observed at the dimer interface constitutes a target for designing potential Mac-1-specific antimicrobial agents. The structures also offer insight into the functional difference between Mac-1 and Mac-2.

The molecular genetic mechanisms used by bacteria to persist in humans are poorly understood. Group A Streptococcus (GAS) causes the majority of bacterial pharyngitis cases in humans and is prone to persistently inhabit the upper respiratory tract. To gain information about how GAS survives in and infects the oropharynx, we analyzed the transcriptome of a serotype M1 strain grown in saliva. The dynamic pattern of changes in transcripts of genes [ spy0874/0875 , herein named sptR and sptS (sptR/S), for saliva persistence] encoding a two-component gene regulatory system of unknown function suggested that SptR/S contributed to persistence of GAS in saliva. Consistent with this idea, an isogenic nonpolar mutant strain (Δ sptR ) was dramatically less able to survive in saliva compared with the parental strain. Iterative expression microarray analysis of bacteria grown in saliva revealed that transcripts of several known and putative GAS virulence factor genes were decreased significantly in the Δ sptR mutant strain. Compared with the parental strain, the isogenic mutant strain also had altered transcripts of multiple genes encoding proteins involved in complex carbohydrate acquisition and utilization pathways. Western immunoblot analysis and real-time PCR analysis of GAS in throat swabs taken from humans with pharyngitis confirmed the findings. We conclude that SptR/S optimizes persistence of GAS in human saliva, apparently by strategically influencing metabolic pathways and virulence factor production. The discovery of a genetic program that significantly increased persistence of a major human pathogen in saliva enhances understanding of how bacteria survive in the host and suggests new therapeutic strategies.

Polymorphonuclear leukocytes (PMNs, or neutrophils) are critical for human innate immunity and kill most invading bacteria. However, pathogens such as Staphylococcus aureus avoid destruction by PMNs to survive, thereby causing human infections. The molecular mechanisms used by pathogens to circumvent killing by the immune system remain largely undefined. To that end, we studied S. aureus pathogenesis and bacteria-PMN interactions using strains originally isolated from individuals with community-acquired (CA) and hospital-acquired infections. Compared with strains from hospital infections (COL and MRSA252), strain MW2 and a methicillin-susceptible relative, MnCop, were significantly more virulent in a mouse model of S. aureus infection, and caused the greatest level of pathology in major vital organs. Although phagocytosis of each strain triggered production of reactive oxygen species and granule-phagosome fusion, those from CA infections were significantly more resistant to killing by human PMNs and caused greater host cell lysis. Microarray analysis of the strains during neutrophil phagocytosis identified genes comprising a global S. aureus response to human innate host defense. Genes involved in capsule synthesis, gene regulation, oxidative stress, and virulence, were up-regulated following ingestion of the pathogen. Notably, phagocytosis of strains from CA infections induced changes in gene expression not observed in the other strains, including up-regulation of genes encoding virulence factors and hypothetical proteins. Our studies reveal a gene transcription program in a prominent human pathogen that likely contributes to evasion of innate host defense.

To better understand the molecular events involved in the origin of new pathogenic bacteria, we studied the evolution of a highly virulent clone of serotype M1 group A Streptococcus (GAS). Genomic, DNA-DNA microarray, and single-nucleotide polymorphism analyses indicated that this clone evolved through a series of horizontal gene transfer events that involved (1) the acquisition of prophages encoding streptococcal pyrogenic exotoxin A and extracellular DNases and (2) the reciprocal recombination of a 36-kb chromosomal region encoding the extracellular toxins NAD+-glycohydrolase (NADase) and streptolysin O (SLO). These gene transfer events were associated with significantly increased production of SLO and NADase. Virtual identity in the 36-kb region present in contemporary serotype M1 and M12 isolates suggests that a serotype M12 strain served as the donor of this region. Multiple horizontal gene transfer events were a crucial factor in the evolutionary origin and emergence of a very abundant contemporary clone of serotype M1 GAS.

agr is a global regulatory system in the staphylococci, operating by a classical two-component signaling module and controlling the expression of most of the genes encoding extracellular virulence factors. As it is autoinduced by a peptide, encoded within the locus, that is the ligand for the signal receptor, it is a sensor of population density or a quorum sensor and is the only known quorum-sensing system in the genus. agr is conserved throughout the staphylococci but has diverged along lines that appear to parallel speciation and subspeciation within the genus. This divergence has given rise to a novel type of interstrain and interspecies cross-inhibition that represents a fundamental aspect of the organism's biology and may be a predominant feature of the evolutionary forces that have driven it. We present evidence, using a newly developed, luciferase-based agr typing scheme, that the evolutionary divergence of the agr system was an early event in the evolution of the staphylococci and long preceded the development of the nucleotide polymorphisms presently used for genotyping. These polymorphisms developed, for the most part, within different agr groups; mobile genetic elements appear also to have diffused recently and, with a few notable exceptions, have come to reside largely indiscriminately within the several agr groups.

