Neisseria meningitidis is the cause of septicemia and meningococcal meningitis. During the course of infection, N. meningitidis encounters multiple environments within its host, which makes rapid adaptation to environmental changes a crucial factor for neisserial pathogenicity. Employing oligonucleotide-based DNA microarrays, we analyzed the transcriptome of N. meningitidis during two key steps of meningococcal infection, i.e., the interaction with epithelial cells (HeLa cells) and endothelial cells (human brain microvascular endothelial cells). Seventy-two genes were differentially regulated after contact with epithelial cells, and 48 genes were differentially regulated after contact with endothelial cells, including a considerable proportion of well-known virulence genes. While a considerable number of genes were in concordance between bacteria adherent to both cell types, we identified several open reading frames that were differentially regulated in only one system. The data obtained with this novel approach may provide insight into the pathogenicity mechanisms of N. meningitidis and could demonstrate the importance of gene regulation on the transcriptional level during different stages of meningococcal infection.

Phospholipases are critical for modification and redistribution of lipid substrates, membrane remodeling and microbial virulence. Among the many different classes of phospholipases, fungal phospholipase B (Plb) proteins show the broadest range of substrate specificity and hydrolytic activity, hydrolyzing acyl ester bonds in phospholipids and lysophospholipids and further catalyzing lysophospholipase-transacylase reactions. The genome of the opportunistic fungal pathogen Candida albicans encodes a PLB multigene family with five putative members; we present the first characterization of this group of potential virulence determinants. CaPLB5, the third member of this multigene family characterized herein is a putative secretory protein with a predicted GPI-anchor attachment site. Real-time RT-PCR gene expression analysis of CaPLB5 and the additional CaPLB gene family members revealed that filamentous growth and physiologically relevant environmental conditions are associated with increased PLB gene activity. The phenotypes expressed by null mutant and revertant strains of CaPLB5 indicate that this lipid hydrolase plays an important role for cell-associated phospholipase A(2) activity and in vivo organ colonization.

Candida albicans is an important fungal pathogen of immunocompromised patients. In cell culture, C. albicans is sensitive to mycophenolic acid (MPA) and mizoribine, both natural product inhibitors of IMP dehydrogenase (IMPDH). These drugs have opposing interactions with the enzyme. MPA prevents formation of the closed enzyme conformation by binding to the same site as a mobile flap. In contrast, mizoribine monophosphate, the active metabolite of mizoribine, induces the closed conformation. Here, we report the characterization of IMPDH from wild-type and MPA-resistant strains of C. albicans. The wild-type enzyme displays significant differences from human IMPDHs, suggesting that selective inhibitors that could be novel antifungal agents may be developed. IMPDH from the MPA-resistant strain contains a single substitution (A251T) that is far from the MPA-binding site. The A251T variant was 4-fold less sensitive to MPA as expected. This substitution did not affect the k cat value, but did decrease the K m values for both substrates, so the mutant enzyme is more catalytically efficient as measured by the value of k cat / K m . These simple criteria suggest that the A251T variant would be the evolutionarily superior enzyme. However, the A251T substitution caused the enzyme to be 40-fold more sensitive to mizoribine monophosphate. This result suggests that A251T stabilizes the closed conformation, and this hypothesis is supported by further inhibitor analysis. Likewise, the MPA-resistant strain was more sensitive to mizoribine in cell culture. These observations illustrate the evolutionary challenge posed by the gauntlet of chemical warfare at the microbial level.

Oligonucleotide-and cDNA-based microarrays comprising a subset of Neisseria meningitidis genes were assessed for study of the meningococcal heat shock response and found to be highly suitable for transcriptional profiling of N. meningitidis. Employing oligonucleotide arrays encompassing the entire genome of N. meningitidis, we analyzed the meningococcal heat shock response on a global scale and identified 55 heat shockderegulated open reading frames (34 induced and 21 repressed).Sequencing of the genomes of Neisseria meningitidis serogroup A and serogroup B strains provided us with a tremendously broad range of information (15,23). The next step is the elucidation of gene expression patterns and gene product function on a genome-wide scale. DNA microarrays offer an ideal tool for high-throughput investigation of gene regulation on the transcriptional level (for review, see references 4, 13, and 17). The two most commonly employed DNA microarray platforms are oligonucleotide and cDNA arrays. Here we performed a comparative analysis of the suitability of both technology platforms for transcriptional profiling of N. meningitidis.The two main features of DNA microarray performance are sensitivity (signal intensity) and specificity (ratio of specific to nonspecific hybridization). Additional care must be taken to standardize experimental conditions and to avoid the detection of false-positive signals (12). In order to validate gene expression modulations of N. meningitidis observed using cDNAbased and oligonucleotide-based microarrays, we first performed parallel hybridizations of identical RNA samples to the same slide. Microarrays containing probes specific for 60 genes selected from the published genome sequence of N. meningitidis serogroup B strain MC58 (23) were produced (Table 1). For cDNA-arrays, internal fragments of each open reading frame (ORF) (300 to 560 bp) were PCR amplified. For oligonucleotide arrays, oligonucleotides (40-mers, three per gene) comprising gene-specific internal fragments (covering 5Ј, central, and 3Ј parts) were designed. All oligonucleotides (manufactured by MWG-Biotech AG, Ebersberg, Germany) carried a C6 amino linker modification at the 5Ј end for covalent attachment to the slide surface. Each probe was spotted 5 (oligonucleotides) or 10 times (PCR products) per array using the Affymetrix 417 Arrayer (MWG-Biotech AG). PCR products were spotted on CMT-GAPS-Coated Slides (Corning, Wiesbaden, Germany), oligonucleotides were spotted on Super Aldehyde Slides (TeleChem International, Sunnyvale, Calif.), and the slides were processed according to the manufacturers' instructions.Cultures of N. meningitidis strain MC58 (24) were grown to mid-logarithmic growth phase (optical density at 600 nm [OD 600 ] ϭ 0.5/5 ϫ 10 8 CFU/ml) at 37°C in supplemented proteose peptone medium and RNA isolated as previously described (5). The RNA was split into two aliquots, and onehalf was labeled with Cy3-dCTP, the other with Cy5-dCTP (Amersham Pharmacia, Freiburg, Germany) during a firststrand reverse transcri...

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