The development of efficient and inexpensive genome sequencing methods has revolutionized the study of human bacterial pathogens and improved vaccine design. Unfortunately, the sequence of a single genome does not reflect how genetic variability drives pathogenesis within a bacterial species and also limits genome-wide screens for vaccine candidates or for antimicrobial targets. We have generated the genomic sequence of six strains representing the five major disease-causing serotypes of Streptococcus agalactiae , the main cause of neonatal infection in humans. Analysis of these genomes and those available in databases showed that the S. agalactiae species can be described by a pan-genome consisting of a core genome shared by all isolates, accounting for ≈80% of any single genome, plus a dispensable genome consisting of partially shared and strain-specific genes. Mathematical extrapolation of the data suggests that the gene reservoir available for inclusion in the S. agalactiae pan-genome is vast and that unique genes will continue to be identified even after sequencing hundreds of genomes.

Streptococcus pneumoniae is one of the leading causes of invasive bacterial disease worldwide. Fragments of the cell wall and the cytolytic toxin pneumolysin have been shown to contribute substantially to inflammatory damage, although the interactions between pneumococcal components and host-cell structures have not been elucidated completely. Results of a previous study indicated that cell-wall components of pneumococci are recognized by Toll-like receptor (TLR)2 but suggested that pneumolysin induces inflammatory events independently of this receptor. In this study we tested the hypothesis that pneumolysin interacts with surface proteins of the TLR family other than TLR2. We found that pneumolysin stimulates tumor necrosis factor-␣ and IL-6 release in wild-type macrophages but not in macrophages from mice with a targeted deletion of the cytoplasmic TLR-adapter molecule myeloid differentiation factor 88, suggesting the involvement of the TLRs in pneumolysin recognition. Purified pneumolysin synergistically activated macrophage responses together with preparations of pneumococcal cell walls or staphylococcal peptidoglycan, which are known to activate TLR2. Furthermore, when compared with wild-type macrophages, macrophages from mice that carry a spontaneous mutation in TLR4 (P712H) were hyporesponsive to both pneumolysin alone and the combination of pneumolysin with pneumococcal cell walls. Finally, these TLR4-mutant mice were significantly more susceptible to lethal infection after intranasal colonization with pneumolysin-positive pneumococci than were control mice. We conclude that the interaction of pneumolysin with TLR4 is critically involved in the innate immune response to pneumococcus.

Newborns are at increased risk of overwhelming infection, yet the mechanisms underlying this susceptibility are incompletely defined. In this study we report a striking 1- to 3-log decrease in sensitivity of monocytes in human neonatal cord blood, compared with monocytes in adult peripheral blood, to the TNF-α-inducing effect of multiple TLR ligands, including bacterial lipopeptides (BLPs), LPS, and the imidazoquinoline compound, imiquimod. In marked contrast, TNF-α release in response to R-848, a TLR ligand that is a congener of imiquimod, was equivalent in newborn and adult blood. Differences in ligand-induced TNF-α release correlated with divergent ligand-induced changes in monocyte TNF-α mRNA levels. Newborn and adult monocytes did not differ in basal mRNA or protein expression of TLRs or mRNA expression of functionally related molecules. Newborn monocytes demonstrated diminished LPS-induced, but equivalent R-848-induced, phosphorylation of p38 mitogen-activated protein kinase and altered BLP- and LPS-induced acute modulation of cognate receptors, suggesting that the mechanism accounting for the observed differences may be localized proximal to ligand recognition by surface TLRs. Remarkably, newborn plasma conferred substantially reduced BLP-, LPS-, and imiquimod-induced TNF-α release on adult monocytes without any effect on R-848-induced TNF-α release, reflecting differences in a plasma factor(s) distinct from soluble CD14. Impaired response to multiple TLR ligands may significantly contribute to immature neonatal immunity. Conversely, relative preservation of responses to R-848 may present unique opportunities for augmenting innate and acquired immunity in the human newborn.

