Polymerizing activity and regulation of group B Streptococcus pilus 2a sortase C1

Group B Streptococcus [GBS; Streptococcus agalactiae] is the leading cause of life-threatening diseases in newborn and is also becoming a common cause of invasive diseases in non-pregnant, elderly and immune-compromised adults. Pili, long filamentous fibers protruding from the bacterial surface, have been discovered in GBS, as important virulence factors and vaccine candidates. Gram-positive bacteria build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates. Despite the availability of several crystal structures, pilus-related C sortases remain poorly characterized to date and their mechanisms of transpeptidation and regulation need to be further investigated. The available three-dimensional structures of these enzymes reveal a typical sortase fold except for the presence of a unique feature represented by an N-terminal highly flexible loop, known as the “lid”. This region interacts with the residues composing the catalytic triad and covers the active site, thus maintaining the enzyme in an auto-inhibited state and preventing the accessibility to the substrate. It is believed that enzyme activation may occur only after lid displacement from the catalytic domain. In this work we provide the first direct evidence of the regulatory role of the lid, demonstrating that it is possible to obtain in vitro an efficient polymerization of pilin subunits using an active C sortase lid mutant carrying a single residue mutation in the lid region. Moreover, biochemical analyses of this recombinant mutant reveal that the lid confers thermodynamic and proteolytic stability to the enzyme. A further characterization of this sortase active mutant showed promiscuity in the substrate recognition, as it is able to polymerize different LPXTG-proteins in vitro.

Structural and functional characterization of Group B Streptococcus pilus 2b

Group B Streptococcus (GBS) is a Gram-positive human pathogen representing one of the most common causes of life-threatening bacterial infections such as sepsis and meningitis in neonates. Covalently polymerized pilus-like structures have been discovered in GBS as important virulence factors as well as vaccine candidates. Pili are protein polymers forming long and thin filamentous structures protruding from bacterial cells, mediating adhesion and colonization to host cells. Gram-positive bacteria, including GBS, build pili on their cell surface via a class C sortase-catalyzed transpeptidation mechanism from pilin protein substrates that are the backbone protein forming the pilus shaft and two ancillary proteins. Also the cell-wall anchoring of the pilus polymers made of covalently linked pilin subunits is mediated by a sortase enzyme. GBS expresses three structurally distinct pilus types (type 1, 2a and 2b). Although the mechanisms of assembly and cell wall anchoring of GBS types 1 and 2a pili have been investigated, those of pilus 2b are not understood until now. Pilus 2b is frequently found in ST-17 strains that are mostly associated with meningitis and high mortality rate especially in infants. In this work the assembly mechanism of GBS pilus type 2b has been elucidated by dissecting through genetic, biochemical and structural studies the role of the two pilus-associated sortases. The most significant findings show that pilus 2b assembly appears “non-canonical”, differing significantly from current pilus assembly models in Gram-positive pathogens. Only sortase-C1 is involved in pilin polymerization, while the sortase-C2 does not act as a pilin polymerase, but it is involved in cell-wall pilus anchoring. Our findings provide new insights into pili biogenesis in Gram-positive bacteria. Moreover, the role of this pilus type during host infection has been investigated. By using a mouse model of meningitis we demonstrated that type 2b pilus contributes to pathogenesis of meningitis in vivo.

Efficacy of a strategy to prevent neonatal early-onset group B streptococcal (GBS) sepsis

BACKGROUND: Existing guidelines recommend different strategies to prevent early-onset neonatal GBS sepsis. In 1997, using our own data on incidence and risk factors, we established a new prevention strategy which includes GBS screening at 36 weeks' gestation and intrapartum antibiotic prophylaxis (IAP) in women with positive or unknown GBS colonization with at least one risk factor. The present study evaluates the efficacy of the new prevention strategy. METHODS: Retrospective study of the incidence of early-onset GBS sepsis among all live births at the University Women's Hospital Basel between 1997 and 2002. Additional analysis of delivery and post partum period of all GBS sepsis cases, including GBS screening, risk factors during labor (prematurity, rupture of membranes (ROM) <12 h, intrapartum signs of infection), and IAP. Comparison of this group's characteristics G2 (9,385 live births, using the new strategy) with the previous group, G1 (1984-1993, 16,126 live births, without GBS screening or routine IAP) was performed. RESULTS: The incidence of early-onset GBS sepsis was reduced from 1/1000 (G1) to 0.53/1000 (G2). We observed a significant reduction of overall intrapartum risk factors in cases of GBS sepsis. CONCLUSION: This study suggests that our new prevention strategy is effective in reducing the incidence of early-onset GBS sepsis in neonates. In comparison, implementation of the CDC's prevention strategy might have prevented 2 additional cases in 9385 live births. However, this would have required treating a much larger number of pregnant women with IAP with consequential increasing costs, side effects and complications.

