The development of vaccines against serogroup B Neisseria meningitidis to reduce the morbidity and mortality of meningococcal disease is a major public health priority. We developed a novel genetic screen for immunogens present on the bacterial surface using human immune sera with bactericidal activity. We found that two mutants lacking nmb1467 survived in high concentrations of sera from two patients, while the wild-type strain was killed. Biochemical assays using purified recombinant NMB1467 indicated that nmb1467 encodes an exopolyphosphatase (PPX) with the ability to hydrolyse inorganic polyphosphate (poly P). In addition, we demonstrated that the Δppx mutant has at least 2-fold more poly P than the wild-type strain. Therefore, we designated NMB1467 as PPX. We showed that N. meningitidis mutant lacking the ppx had an increased resistance against normal human complement system. Substitution of the glutamic acid at residue 147 of PPX with an alanine significantly reduced the enzymatic activity in vitro, and contributed to increased level of poly P in N. meningitidis and the resistance of bacteria against the complement-mediated killing. Levels of polysaccharide capsule and lipopolysaccharide (LPS) sialylation, two important virulence factors, were not affected by the loss of ppx in N. meningitidis. Using flow cytometry, we demonstrated that binding of factor H (fH), the negative regulator of the alternative pathway of complement activation, to the bacterial surface was increased in the strain lacking PPX. By Western blot analysis, we did not detect a significant change in the expression of the fH receptor, indicting another mechanism is involved in the fH binding to the bacterial surface and resistance of bacteria against complement-mediated killing.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:505458 |
| Date | January 2009 |
| Creators | Zhang, Qian |
| Contributors | Tang, Christoph |
| Publisher | Imperial College London |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/10044/1/4340 |
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