Group A Streptococcus (GAS) commonly infects the human oropharynx, but the initial molecular events governing this process are poorly understood. Saliva is a major component of the innate and acquired immune defense in this anatomic site. Although landmark studies were done more than 60 years ago, investigation of GAS-saliva interaction has not been addressed extensively in recent years. Serotype M1 GAS strain MGAS5005 cultured in human saliva grew to ∼10 7 CFU/ml and, remarkably, maintained this density for up to 28 days. Strains of several other M-protein serotypes had similar initial growth patterns but did not maintain as high a CFU count during prolonged culture. As revealed by analysis of the growth of isogenic mutant strains, the ability of GAS to maintain high numbers of CFU/ml during the prolonged stationary phase in saliva was dependent on production of streptococcal inhibitor of complement (Sic) and streptococcal pyrogenic exotoxin B (SpeB). During cultivation in human saliva, GAS had growth-phase-dependent production of multiple proven and putative extracellular virulence factors, including Sic, SpeB, streptococcal pyrogenic exotoxin A, Mac protein, and streptococcal phospholipase A 2 . Our results clearly show that GAS responds in a complex fashion to growth in human saliva, suggesting that the molecular processes that enhance colonization and survival in the upper respiratory tract of humans are well under way before the organism reaches the epithelial cell surface.

Genome sequencing of group A Streptococcus (GAS) has revealed that prophages account for the vast majority of gene content differences between strains. Serotype M28 strains are a leading cause of pharyngitis and invasive infections, but little is known about genetic diversity present in natural populations of these organisms. To study this issue, population-based samples of 568 strains from Ontario, Canada; Finland; and Houston, Texas, were analyzed. Special attention was given to analysis of variation in prophage-encoded virulence gene content by a PCR-based method. Thirty and 29 distinct prophage-encoded virulence gene profiles were identified among pharyngitis and invasive infection isolates. Thirteen profiles, representing the majority of the strains, were shared between these two classes of isolates. Significant differences were observed in the frequency of occurrence of certain prophage toxin gene profiles and infection type. M28 strains are highly diverse in prophage-encoded virulence gene content and integration site, supporting the key concept that prophages are critical contributors to GAS genetic diversity and population biology. Nucleotide sequence variation in the emm gene (encodes M protein) was also examined. Only three allelic variants were identified in the hypervariable portion of the emm28 gene. All but one strain had the same inferred amino acid sequence in the first 100 amino acids of the mature M28 protein. In contrast, size differences in the emm28 gene and inferred protein due to variable numbers of C-terminal repeats were common. The presence of macrolide resistance genes ( mefA , ermB , and ermTR ) was analyzed by PCR, and less than 2% of the strains were positive.

The molecular basis for bacterial responses to host signals during natural infections is poorly understood. The gram-positive bacterial pathogen group A Streptococcus (GAS) causes human mucosal, skin, and life-threatening systemic infections. During the transition from a throat or skin infection to an invasive infection, GAS must adapt to changing environments and host factors. To better understand how GAS adapts, we used transcript profiling and functional analysis to investigate the transcriptome of a wild-type serotype M1 GAS strain in human blood. Global changes in GAS gene expression occur rapidly in response to human blood exposure. Increased transcription was observed for many genes that likely enhance bacterial survival, including those encoding superantigens and host-evasion proteins regulated by a multiple gene activator called Mga. GAS also coordinately expressed genes involved in proteolysis, transport, and catabolism of oligopeptides to obtain amino acids in this protein-rich host environment. Comparison of the transcriptome of the wild-type strain to that of an isogenic deletion mutant (DeltacovR) mutated in the two-component regulatory system designated CovR-CovS reinforced the hypothesis that CovR-CovS has an important role linking key biosynthetic, catabolic, and virulence functions during transcriptome restructuring. Taken together, the data provide crucial insights into strategies used by pathogenic bacteria for thwarting host defenses and surviving in human blood.

In a series of four articles published between 1916 and 1919 in The Journal of Medical Research , precursor to The American Journal of Pathology , the investigative pathologist S. Burt Wolbach unambiguously showed that Rocky Mountain spotted fever has a tick-borne mode of transmission, the causative agent replicates intracellularly, and the disease is fundamentally a vasculitis. Although underappreciated, Wolbach's tour-de-force work epitomized investigative pathology. These four articles should be mandatory reading for young investigators and are recommended also to seasoned investigators who seek reinvigoration in the beauty in their craft.

Human polymorphonuclear leukocytes (PMNs) are the first line of defense against invading microorganisms. Although most invading bacteria are eliminated by PMNs, some have evolved complex strategies to prevent normal PMN function. This review focuses on the interaction of human PMNs with Streptococcus pyogenes as a paradigm for successful pathogen evasion mechanisms.

