Table 11.2 Continued.

Virulence gene N. gonorrhoeae N. meningitidis

Serogroup A Serogroup B Serogroup C Strain FA1090 Strain Z2491 Strain MC58 Strain FAM18

a Unless stated otherwise, the corresponding N. meningitidis B MC58 encoded proteins were used as query and the genomes sequences of N. gonorrhoeae FA1090 [11], N. meningitidis A Z2491 [12], and N. meningitidis C FAM18 [14] as databases. Sequences were considered to be homologous if they shared a minimum sequence identity of 60% over at least 60% of the query length with expect values E < e-70. b TBLASTN search with the corresponding FA1090 encoded protein.

c TBLASTN search with the corresponding Z2491 encoded protein.

Whole-genome sequences in combination with bioinformatic tools provided a way of discovering previously unknown proteins. In a first approach of so-called "reverse vaccinology" the whole genome of meningococcal serogroup B strain MC58 was screened for the identification of new vaccine candidates [87]. Several of the identified proteins shared homologies with known virulence factors of other bacteria. To verify whether the previously unknown proteins had a role in the pathogenesis of meningococci, some of these proteins were further characterized biochemically and functionally.

The genome-derived Neisseria antigen (GNA) 33 is a membrane-bound lipopro-tein with murein hydrolase activity that is present in all Neisseria species and well conserved in different meningococcal isolates [88]. Functional analyses of the gene revealed that it plays an important role in peptidoglycan metabolism, cell separation, and membrane architecture. The inability of a knockout mutant to cause bacteremia in the infant rat model also indicated a role in virulence.

In GNA992, a putative surface-exposed protein homologue to two adhesins (Hsfand Hia) of Haemophilus influenzae was identified. Secondary structure anal ysis of GNA992 predicted a specific folding which has been observed in eukaryotic molecules involved in cell-cell adhesion and also in bacterial invasins, suggesting that this structure could be functionally relevant for the interaction with the host cell [89].

App (adhesion and penetration protein) (NMB1985) is a member of the autotransporter family of proteins and a homologue of the Hap (Haemophilus adhesion and penetration) protein of H. influenzae that plays a role in the interaction with human epithelial cells. This function could also be assigned to App. However, in comparison to the wildtype strain, a knockout mutant showed only a reduction of adhesion to Chang epithelial cells, whereas no difference was observed with Hep2 epithelial cells and HUVEC endothelial cells, respectively [90, 91].

NadA (Neisseria adhesion A) (NMB1994) is a surface-exposed protein which is able to elicit bactericidal antibodies. Fifty percent of the strains isolated from patients harbor the nadA gene, versus only 16.2% of the strains isolated from healthy carriers. NadA is present in strains of the ST-8, ST-11, and ST-32 complex, whereas it is absent in strains of the ST-41/44 complex as well as in gonococci and in the commensal species N.lactamica and N.cinerea [92, 93]. Meningococcal NadA knockout mutants showed a reduced ability to adhere to and invade Chang epithelial cells in vitro. Furthermore, NadA is able to promote adhesion of E. coli to human epithelial cells but not to endothelial cells [94].

NarE (Neisseria ADP-ribosylating enzyme) (NMB1343) was identified as a structural homologue of cholera toxin in meningococci by an in silico approach. In biochemical studies it was verified that NarE possesses ADP-ribosylation and NAD-glycohydrolase activity. The protein is exported into the periplasm of meningococci. The narA gene is always present in ST-32 and St-41/44 strains but absent in ST-8 and ST-11 strains. In gonococci, NarE is not expressed because of a frame-shift in the narE gene [95, 96].

By microarray analysis of meningococcal gene expression in response to growth with iron, a Fur-dependent, up-regulated gene was identified which belonged to a putative operon comprising three genes encoding proteins so far of unknown function [97]. Further characterization of this operon showed the necessity of this operon for protection of meningococci against hydrogen peroxide-mediated killing. The deletion mutant exhibited increased sensitivity to reactive oxygen species-producing cells [98].

The screening of the above-mentioned genome-wide collection of defined mutants [73] identified a mutant which presented reduced adherence to both human endothelial cells and human epithelial cells. The adherence of the so-called pilX mutant was as low as that of a nonpiliated mutant. The pilin-like protein PilX copurified with the pilus fiber and was found to be essential for bacterial aggregation, i.e., for formation of bacterial microcolonies. For this reason, the reduced number of adherent bacteria of the mutant resulted from the absence of interbacterial interactions [99].

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