Cell Invasion by Neisseria meningitidis Requires a Functional Interplay between the Focal Adhesion Kinase, Src and Cortactin

Entry of Neisseria meningitidis (the meningococcus) into human brain microvascular endothelial cells (HBMEC) is mediated by fibronectin or vitronectin bound to the surface protein Opc forming a bridge to the respective integrins. This interaction leads to cytoskeletal rearrangement and uptake of meningococci. In this study, we determined that the focal adhesion kinase (FAK), which directly associates with integrins, is involved in integrin-mediated internalization of N. meningitidis in HBMEC. Inhibition of FAK activity by the specific FAK inhibitor PF 573882 reduced Opc-mediated invasion of HBMEC more than 90%. Moreover, overexpression of FAK mutants that were either impaired in the kinase activity or were not capable of autophosphorylation or overexpression of the dominant-negative version of FAK (FRNK) blocked integrin-mediated internalization of N. meningitidis. Importantly, FAK-deficient fibroblasts were significantly less invaded by N. meningitidis. Furthermore, N. meningitidis induced tyrosine phosphorylation of several host proteins including the FAK/Src complex substrate cortactin. Inhibition of cortactin expression by siRNA silencing and mutation of critical amino acid residues within cortactin, that encompass Arp2/3 association and dynamin binding, significantly reduced meningococcal invasion into eukaryotic cells suggesting that both domains are critical for efficient uptake of N. meningitidis into eukaryotic cells. Together, these results indicate that N. meningitidis exploits the integrin signal pathway for its entry and that FAK mediates the transfer of signals from activated integrins to the cytoskeleton. A cooperative interplay between FAK, Src and cortactin then enables endocytosis of N. meningitidis into host cells

Phosphorylcholine Allows for Evasion of Bactericidal Antibody by Haemophilus influenzae

The human pathogen Haemophilus influenzae has the ability to quickly adapt to different host environments through phase variation of multiple structures on its lipooligosaccharide (LPS), including phosphorylcholine (ChoP). During colonization with H. influenzae, there is a selection for ChoP+ phase variants. In a murine model of nasopharyngeal colonization, this selection is lost in the absence of adaptive immunity. Based on previous data highlighting the importance of natural antibody in limiting H. influenzae colonization, the effect of ChoP expression on antibody binding and its bactericidal activity was investigated. Flow cytometric analysis revealed that ChoP+ phase variants had decreased binding of antibody to LPS epitopes compared to ChoP− phase variants. This difference in antibody binding correlated with increased survival of ChoP+ phase variants in the presence of antibody-dependent, complement-mediated killing. ChoP+ phase variants were also more resistant to trypsin digestion, suggesting a general effect on the physical properties of the outer membrane. Moreover, ChoP-mediated protection against antibody binding correlated with increased resilience of outer membrane integrity. Collectively, these data suggest that ChoP expression provides a selective advantage during colonization through ChoP-mediated effects on the accessibility of bactericidal antibody to the cell surface

Functional genomics of Neisseria meningitidis pathogenesis.

The pathogenic bacterium Neisseria meningitidis is an important cause of septicemia and meningitis, especially in childhood. The establishment and maintenance of bacteremic infection is a pre-requisite for all the pathological sequelae of meningococcal infection. To further understand the genetic basis of this essential step in pathogenesis, we analyzed a library of 2,850 insertional mutants of N. meningitidis for their capacity to cause systemic infection in an infant rat model. The library was constructed by in vitro modification of Neisseria genomic DNA with the purified components of Tn10 transposition. We identified 73 genes in the N. meningitidis genome that are essential for bacteremic disease. Eight insertions were in genes encoding known pathogenicity factors. Involvement of the remaining 65 genes in meningocoocal pathogenesis has not been demonstrated previously, and the identification of these genes provides insights into the pathogenic mechanisms that underlie meningococcal infection. Our results provide a genome-wide analysis of the attributes of N. meningitidis required for disseminated infection, and may lead to new interventions to prevent and treat meningococcal infection

Effect of smoking and alcohol use during pregnancy on the occurrence of low birthweight in a farming region in South Africa

The aim of this case–control study was to determine the risk factors for low birthweight in a farming region in South Africa, with particular attention to maternal alcohol use and smoking, both independently and in combination. Data collection was via structured postpartum interviews and review of antenatal and delivery records. The study setting was a regional referral hospital in a farming region. The study subjects were 200 infants with birthweight <2500g (cases) and 200 unmatched control infants of normal weight born during the same period as the cases. The outcome measure was low birthweight, i.e. infant birthweight <2500 g. Results showed the contribution of term low birthweight (as a measure of intrauterine growth retardation) to the total low-birthweight incidence was almost 50%, indicating a substantial intrauterine growth retardation component in this population. Sociodemographic factors were not as predictive of low birthweight in this predominantly low income population. Smoking (adjusted OR 2.67, [95% CI 1.69, 4.20]) was the strongest life style-related predictor of low birthweight. The alcohol low-birthweight relationship was not significant when adjusted for smoking status (crude OR 2.15, [95% CI 1.37, 3.39]; adjusted OR 1.32, [95% CI 0.80, 2.20]). However, there appeared to be an interaction with combined use of these two substances during pregnancy that increased the risk of low birthweight (adjusted OR increased to 4.24, [95% CI 1.01, 17.76]. It is clear that life style factors such as smoking and drinking are contributing to the occurrence of low birthweight in the target region. A comprehensive health promotion programme needs to be implemented as an integral part of antenatal and family planning services, to reduce smoking and drinking by women in this community.Funding for this research was provided by the South African National Research Foundation Grant #2050641 and the University of the Western Cape Research Departmen