We describe the genome sequence of a macrolide-resistant strain (MGAS10394) of serotype M6 group A Streptococcus (GAS). The genome is 1,900,156 bp in length, and 8 prophage-like elements or remnants compose 12.4% of the chromosome. A 8.3-kb prophage remnant encodes the SpeA4 variant of streptococcal pyrogenic exotoxin A. The genome of strain MGAS10394 contains a chimeric genetic element composed of prophage genes and a transposon encoding the mefA gene conferring macrolide resistance. This chimeric element also has a gene encoding a novel surface-exposed protein (designated "R6 protein"), with an LPKTG cell-anchor motif located at the carboxyterminus. Surface expression of this protein was confirmed by flow cytometry. Humans with GAS pharyngitis caused by serotype M6 strains had antibody against the R6 protein present in convalescent, but not acute, serum samples. Our studies add to the theme that GAS prophage-encoded extracellular proteins contribute to host-pathogen interactions in a strain-specific fashion.

Neutrophils are a critical component of human innate host defense and efficiently kill the vast majority of invading microorganisms. However, bacterial pathogens such as group A Streptococcus (GAS) successfully avert destruction by neutrophils to cause human infections. Relatively little is known about how pathogens detect components of the innate immune system to respond and survive within the host. In this study, we show that inactivation of a two-component gene regulatory system designated Ihk-Irr significantly attenuates streptococcal virulence in mouse models of soft tissue infection and bacteremia. Microarray analysis of wild-type and irr-negative mutant (irr mutant) GAS strains revealed that Ihk-Irr influenced expression of 20% of all transcripts in the pathogen genome. Notably, at least 11 genes involved in cell wall synthesis, turnover, and/or modification were down-regulated in the irr mutant strain. Compared with the wild-type strain, significantly more of the irr mutant strain was killed by human neutrophil components that destroy bacteria by targeting the cell envelope (cell wall and/or membrane). Unexpectedly, expression of ihk and irr was dramatically increased in the wild-type strain exposed to these same neutrophil products under conditions that favored cell envelope damage. We report a GAS mechanism for detection of innate host defense that initiates the pathogen survival response, in which cell wall synthesis is critical. Importantly, our studies identify specific genes in the pathogen survival response as potential targets to control human infections.

Different polymorphisms have been described in the minimal promoter region (MPR) of the interferon-γ receptor 1 (IFNGR1), a molecule that plays a critical role in mycobacterial control. We sequenced the IFNGR1 MPR from African American, Caucasian and Korean controls, and from mycobacteria-infected patients. Six different single nucleotide polymorphisms (SNPs) were detected in the IFNGR1 MPR. The three ethnic groups showed different SNP distribution patterns, but no significant differences were detected between mycobacterial cases and controls. Two polymorphisms were found in all populations (G-611A, T-56C). We cloned the four allelic variants (var) of haplotype G-611A/T-56C into a luciferase reporter vector and determined their promoter activity. Polymorphisms at position -611 had a stronger effect on the promoter activity than those at position -56, and constructs carrying G-611 produced a stronger promoter activity than -611A constructs. The IFNGR1 MPR is a polymorphic region with at least two SNPs influencing its activity, but these are not associated with increased mycobacterial susceptibility.

Objective. Our objective was to confirm an observed temporal association of increased numbers of rheumatic fever cases concomitant with the appearance of an increased prevalence of mucoid strains of Streptococcus pyogenes. During the resurgence of rheumatic fever (RF) that has occurred in the Intermountain area surrounding Salt Lake City, Utah, since 1985, the largest number of cases occurred in 1985 and 1986 and 12 years later in 1997 and 1998. During the initial outbreak and the later exacerbation of the resurgence, an increased number of mucoid strains of S pyogenes were present in the community.Methods. The referred cases of RF that fulfilled Jones criteria have been systematically reviewed by the medical staff at Primary Children’s Medical Center yearly since 1985. Before the resurgence of RF, a program was initiated by the microbiology laboratory at Primary Children’s Medical Center to store frozen isolates of S pyogenes. All frozen specimens were randomly selected and entered into a log; the coded entry allowed for comments regarding the origin of the isolate and whether the isolate had a mucoid appearance on the blood agar culture plate. This log was reviewed in October 2002 to determine whether the percentage of frozen mucoid isolates stored during the resurgence of RF would support the clinical and epidemiologic suspicion that mucoid isolates seemed to be present with a higher frequency during the 2 periods of high incidence of RF. The percentage of mucoid isolates was compared with the yearly number of cases of RF. A Pearson r correlation analysis was completed to determine whether there was a significant association between the percentage of mucoid isolates and the number of cases of RF.Results. The highest number of cases of RF was temporally associated with the highest percentage of mucoid isolates. There was statistically significant correlation between percentage of mucoid strains and the number of RF cases.Conclusions. The Utah experience with the resurgence of RF in a civilian population during the last decade and a half of the 20th century confirmed the temporal association of mucoid strains of S pyogenes, primarily M-type 18, with a high incidence of RF.

We have identified a Crp/Fnr-like transcriptional regulator of Streptococcus pyogenes that when inactivated attenuates virulence. The gene, named srv for s treptococcal regulator of virulence, encodes a 240-amino-acid protein with 53% amino acid similarity to PrfA, a transcriptional activator of virulence in Listeria monocytogenes .

