Abstract
Neisseria meningitidis is a Gram-negative bacterial pathogen that remains a leading cause of systemic meningococcal infection ranging from bacteraemia, meningitis, and fulminant meningococcal septicaemia world-wide. The ability to adhere and invade a range of human origin cells is an absolute necessity for N. meningitidis to colonize and disseminate inside its host and so to cause the rapid and damaging invasive form of the disease. From previous studies it has been established that two-component regulatory systems such as PhoP/PhoQ are involved in processes which are crucial for bacterial pathogenesis. We focused on investigation of the specific role played by the N. meningitidis PhoP regulator in the interaction of meningococci with human epithelial cells. In this study, to facilitate confocal microscope studies, bacteria were made fluorescent by transforming them with a Green Fluorescent Protein plasmid. A549 epithelial cells, a respiratory human cell line were challenged with the fluorescent N. meningitidis strain wild-type (L91543) and N. meningitidis strain phoP mutant (L91543/NMB0595 knockout mutant). Thus an epithelial colonization model was developed to compare the binding and uptake ability of the phoP mutant with the parental wild-type via adhesion and invasion assays. The influence of colonization on the host cell cytoskeleton during bacterial adhesion and invasion was also examined by confocal microscopy. Finally we analysed the transcriptome of N. meningitidis wild-type and the phoP mutant with and without epithelial cell interaction via a comparative genome hybridisation approach using microarray technology. Our findings demonstrated that the N. meningitidis phoP mutant shows a defect in adherence to and invasion into epithelial cells and this is independent of numbers or replications in cultures. The active nature of the invasion was confirmed by inhibition by cytocholasin D and less damaging of the cell membrane by the phoP mutant was observed by confocal microscopy. Our data suggested that PhoP regulator of the two-component system is involved in the colonization process of the N. meningitidis. This fits with our hypothesis that some of the genes controlled or influenced by the PhoP regulator are involved in the N. meningitidis virulence. We have identified some important virulence genes including, nspA, tbpA, tbpB and clpB that were differentially significantly regulated in the phoP mutant as revealed by the transcriptomic microarray data. These findings may lead to further identification and characterization of the novel targets for drug design and future therapeutics.