The 2,160,267 bp genome sequence of Streptococcus agalactiae, the leading cause of bacterial sepsis, pneumonia, and meningitis in neonates in the U.S. and Europe, is predicted to encode 2,175 genes. Genome comparisons among S. agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, and the other completely sequenced genomes identified genes specific to the streptococci and to S. agalactiae. These in silico analyses, combined with comparative genome hybridization experiments between the sequenced serotype V strain 2603 V͞R and 19 S. agalactiae strains from several serotypes using whole-genome microarrays, revealed the genetic heterogeneity among S. agalactiae strains, even of the same serotype, and provided insights into the evolution of virulence mechanisms. Streptococcus agalactiae, or group B Streptococcus, is the leading cause of bacterial sepsis, pneumonia, and meningitis in neonates in the U.S. and Europe. Although S. agalactiae usually behaves as a commensal organism that colonizes the gastrointestinal or genital tract of 25-40% of healthy women, it can cause life-threatening invasive infection in susceptible hosts: newborn infants, pregnant women, and nonpregnant adults with underlying chronic illnesses (1, 2). Since guidelines recommending intrapartum antibiotic prophylaxis for high-risk or colonized women were issued in 1996 (3), the incidence of neonatal infections has decreased, and invasive S. agalactiae infections in immunocompromised adults have become more common. Adult disease now accounts for the majority of serious S. agalactiae infections. First recognized as a pathogen in bovine mastitis, S. agalactiae is distinguished from other pathogenic streptococci by the cell wall-associated group B carbohydrate.Another polysaccharide constitutes the organism's capsule, an important S. agalactiae virulence determinant. S. agalactiae strains of capsular serotype V were rarely isolated before the mid-1980s but now account for approximately one-third of clinical isolates in the U.S. (4-6). Type V is the most common capsular serotype associated with invasive infection in nonpregnant adults, and the emergence of type V strains over the past decade has been temporally linked to an increase in S. agalactiae disease in this population (7). As a species, S. agalactiae shares certain features with other pathogenic streptococci; however, the precise repertoire of shared and unique attributes that account for the emergence of S. agalactiae as a major pathogen for specific human populations remains undefined. To elucidate the molecular basis for S. agalactiae virulence, we determined the complete genome sequence (8) of the recent clinical type V isolate 2603 V͞R (www.tigr.org) and performed comparative analyses with other S. agalactiae strains and with other species of pathogenic streptococci. Methods ORF Prediction and Gene Identification.ORFs were predicted by GLIMMER (9, 10) trained with ORFs larger than 600 bp from the genomic sequence and S. agalactiae genes available in GenBank. All predicted proteins la...

Group B streptococci (GBS) cause sepsis and meningitis in neonates and serious infections in adults with underlying chronic illnesses. Specific antibodies have been shown to be an important factor in protective immunity for neonates, but the role of serum complement is less well defined. To elucidate the function of the complement system in immunity to this pathogen, we have used the approach of gene targeting in embryonic stem cells to generate mice totally deficient in complement component C3. Comparison of C3-deficient mice with mice deficient in complement component C4 demonstrated that the 50% lethal dose for GBS infection was reduced by "50-fold and 25-fold, respectively, compared to control mice. GBS were effectively killed in vitro by human blood leukocytes in the presence of specific antibody and C4-deficient serum but not C3-deficient serum. The defective opsonization by C3-deficient serum in vitro was corroborated by in vivo studies in which passive immunization of pregnant dams with specific antibodies conferred protection from GBS challenge to normal and C4-deficient pups but not C3-deficient pups. These results indicate that the alternative pathway is sufficient to mediate effective opsonophagocytosis and protective immunity to GBS in the presence of specific antibody. In contrast, the increased susceptibility to infection of nonimmune mice deficient in either C3 or C4 implies that the classical pathway plays an essential role in host defense against GBS infection in the absence of specific immunity.

SummaryThe hyaluronic acid capsule of group A Streptococcus (GAS) is an important virulence factor, but little is known about mechanisms that regulate capsule expression. Transposon Tn916 mutagenesis of the poorly encapsulated M-type 3 GAS strain DLS003 produced a transconjugant that exhibited a mucoid colony morphology, reflecting increased hyaluronic acid capsule production. Analysis of chromosomal DNA sequence immediately downstream of the transposon insertion identified two open reading frames, designated csrR and csrS, which exhibited sequence similarity to bacterial two-component regulatory systems. We constructed an in-frame deletion mutation within csrR, which encodes the putative response component. Replacement of the native csrR gene in the DLS003 chromosome with the mutant allele resulted in a sixfold increase in capsule production and a corresponding increase in transcription of the has operon, which contains the essential genes for hyaluronic acid synthesis. Increased capsule production by the csrR mutant strain was associated with enhanced resistance to complement-mediated opsonophagocytic killing in vitro and with a 500-fold increase in virulence in mice. These results establish CsrR as a negative regulator of hyaluronic acid capsule synthesis and suggest that it is part of a twocomponent regulatory system that influences capsule expression and virulence.