Strain typing of microbial pathogens has two major aims: (i) to index genetic microvariation for use in outbreak investigations and (ii) to index genetic macrovariation for use in phylogenetic and population-based analyses. Until now, there has been no clear indication that one genetic marker can efficiently be used for both purposes. Previously, we had shown that DNA sequence analysis of the protein A gene variable repeat region ( spa typing) provides a rapid and accurate method to discriminate Staphylococcus aureus outbreak isolates from those deemed epidemiologically unrelated. Here, using the hypothesis that the genetic macrovariation within a low-level recombinogenic species would accurately be characterized by a single-locus marker, we tested whether spa typing could congruently index the extensive genetic variation detected by a whole-genome DNA microarray in a collection of 36 isolates, which was recovered from 10 countries on four continents over a period of four decades, that is representative of the breadth of diversity within S. aureus. Using spa and coa typing, pulsed-field gel electrophoresis (PFGE), and microarray and multilocus enzyme electrophoresis (MLEE) data in molecular epidemiologic and evolutionary analyses, we determined that S. aureus likely has a primarily clonal population structure and that spa typing can singly index genetic variation with 88% direct concordance with the microarray and can correctly assign isolates to phylogenetic lineages. spa typing performed better than MLEE, PFGE, and coa typing in discriminatory power and in the degree of agreement with the microarray at various phylogenetic depths. This study showed that genetic analysis of the repeat region of protein A comprehensively characterizes both micro- and macrovariation in the primarily clonal population structure of S. aureus .

The resistance of group A Streptococcus (GAS) to macrolide antibiotics is now a worldwide problem. Preliminary sequencing of the genome of an erythromycin-resistant serotype M6 clone that was responsible for a pharyngitis outbreak in Pittsburgh, Pennsylvania, was conducted to determine the structure of the genetic element containing the mefA gene, which encodes a macrolide efflux protein. The mefA gene is associated with a 58.8-kb chimeric genetic element composed of a transposon inserted into a prophage. This element also encodes a putative extracellular protein with a cell-wall anchoring motif (LPKTG) located at the carboxyterminus. The mefA element was present in phylogenetically diverse GAS strains isolated throughout the United States. Culture supernatants, prepared after mitomycin C treatment, of a strain representing the outbreak clone contained mefA element DNA in a DNAse-resistant form. Together, these data provide new information about the molecular genetic basis of macrolide resistance and dissemination in GAS strains.

Leucine-rich repeats (LRR) characterize a diverse array of proteins and function to provide a versatile framework for protein-protein interactions. Importantly, each of the bacterial LRR proteins that have been well described, including those of Listeria monocytogenes , Yersinia pestis , and Shigella flexneri , have been implicated in virulence. Here we describe an 87.4-kDa group A Streptococcus (GAS) protein (designated Slr, for streptococcal leucine-rich) containing 10 1/2 sequential units of a 22-amino-acid C-terminal LRR homologous to the LRR of the L. monocytogenes internalin family of proteins. In addition to the LRR domain, slr encodes a gram-positive signal secretion sequence characteristic of a lipoprotein and a putative N-terminal domain with a repeated histidine triad motif (HxxHxH). Real-time reverse transcriptase PCR assays indicated that slr is transcribed abundantly in vitro in the exponential phase of growth. Flow cytometry confirmed that Slr was attached to the GAS cell surface. Western immunoblot analysis of sera obtained from 80 patients with invasive infections, noninvasive soft tissue infections, pharyngitis, and rheumatic fever indicated that Slr is produced in vivo. An isogenic mutant strain lacking slr was significantly less virulent in an intraperitoneal mouse model of GAS infection and was significantly more susceptible to phagocytosis by human polymorphonuclear leukocytes. These studies characterize the first GAS LRR protein as an extracellular virulence factor that contributes to pathogenesis and may participate in evasion of the innate host defense.

A secreted, hypervariable virulence factor called the streptococcal inhibitor of complement (Sic) has been linked to the reemergence of epidemics due to the human pathogenic bacterium Group A Streptococcus. This paper describes a method for expressing and purifying Sic from an attenuated GAS strain using a chemically defined growth medium. This method was used to label specific amino acid residue types in Sic with forms containing the magnetically active isotope (15)N, at the amide nitrogen. The (15)N-labeling of Sic permits a detailed investigation of the structure and dynamics of the protein using nuclear magnetic resonance spectroscopy. The level of stable isotope incorporation was established using mass spectrometry and nuclear magnetic resonance spectroscopy.