Expression of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1beta in the brain during experimental group B streptococcal meningitis

We performed mRNA in situ hybridization for TNF-alpha and IL-1beta from infant rats with group B streptococcal meningitis. Induction of both cytokines was seen in the ependyma and the meninges at 4 h. Both cytokines were expressed in the brain parenchyma at 12 h. Induction of IL-1beta mRNA was seen in vessels within the brain cortex. Neutrophilic infiltrate at all time points examined was minimal and could not account for the observed cytokine expression.

Tumor necrosis factor-alpha contributes to apoptosis in hippocampal neurons during experimental group B streptococcal meningitis

To evaluate the role of tumor necrosis factor-alpha (TNF-alpha) in neuronal injury in experimental group B streptococcal meningitis, infected neonatal rats were treated with a monoclonal antibody against TNF-alpha (20 mg/kg intraperitoneally) or saline given at the time of infection. Histopathology after 24 h showed necrosis in the cortex and apoptosis in the hippocampal dentate gyrus. Treated animals had significantly less hippocampal injury than did controls (P < .001) but had similar cortical injury and cerebrospinal fluid (CSF) inflammation. The antibody was then administered directly intracisternally (170 microg) to test whether higher CSF concentrations reduced inflammation or cortical injury. Again, hippocampal apoptosis was significantly reduced (P < .01), while cortical injury and inflammation were not. Thus, TNF-alpha played a critical role in neuronal apoptosis in the hippocampus, while it was not essential for the development of inflammation and cortical injury in this model.

[Perianal erythema in a child--group A streptococci?!]

Perianal streptococcal dermatitis is a common disease. The typical clinical picture includes perianal erythema, pruritus, painful defaecation and bloody stools. The diagnosis is made by a swab taken from the affected skin with bacterial culture. Therapy consists of penicillin for 10 days. Screening for affected persons in contact with the patient is indicated because perianal streptococcal dermatitis is known to be highly contagious. Relapse is common and therefore follow-up visits are recommended. In case of relapse, a first or second generation cephalosporin may be considered.

Bacterial phenotype variants in group B streptococcal toxic shock syndrome

We conducted genetic and functional analyses of isolates from a patient with group B streptococcal (GBS) necrotizing fasciitis and toxic shock syndrome. Tissue cultures simultaneously showed colonies with high hemolysis (HH) and low hemolysis (LH). Conversely, the HH and LH variants exhibited low capsule (LC) and high capsule (HC) expression, respectively. Molecular analysis demonstrated that the 2 GBS variants were of the same clonal origin. Genetic analysis found a 3-bp deletion in the covR gene of the HH/LC variant. Functionally, this isolate was associated with an increased growth rate in vitro and with higher interleukin-8 induction. However, in whole blood, opsonophagocytic and intracellular killing assays, the LH/HC phenotype demonstrated higher resistance to host phagocytic killing. In a murine model, LH/HC resulted in higher levels of bacteremia and increased host mortality rate. These findings demonstrate differences in GBS isolates of the same clonal origin but varying phenotypes.

Clinical decisions related to CBC screening of asymptomatic full-term infants at risk for group B streptococcus disease

Objective. Although complete blood count (CBC) changes occur with the development of clinical sepsis in newborns, the CBC has not been reported to be a sensitive predictor of sepsis in asymptomatic full-term newborn infants, nor has it been reported to be related to risk factors for sepsis or clinical decisions. The objective of this study was to evaluate the relationship between the WBC/I:T (immature:total neutrophil) ratio and maternal group B streptococcal (GBS) risk factors (rupture of membranes ≥18 hours, maternal temperature ≥100.4°F, maternal age ≤20 years, previous infant with invasive GBS disease, maternal GBS bacteriuria, and black ethnicity); and to evaluate the relationship between the WBC/I:T ratios and providers' clinical decisions (observe versus repeat the CBC or complete sepsis evaluation) in the asymptomatic full-term newborn at risk for early-onset GBS sepsis. ^ Methods. Medical records of infants admitted to the well baby nursery at a tertiary care teaching hospital in Houston, TX between 1/1/99 and 12/31/00 whose gestational ages were ≥35 weeks; who had mothers with GBS positive or unknown culture status and inadequate intrapartum antibiotic prophylaxis; and who had screening CBCs performed in the first 30 hours of life because of GBS risk were reviewed (n = 412). Demographic information, maternal GBS risk factors, CBC results, clinical decisions, and rationales for clinical decisions were collected. ^ Results. With the exception of black ethnicity (p = .0000, odds ratio = 0.213), no statistically significant differences in risk factors between infants with normal and abnormal WBC counts or normal and abnormal I:T ratios were found. Infants with abnormal WBCs had a significantly higher likelihood of having a CBC repeated (p = 0.002 for WBC). Providers documented the CBC result in the rationale for clinical decisions in 62% of the cases. ^ Conclusion. The CBC results were not related to maternal risk factors for GBS except for ethnicity. Black infants had significantly lower WBC levels than infants of other ethnicities, although this difference was clinically insignificant. Infants with abnormal WBCs had a significantly higher likelihood of undergoing repeat CBCs but not sepsis evaluations. Provider rationale was difficult to evaluate due to insufficient documentation. The screening CBC result did not impact the clinicians' decisions to initiate sepsis evaluations in this population. ^