Accumulation of (5 ` S)-8,5 `-cyclo-2 `-deoxyadenosine in organs of Cockayne syndrome complementation group B gene knockout mice

Cockayne syndrome (CS) is a human genetic disorder characterized by sensitivity to UV radiation, neurodegeneration, premature aging among other phenotypes. CS complementation group B (CS-B) gene (csb) encodes the CSB protein (CSB) that is involved in base excision repair of a number of oxidatively induced lesions in genomic DNA in vivo. We hypothesized that CSB may also play a role in cellular repair of the DNA helix-distorting tandem lesion (5`S)-8,5`-cyclo-2`-deoxyadenosine (S-cdA). Among many DNA lesions. S-cdA is unique in that it represents a concomitant damage to both the sugar and base moieties of the same nucleoside. Because of the presence of the C8-C5` covalent bond, S-cdA is repaired by nucleotide excision repair unlike most of other oxidatively induced lesions in DNA, which are subject to base excision repair. To test our hypothesis, we isolated genomic DNA from brain, kidney and liver of wild type and csb knockout (csb(-/-)) mice. Animals were not exposed to any exogenous oxidative stress before the experiment. DNA samples were analysed by liquid chromatography/mass spectrometry with isotope-dilution. Statistically greater background levels of S-cdA were observed in all three organs of csb(-/-) mice than in those of wild type mice. These results suggest the in vivo accumulation of S-cdA in genomic DNA due to lack of its repair in csb(-/-) mice. Thus, this study provides, for the first time, the evidence that CSB plays a role in the repair of the DNA helix-distorting tandem lesion S-cdA. Accumulation of unrepaired S-cdA in vivo may contribute to the pathology associated with CS. Published by Elsevier B.V.

Group B streptococci colonization in pregnant women: risk factors and evaluation of the vaginal flora

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Curvature versus V-bends in a group B titanium T-loop spring

Objective: To compare the system of forces acting on curvature and preactivated V-bends in titanium T-loop springs (TTLSs) made of 0.017- X 0.025-inch TMA (titanium molibdenium alloy) wire.Materials and Methods: Pictures of TTLSs preactivated by curvature and V-bends were inserted in the LOOP software program to design both TTLSs. Symmetry was assured using the program. Both TTLSs used the same amount (length) of wire and had the same angulation between their anterior and posterior extremities when passive. The loops were activated 7 mm, and forces and moments were registered after each 0.5 mm of deactivation. The brackets were at the same height, separated by 23 mm and angulated 0 degrees.Results: The preactivated curvature TTLS delivered horizontal forces ranging from 34 gF to 456 gF, while the TTLS preactivated by V-bends delivered forces ranging from 54 gF to 517 gF. The forces decreased more (30 gF vs 33 gF) with every 0.5 mm of activation on the preactivated V-bend TTLS than on the preactivated curvature TTLS. Vertical forces were low and clinically insignificant for both TTLSs. The moment to force (MF) ratios were systematically higher on the preactivated curvature than on the preactivated V-bend TTLS (from 5.8 mm to 38.8 mm vs 4.7 mm to 28.3 mm).Conclusions: Although both loops show symmetrical moments in their anterior and posterior extremities and can be used for group B anchorage, the curvature preactivated TTLS delivers lower horizontal forces and higher MF ratios than the acute preactivated V-bend TTLS.

Structural and functional characterization of Group B Streptococcus class C sortases

In Group B Streptococcus (GBS) three structurally distinct types of pili have been discovered as potential virulence factors and vaccine candidates. The pilus-forming proteins are assembled into high-molecular weight polymers via a transpeptidation mechanism mediated by specific class C sortases. Using a multidisciplinary approach including bioinformatics, structural and biochemical studies and in vivo mutagenesis we performed a broad characterization of GBS sortase C. The high resolution X-ray structure of the enzymes revealed that the active site, located into the β-barrel core of the enzyme, is made of the catalytic triad His157-Cys219-Arg228 and covered by a loop, known as the “lid”. We show that the catalytic triad and the predicted N- and C-terminal trans-membrane regions are required for the enzyme activity. Interestingly, by in vivo complementation mutagenesis studies we found that the deletion of the entire lid loop or mutations in specific lid key residues had no effect on catalytic activity of the enzyme. In addition, kinetic characterizations of recombinant enzymes indicate that the lid mutants can still recognize and cleave the substrate-mimicking peptide at least as well as the wild type protein.

Analysis of Two Component Systems in Group B Streptococcus Shows that RgfAC and the Novel FspSR Modulate Virulence and Bacterial Fitness

Group B Streptococcus (GBS), in its transition from commensal to pathogen, will encounter diverse host environments and thus require coordinately controlling its transcriptional responses to these changes. This work was aimed at better understanding the role of two component signal transduction systems (TCS) in GBS pathophysiology through a systematic screening procedure. We first performed a complete inventory and sensory mechanism classification of all putative GBS TCS by genomic analysis. Five TCS were further investigated by the generation of knock-out strains, and in vitro transcriptome analysis identified genes regulated by these systems, ranging from 0.1-3% of the genome. Interestingly, two sugar phosphotransferase systems appeared differently regulated in the knock-out mutant of TCS-16, suggesting an involvement in monitoring carbon source availability. High throughput analysis of bacterial growth on different carbon sources showed that TCS-16 was necessary for growth of GBS on fructose-6-phosphate. Additional transcriptional analysis provided further evidence for a stimulus-response circuit where extracellular fructose-6-phosphate leads to autoinduction of TCS-16 with concomitant dramatic up-regulation of the adjacent operon encoding a phosphotransferase system. The TCS-16-deficient strain exhibited decreased persistence in a model of vaginal colonization and impaired growth/survival in the presence of vaginal mucoid components. All mutant strains were also characterized in a murine model of systemic infection, and inactivation of TCS-17 (also known as RgfAC) resulted in hypervirulence. Our data suggest a role for the previously unknown TCS-16, here named FspSR, in bacterial fitness and carbon metabolism during host colonization, and also provide experimental evidence for TCS-17/RgfAC involvement in virulence.

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.

Brain injury in experimental neonatal meningitis due to group B streptococci

We have characterized the pattern of brain injury in a rat model of meningitis caused by group B streptococci (GBS). Infant rats (12-14 days old; n = 69) were infected intracisternally with 10 microliters of GBS (log10(2.3) to 4.5 colony-forming units). Twenty hours later, illness was assessed clinically and cerebrospinal fluid was cultured. Animals were either immediately euthanized for brain histopathology or treated with antibiotics and examined later. Early GBS meningitis was characterized clinically by severe obtundation and seizures, and histopathologically by acute inflammation in the subarachnoid space and ventricles, a vasculopathy characterized by vascular engorgement, and neuronal injury that was most prominent in the cortex and often followed a vascular pattern. Incidence of seizures, vasculopathy and neuronal injury correlated with the inoculum size (p < 0.01). Early injury was almost completely prevented by treatment with dexamethasone. Within days after meningitis, injured areas became well demarcated and showed new cellular infiltrates. Thirty days post-infection, brain weights of infected animals treated with antibiotics were decreased compared to uninfected controls (1.39 +/- 0.18 vs 1.64 +/- 0.1 g; p < 0.05). Thus, GBS meningitis in this model caused extensive cortical neuronal injury resembling severe neonatal meningitis in humans.