In bacteria, translation initiates with formyl-methionine; however, the N-terminal formyl group is usually removed by peptide deformylase, an enzymatic activity requiring iron. Staphylococcus aureus δ-toxin is a 26-amino-acid polypeptide secreted predominantly with a formylated N-terminal methionine, which led us to investigate regulation of δ-toxin deformylation. We observed that during exponential and early postexponential growth, δ-toxin accumulated in the culture medium in formylated and deformylated forms. In contrast, only formylated δ-toxin accumulated after the early postexponential phase. The transition from producing both species of δ-toxin to producing only formyl-methionine-containing δ-toxin coincided with increased tricarboxylic acid (TCA) cycle activity. The TCA cycle contains several iron-requiring enzymes, which led us to hypothesize that TCA cycle induction depletes the iron in the culture medium, thereby inhibiting peptide deformylase activity. As expected, S. aureus depletes the iron in the culture medium between the postexponential and stationary phases of growth. Inhibition of δ-toxin deformylation was relieved by TCA cycle inactivation or by addition of supplemental iron to the culture medium. Of interest, peptides containing formyl-methionine are potent chemoattractants for neutrophils, suggesting that δ-toxin deformylation may have functional consequences. We found neutrophil chemotactic activity only with formylated δ-toxin. The S. aureus TCA cycle is derepressed upon depletion of rapidly catabolizable carbon sources; this coincides with the transition to producing only formylated δ-toxin and results in an increased inflammatory response. The proinflammatory response should increase host cell damage and result in the release of nutrients. Taken together, these results establish that there is an important linkage between bacterial metabolism and pathogenesis.

Group A streptococci (GAS) can use heme and hemoproteins as sources of iron. However, the machinery for heme acquisition in GAS has not been firmly revealed. Recently, we identified a novel heme-associated cell surface protein (Shp) made by GAS. Theshpgene is cotranscribed with eight downstream genes, includingspy1795,spy1794, andspy1793encoding a putative ABC transporter (designated HtsABC). In this study,spy1795(designatedhtsA) was cloned from a serotype M1 strain, and recombinant HtsA was overexpressed inEscherichia coliand purified to homogeneity. HtsA binds 1 heme molecule per molecule of protein. HtsA was produced in vitro and localized to the bacterial cell surface. GAS up-regulated transcription ofhtsAin human blood compared with that in Todd-Hewitt broth supplemented with 0.2% yeast extract. The level of thehtsAtranscript dramatically increased under metal cation-restricted conditions compared with that under metal cation-replete conditions. The cation content, cell surface location, and gene transcription of HtsA were also compared with those of MtsA and Spy0385, the lipoprotein components of two other putative iron acquisition ABC transporters of GAS. Our results suggest that HtsABC is an ABC transporter that may participate in heme acquisition in GAS.

Interleukin (IL)-8 is involved in the pathogenesis of human tuberculosis (TB). However, the contribution of polymorphisms of the IL-8 gene and its receptor genes CXCR-I and CXCR-2 to human TB susceptibility remains untested. In a case-control study, white subjects with TB disease were more likely to be homozygous for the IL-8 -251A allele, compared with control subjects (odds ratio [OR], 3.41; 95% confidence interval [CI], 1.52-7.64). African Americans with TB also showed an increased odds of being homozygous for this allele (OR, 3.46; 95% Cl, 1.48-8.08). To exclude population artifacts in the case-control study, a separate analysis that used a transmission-disequilibrium test with 76 informative families confirmed that the IL-8 -251A allele was preferentially transmitted to TB-infected children (P = .02). CXCR-I and CXCR-2 did not demonstrate significant associations with TB susceptibility. These data suggest that IL-8 is important in the genetic control of human TB susceptibility.

Tuberculosis and malaria together result in an estimated 5 million deaths annually. The spread of multidrug resistance in the most pathogenic causative agents, Mycobacterium tuberculosis and Plasmodium falciparum, underscores the need to identify active compounds with novel inhibitory properties. Although genetically unrelated, both organisms use a type II fatty-acid synthase system. Enoyl acyl carrier protein reductase (ENR), a key type II enzyme, has been repeatedly validated as an effective antimicrobial target. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the M. tuberculosis and P. falciparum enzyme (referred to as InhA and PfENR, respectively). The crystal structure of InhA complexed with NAD+ and one of the inhibitors was determined to elucidate the mode of binding. Structural analysis of InhA with the broad spectrum antimicrobial triclosan revealed a unique stoichiometry where the enzyme contained either a single triclosan molecule, in a configuration typical of other bacterial ENR:triclosan structures, or harbored two triclosan molecules bound to the active site. Significantly, these compounds do not require activation and are effective against wild-type and drug-resistant strains of M. tuberculosis and P. falciparum. Moreover, they provide broader chemical diversity and elucidate key elements of inhibitor binding to InhA for subsequent chemical optimization.

Forty-eight Mycobacterium tuberculosis strains were obtained from patients living in metropolitan Manila, Republic of the Philippines. Three molecular typing methods, IS 6110 restriction fragment length polymorphism, spoligotyping, and DNA sequencing of the oxyR , gyrA , and katG loci, established that these strains have restricted diversity and are members of a related genetic group of organisms. Comparison of the DNA fingerprint patterns with those in international databases confirmed the uniqueness of this group of isolates, which we designate the Manila family of M. tuberculosis .

