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.
Steller
Sigrid
S
Max-Planck-Institute for Molecular Genetics, Berlin, Germany.
Angenendt
Philipp
P
Cahill
Dolores J
DJ
Heuberger
Sigrid
S
Lehrach
Hans
H
Kreutzberger
Jürgen
J
D001426:Bacterial Proteins / Q000032:analysis*; D015415:Biomarkers / Q000097:blood; D015153:Blotting, Western; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D009345:Neisseria meningitidis / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016870:Neisseriaceae Infections / Q000097:blood / Q000175:diagnosis* / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D016133:Polymerase Chain Reaction; D040081:Protein Array Analysis / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012703:Serotyping
D001426:Bacterial Proteins; D015415:Biomarkers; D011993:Recombinant Fusion Proteins; D006639:Histidine