Bacterial protein microarrays for identification of new potential diagnostic markers for Neisseria meningitidis infections.

Neisseria meningitidis is the most common cause of meningitis and causes epidemic outbreaks. One trait of N. meningitidis, which is associated with most of the currently recognized virulence determinants, is the presence of phase-variable genes that are suspected to enhance its ability to cause an invasive disease. To detect the immune responses to phase-variable expressed proteins, we applied protein microarray technology for the screening of meningitis patient sera. We amplified all 102 known phase-variable genes from N. meningitidis serogroup B strain MC58 by polymerase chain reaction and subcloned them for expression in Escherichia coli. With this approach, we were able to express and purify 67 recombinant proteins representing 66% of the annotated genes. These were spotted robotically onto coated glass slides to generate protein microarrays, which were screened using 20 sera of patients suffering from meningitis, as well as healthy controls. From these screening experiments, 47 proteins emerged as immunogenic, exhibiting a variable degree of seroreactivity with some of the patient sera. Nine proteins elicited an immune response in more than three patients, with one of them, the phase-variable opacity protein OpaV (NMB0442), showing responses in 11 patient sera. This is the first time that protein microarray technology has been applied for the investigation of genetic phase variation in pathogens. The identification of disease-specific proteins is a significant target in biomedical research, as such proteins may have medical, diagnostic, and commercial potential as disease markers.

Initiation of surfactin biosynthesis and the role of the SrfD-thioesterase protein.

In this paper, the initiation reactions in surfactin biosynthesis by Bacillus subtilis OKB 105 were investigated. Evidence for a specific role of the SrfD protein, the external thioesterase enzyme in surfactin biosynthesis, was obtained for the first time. The action of SrfD was investigated both with the native, but only partially purified, enzyme and the highly purified, His-tagged protein overexpressed in Escherichia coli. Surfactin can be formed by the interaction of the three amino acid activating components of surfactin synthetase SrfA, B and C alone. This process is stimulated by SrfD. In the initiation reactions, the beta-hydroxy fatty acid substrate is transferred from beta-hydroxymyristoyl-coenzyme A to the start enzyme SrfA followed by formation of beta-hydroxymyristoyl-glutamate. The same reactions were also observed with the recombinant L-Glu-activating module of surfactin synthetase. Lipopeptide formation can be initiated by these function units alone, but SrfD efficiently supports and stimulates the formation of initiation products. From these results, we infer that SrfD functions as the thioesterase/acyltransferase enzyme in the initiation process previously postulated by Menkhaus et al. [Menkhaus et al. (1993) J. Biol. Chem. 268, 7678-7684], thus enhancing surfactin formation.