Recent genomic studies have revealed extensive variation in natural populations of many pathogenic bacteria. However, the evolutionary processes which contribute to much of this variation remain unclear. A previous whole-genome DNA microarray study identified variation at a large chromosomal region (RD13) of Staphylococcus aureus which encodes a family of proteins with homology to staphylococcal and streptococcal superantigens, designated staphylococcal exotoxin-like (SET) proteins. In the present study, RD13 was found in all 63 S. aureus isolates of divergent clonal, geographic, and disease origins but contained a high level of variation in gene content in different strains. A central variable region which contained from 6 to 10 different set genes, depending on the strain, was identified, and DNA sequence analysis suggests that horizontal gene transfer and recombination have contributed to the diversification of RD13. Phylogenetic analysis based on the RD13 DNA sequence of 18 strains suggested that loss of various set genes has occurred independently several times, in separate lineages of pathogenic S. aureus , providing a model to explain the molecular variation of RD13 in extant strains. In spite of multiple episodes of set deletion, analysis of the ratio of silent substitutions in set genes to amino acid replacements in their products suggests that purifying selection (selective constraint) is acting to maintain SET function. Further, concurrent transcription in vitro of six of the seven set genes in strain COL was detected, indicating that the expression of set genes has been maintained in contemporary strains, and Western immunoblot analysis indicated that multiple SET proteins are expressed during the course of human infections. Overall, we have shown that the chromosomal region RD13 has diversified extensively through episodes of gene deletion and recombination. The coexpression of many set genes and the production of multiple SET proteins during human infection suggests an important role in host-pathogen interactions.

The molecular mechanisms used by group A Streptococcus (GAS) to survive on the host mucosal surface and cause acute pharyngitis are poorly understood. To provide new information about GAS host-pathogen interactions, we used real-time reverse transcription-PCR (RT-PCR) to analyze transcripts of 17 GAS genes in throat swab specimens taken from 18 pediatric patients with pharyngitis. The expression of known and putative virulence genes and regulatory genes (including genes in seven two-component regulatory systems) was studied. Several known and previously uncharacterized GAS virulence gene regulators were highly expressed compared to the constitutively expressed control gene proS . To examine in vivo gene transcription in a controlled setting, three cynomolgus macaques were infected with strain MGAS5005, an organism that is genetically representative of most serotype M1 strains recovered from pharyngitis and invasive disease episodes in North America and Western Europe. These three animals developed clinical signs and symptoms of GAS pharyngitis and seroconverted to several GAS extracellular proteins. Real-time RT-PCR analysis of throat swab material collected at intervals throughout a 12-day infection protocol indicated that expression profiles of a subset of GAS genes accurately reflected the profiles observed in the human pediatric patients. The results of our study demonstrate that analysis of in vivo GAS gene expression is feasible in throat swab specimens obtained from infected human and nonhuman primates. In addition, we conclude that the cynomolgus macaque is a useful nonhuman primate model for the study of molecular events contributing to acute pharyngitis caused by GAS.

Group A Streptococcus (GAS) evades polymorphonuclear leukocyte (PMN) phagocytosis and killing to cause human disease, including pharyngitis and necrotizing fasciitis (flesh-eating syndrome). We show that GAS genes differentially regulated during phagocytic interaction with human PMNs comprise a global pathogen-protective response to innate immunity. GAS prophage genes and genes involved in virulence, oxidative stress, cell wall biosynthesis, and gene regulation were up-regulated during PMN phagocytosis. Genes encoding novel secreted proteins were up-regulated, and the proteins were produced during human GAS infections. We discovered an essential role for the Ihk-Irr two-component regulatory system in evading PMN-mediated killing and promoting host–cell lysis, processes that would facilitate GAS pathogenesis. Importantly, the irr gene was highly expressed during human GAS pharyngitis. We conclude that a complex pathogen genetic program circumvents human innate immunity to promote disease. The gene regulatory program revealed by our studies identifies previously undescribed potential vaccine antigens and targets for therapeutic interventions designed to control GAS infections.

Human polymorphonuclear leukocytes (PMNs) are an essential part of innate immunity and contribute significantly to inflammation. Although much is nderstood about the inflammatory response, the molecular basis for termination of inflammation in humans is largely undefined. We used human oligonucleotide microarrays to identify genes differentially regulated during the onset of apoptosis occurring after PMN phagocytosis. Genes encoding proteins that regulate cell metabolism and vesicle trafficking comprised 198 (98 genes induced, 100 genes repressed) of 867 differentially expressed genes. We discovered that complex cellular pathways involving glutathione and thioredoxin detoxification systems, heme catabolism, ubiquitin-proteasome degradation, purine nucleotide metabolism, and nuclear import were regulated at the level of gene expression during the initial stages of PMN apoptosis. Eleven genes encoding key regulators of glycolysis, the hexose monophosphate shunt, the glycerol-phosphate shuttle, and oxidative phosphorylation were induced. Increased levels of cellular reduced glutathione and γ-glutamyltransferase and glycolytic activity confirmed that several of these metabolic pathways were up-regulated. In contrast, seven genes encoding critical enzymes involved in fatty acid β-oxidation, which can generate toxic lipid peroxides, were down-regulated. Our results indicate that energy metabolism and oxidative stress-response pathways are gene-regulated during PMN apoptosis. We propose that changes in PMN gene expression leading to programmed cell death are part of an apoptosis-differentiation program, a final stage of transcriptionally regulated PMN maturation that is accelerated significantly by phagocytosis. These findings provide new insight into the molecular events that contribute to the resolution of inflammation in humans.