Mucoid strains of group A Streptococcus have been associated with recent outbreaks ofacute rheumatic fever. The mucoid colony morphology of these strains is a result of abundant production of capsular polysaccharide, which is composed of hyaluronic acid. To study the role of the hyaluronic acid capsule in virulence, we derived an acapsular mutant from a mucoid strain of group A Streptococcus by transposon mutagenesis. M protein expression was not altered in the mutant strain. The mucoid wild-type strain grew in fresh human blood and was resistant to phagocytic killing in vitro. In contrast, the acapsular mutant failed to grow in fresh human blood and was sensitive to phagocytic killing in vitro. Loss of capsule was associated with a 100-fold reduction in virulence of the organisms in mice. We conclude that the hyaluronic acid capsule protects mucoid group A streptococci from phagocytosis and has an important role in virulence.Group A Streptococcus (GAS) continues to be a major cause of morbidity and mortality throughout the world, both from infections and from the postinfectious sequelae of acute rheumatic fever and poststreptococcal glomerulonephritis. Recent reports suggest that after declining in frequency in developed countries for the past half-century, the incidence of life-threatening infections due to GAS and of acute rheumatic fever is increasing (1-5). The reasons for the resurgence of serious human diseases due to GAS are not known; however, it has been suggested that the GAS strains prevalent today may have increased expression of one or more virulence factors (1, 6). One such factor may be the hyaluronic acid capsule. Occasional strains of GAS isolated from clinical sources grow as large, spreading, wet colonies on solid media; this characteristic mucoid colony morphology is due to abundant production by these strains of the GAS capsular polysaccharide, which is composed of hyaluronic acid, a high molecular weight polymer consisting of alternating residues of N-acetylglucosamine and glucuronic acid (7,8). Mucoid strains of GAS have been implicated as causing unusually severe infections and frequently have been associated with individual cases or community outbreaks of rheumatic fever, including clusters of rheumatic fever cases reported recently from several regions of the U.S.A. (3, 5, 9, 10). Kaplan et al. (6) studied 42 GAS strains isolated from sibling contacts or patients with acute rheumatic fever during several outbreaks in the mid-1980s and found 45% to be mucoid.Mucoid isolates are generally rich in M protein and are highly virulent in experimental animals (9, 11). M protein has been considered to be the major surface component responsible for resistance of GAS to phagocytosis (12). The fact that highly virulent, M protein-rich strains usually appear mucoid suggests that the presence of a large hyaluronic acid capsule may also confer special virulence properties on these strains. Hirst (13) attempted to define the role of the hyaluronic acid capsule in virulence by experimentally in...

The nervous system, the immune system, and microbial pathogens interact closely at barrier tissues. Here, we find that a bacterial pathogen, Streptococcus pyogenes, hijacks pain and neuronal regulation of the immune response to promote bacterial survival. Necrotizing fasciitis is a life-threatening soft tissue infection in which "pain is out of proportion" to early physical manifestations. We find that S. pyogenes, the leading cause of necrotizing fasciitis, secretes streptolysin S (SLS) to directly activate nociceptor neurons and produce pain during infection. Nociceptors, in turn, release the neuropeptide calcitonin gene-related peptide (CGRP) into infected tissues, which inhibits the recruitment of neutrophils and opsonophagocytic killing of S. pyogenes. Botulinum neurotoxin A and CGRP antagonism block neuron-mediated suppression of host defense, thereby preventing and treating S. pyogenes necrotizing infection. We conclude that targeting the peripheral nervous system and blocking neuro-immune communication is a promising strategy to treat highly invasive bacterial infections. VIDEO ABSTRACT.