Figure 1  Leptospira  gene clusters predict nonulosonic RAD001 acid biosynthesis A. The sequenced genome of L. interrogans serovar Copenhageni L1-130 (top) and L. interrogans serovar Lai strain 56601 (bottom) encode a cluster of genes with predicted activities in the synthesis of sialic acids (N-acetylneuraminic acid) or related molecules. B. PCR of sialic acid cluster genes shows DNA amplification in pathogenic Leptospira

species. Integrity of DNA was confirmed by amplification of the 16 S rRNA gene. C. Southern blots probed for the NeuA-2 region of the gene cluster using a DIG-labeled oligonucleotide. Genomic DNAs from the following bacteria were probed as described in materials and methods: DNA Synthesis inhibitor 1) S. enterica, 2) L. interrogans serovar Lai strain 55601, 3) L. interrogans serovar Copenhageni strain L1-130, 4) L. biflexa serovar Patoc, 5) L. licerasiae (rat isolate CEH 008), 6) L. licerasiae isolate MMD4847), 7) L. interrogans serovar Icterohaemorrhagiae (isolate MMD 3731), 8) L. fainei serovar Hurstbridge, 9) S. enterica. DMB-derivatization and HPLC-MS analysis reveals multiple varieties of nonulosonic acids expressed by Leptospira Strains were evaluated biochemically to determine

whether nonulosonic acid biosynthetic pathways were functional in different species and strains of Leptospira. Bacteria were hydrolyzed with mild acetic acid to release nonulosonic acid species, and low molecular weight fractions were fluorescently derivatized with 1,2-diamino-4,5-methylene dioxybenzene (DMB),

a molecule that specifically reacts with alpha keto acids, including NulOs. DMB-derivatized reaction products were separated by high performance liquid chromatography (HPLC) with a tandem electrospray ionization mass spectrometer. As expected by the Gram-negative-like structure of Leptospira, all samples displayed an early-eluting HPLC peak corresponding to the retention time and mass of 2-keto-3-deoxy-D-manno-octulosonic acid the (Kdo). Kdo is an 8-carbon α-keto acid present in the core region of lipopolysaccharide in most Gram-negative bacteria. It serves as an internal positive control in these assays (Figure 2 peak b, m/z 355) and allowed comparison between different HPLC runs. Masses of some DMB-derivatized peaks did not readily correspond to masses of known varieties of nonulosonic acids (for example Figure 2 peak a, 407 and peak d, 440). It is not known whether these masses represent nonulosonic acids. In contrast, a consistent m/z of 433 (peak c) indicates the presence of di-N-acetylated nonulosonic acids and was found in pathogenic L. interrogans serovar Lai and L. alexanderi, and intermediate strain L. fainei. In all cases, the DMB-derivatized di-N-acetylated masses were accompanied with characteristic masses corresponding to the hydrated and hydrated sodium salt (m/z 451 and 473 respectively).