Mycobacterium tuberculosis KatG catalyzes the activation of the antitubercular agent isoniazid to yield an inhibitor targeting enoyl reductase (InhA). However, no firm biochemical link between many KatG variants and isoniazid resistance has been established. In the present study, six distinct KatG variants identified in clinical Mycobacterium tuberculosis isolates resistant to isoniazid were generated by site-directed mutagenesis, and the recombinant mutant proteins (KatG A110V , KatG A139P , KatG S315N , KatG L619P , KatG L634F , and KatG D735A ) were purified and characterized with respect to their catalase-peroxidase activities (in terms of k cat / K m ), rates of free-radical formation from isoniazid oxidation, and, moreover, abilities to activate isoniazid. The A110V amino acid replacement did not result in significant alteration of KatG activities except that the peroxidase activity was enhanced. The other mutations, however, resulted in modestly reduced catalase and peroxidase catalytic efficiencies and, for the four mutants tested, significantly lower activities to oxidize isoniazid. Compared to the wild-type enzyme, the ability of the KatG L634F , KatG A139P , and KatG D735A variants to activate isoniazid decreased by 36%, 76%, and 73%, respectively, whereas the KatG S315N and KatG L619P variants completely lost their abilities to convert isoniazid into the InhA inhibitor. In addition, the inclusion of exogenous Mn 2+ to the isoniazid activation reaction mix significantly improved the ability of wild-type and KatG mutants to produce the InhA inhibitor.

Genomic analysis of type emm59 group A Streptococcus invasive strains isolated in the United States discovered higher than anticipated genetic heterogeneity among strains and identified a heretofore unrecognized monoclonal cluster of invasive infections in the San Francisco Bay area. Heightened monitoring for a potential shift in the epidemic behavior of emm59 group A Streptococcus is warranted.

Recently, it was reported that a streptococcal Mac protein (designated Mac 5005 ) made by serotype M1 group A Streptococcus (GAS) is a homologue of human CD11b that inhibits opsonophagocytosis and killing of GAS by human polymorphonuclear leukocytes (PMNs) (B. Lei, F. R. DeLeo, N. P. Hoe, M. R. Graham, S. M. Mackie, R. L. Cole, M. Liu, H. R. Hill, D. E. Low, M. J. Federle, J. R. Scott, and J. M. Musser, Nat. Med. 7: 1298-1305, 2001). To study mac variation and expression of the Mac protein, the gene in 67 GAS strains representing 36 distinct M protein serotypes was sequenced. Two distinct genetic complexes were identified, and they were designated complex I and complex II. Mac variants in each of the two complexes were closely related, but complex I and complex II variants differed on average at 50.66 ± 5.8 amino acid residues, most of which were located in the middle one-third of the protein. Complex I Mac variants have greater homology with CD11b than complex II variants. GAS strains belonging to serotypes M1 and M3, the most abundant M protein serotypes responsible for human infections in many case series, have complex I Mac variants. The mac gene was cloned from representative strains assigned to complexes I and II, and the Mac proteins were purified to apparent homogeneity. Both Mac variants had immunoglobulin G (IgG)-endopeptidase activity. In contrast to Mac 5005 (complex I), Mac 8345 (complex II) underwent autooxidation of its cysteine residues, resulting in the loss of IgG-endopeptidase activity. A Mac 5005 Cys94Ala site-specific mutant protein was unable to cleave IgG but retained the ability to inhibit IgG-mediated phagocytosis by human PMNs. Thus, the IgG-endopeptidase activity was not essential for the key biological function of Mac 5005 . Although Mac 5005 and Mac 8345 each have an Arg-Gly-Asp (RGD) motif, the proteins differed in their interactions with human integrins α v β 3 and α IIb β 3 . Binding of Mac 5005 to integrins α v β 3 and α IIb β 3 was mediated primarily by the RGD motif in Mac 5005 , whereas binding of Mac 8345 involved the RGD motif and a region in the middle one-third of the molecule whose sequence is different in Mac 8345 and Mac 5005 . Taken together, the data add to the emerging theme in GAS pathogenesis that allelic variation in virulence genes contributes to fundamental differences in host-pathogen interactions among strains.

The pathogenesis of acute rheumatic fever (ARF) is poorly understood. We identified two contiguous bacteriophage genes, designatedspeLandspeM, encoding novel inferred superantigens in the genome sequence of an ARF strain of serotype M18 group A streptococcus (GAS).speLandspeMwere located at the same genomic site in 33 serotype M18 isolates, and no nucleotide sequence diversity was observed in the 33 strains analyzed. Furthermore, the genes were absent in 13 non-M18 strains tested. These data indicate a recent acquisition event by a distinct clone of serotype M18 GAS.speLandspeMwere transcribed in vitro and upregulated in the exponential phase of growth. Purified SpeL and SpeM were pyrogenic and mitogenic for rabbit splenocytes and human peripheral blood mononuclear cells in picogram amounts. SpeL preferentially expanded human T cells expressing T-cell receptors Vβ1, Vβ5.1, and Vβ23, and SpeM had specificity for Vβ1 and Vβ23 subsets, indicating that both proteins had superantigen activity. SpeL was lethal in two animal models of streptococcal toxic shock, and SpeM was lethal in one model. Serologic studies indicated that ARF patients were exposed to serotype M18 GAS, SpeL, and SpeM. The data demonstrate that SpeL and SpeM are pyrogenic toxin superantigens and suggest that they may participate in the host-pathogen interactions in some ARF patients.

Two-component gene regulatory systems composed of a membrane-bound sensor and cytoplasmic response regulator are important mechanisms used by bacteria to sense and respond to environmental stimuli. Group AStreptococcus, the causative agent of mild infections and life-threatening invasive diseases, produces many virulence factors that promote survival in humans. A two-component regulatory system, designatedcovRS(cov, control of virulence; csrRS), negatively controls expression of five proven or putative virulence factors (capsule, cysteine protease, streptokinase, streptolysin S, and streptodornase). Inactivation ofcovRSresults in enhanced virulence in mouse models of invasive disease. Using DNA microarrays and quantitative RT-PCR, we found that CovR influences transcription of 15% (n= 271) of all chromosomal genes, including many that encode surface and secreted proteins mediating host–pathogen interactions. CovR also plays a central role in gene regulatory networks by influencing expression of genes encoding transcriptional regulators, including other two-component systems. Differential transcription of genes influenced bycovRalso was identified in mouse soft-tissue infection. This analysis provides a genome-scale overview of a virulence gene network in an important human pathogen and adds insight into the molecular mechanisms used by group AStreptococcusto interact with the host, promote survival, and cause disease.

Analysis of the genome sequence of a serotype M1 group A Streptococcus (GAS) strain identified a gene encoding a previously undescribed putative cell surface protein. The gene was cloned from a serotype M1 strain, and the recombinant protein was overexpressed in Escherichia coli and purified to homogeneity. The purified protein was associated with heme in a 1:1 stoichiometry. This streptococcal heme-associated protein, designated Shp, was produced in vitro by GAS, located on the bacterial cell surface, and accessible to specific antibody raised against the purified recombinant protein. Mice inoculated subcutaneously with GAS and humans with invasive infections and pharyngitis caused by GAS seroconverted to Shp, indicating that Shp was produced in vivo. The blood of mice actively immunized with Shp had significantly higher bactericidal activity than the blood of unimmunized mice. The shp gene was cotranscribed with eight contiguous genes, including homologues of an ABC transporter involved in iron uptake in gram-negative bacteria. Our results indicate that Shp is a novel cell surface heme-associated protein.

Genome sequences are available for many bacterial strains, but there has been little progress in using these data to understand the molecular basis of pathogen emergence and differences in strain virulence. Serotype M3 strains of group AStreptococcus(GAS) are a common cause of severe invasive infections with unusually high rates of morbidity and mortality. To gain insight into the molecular basis of this high-virulence phenotype, we sequenced the genome of strain MGAS315, an organism isolated from a patient with streptococcal toxic shock syndrome. The genome is composed of 1,900,521 bp, and it shares ≈1.7 Mb of related genetic material with genomes of serotype M1 and M18 strains. Phage-like elements account for the great majority of variation in gene content relative to the sequenced M1 and M18 strains. Recombination produces chimeric phages and strains with previously uncharacterized arrays of virulence factor genes. Strain MGAS315 has phage genes that encode proteins likely to contribute to pathogenesis, such as streptococcal pyrogenic exotoxin A (SpeA) and SpeK, streptococcal superantigen (SSA), and a previously uncharacterized phospholipase A2(designated Sla). Infected humans had anti-SpeK, -SSA, and -Sla antibodies, indicating that these GAS proteins are madein vivo. SpeK and SSA were pyrogenic and toxic for rabbits. Serotype M3 strains with the phage-encodedspeKandslagenes increased dramatically in frequency late in the 20th century, commensurate with the rise in invasive disease caused by M3 organisms. Taken together, the results show that phage-mediated recombination has played a critical role in the emergence of a new, unusually virulent clone of serotype M3 GAS.

Staphylococcus aureus is a major cause of mastitis in bovine and other ruminant species. We here present the results of a comparative genomic analysis between a bovine mastitis-associated clone, RF122, and the recently sequenced human-associated clones, Mu50 and N315, of Staphylococcus aureus . A shotgun sequence survey of ∼10% of the RF122 genome identified numerous unique sequences and those with elevated rates of nonsynonymous substitution. Taken together, these analyses show that there are notable differences in the genomes of bovine mastitis-associated and human clones of S. aureus and provide a framework for the identification of specific factors associated with host specificity in this major human and animal pathogen.