SummaryThe Arg-gingipains (RgpsA and B) of Porphyromonas gingivalis are a family of extracellular cysteine proteases and are important virulence determinants of this periodontal bacterium. A monoclonal antibody, MAb1B5, which recognizes an epitope on glycosylated monomeric RgpAs also cross-reacts with a cellsurface polysaccharide of P. gingivalis W50 suggesting that the maturation pathway of the Arg-gingipains may be linked to the biosynthesis of a surface carbohydrate. We report the purification and structural characterization of the cross-reacting anionic polysaccharide (APS), which is distinct from both the lipopolysaccharide and serotype capsule polysaccharide of P. gingivalis W50. The structure of APS was determined by 1D and 2D NMR spectroscopy and methylation analysis, which showed it to be a phosphorylated branched mannan. The backbone is built up of α α α α -1,6-linked mannose residues and the sidechains contain α α α α -1,2-linked mannose oligosaccharides of different lengths (one to two sugar residues) attached to the backbone via 1,2-linkage. One of the side-chains in the repeating unit contains Man α α α α 1-2Man α α α α 1-phosphate linked via phosphorus to a backbone mannose at position 2. De-O -phosphorylation of APS abolished cross-reactivity suggesting that Man α α α α 1-2Man α α α α 1-phosphate fragment forms part of the epitope recognized by MAb1B5. This phosphorylated branched mannan represents a novel polysaccharide that is immunologically related to the post-translational additions of Arg-gingipains.

Protein glycosylation has an important influence on a broad range of molecular interactions in eukaryotes, but is comparatively rare in bacteria. Several antigens from Mycobacterium tuberculosis, the causative agent of human tuberculosis, have been identified as glycoproteins on the basis of lectin binding, or by detailed structural analysis. By production of a set of alkaline phosphatase (PhoA) hybrid proteins in a mycobacterial expression system, the peptide region required for glycosylation of the 19 kDa lipoprotein antigen from M.tuberculosis was defined. Mutagenesis of two threonine clusters within this region abolished lectin binding by PhoA hybrids and by the 19 kDa protein itself. Substitution of the threonine residues also resulted in generation of a series of smaller forms of the protein as a result of proteolysis. In a working model to account for these observations, we propose that the role of glycosylation is to regulate cleavage of a proteolytically sensitive linker region close to the acylated N‐terminus of the protein.

Mycobacterium tuberculosis has innate resistance to a range of broadspectrum antimicrobial agents. This may in part reflect the relative impermeability of the mycobacterial cell wall, but additional specific mechanisms may also be important. In the case of fosfomycin, it has been suggested that a key difference in the active site of the M. tuberculosis MurA enzyme might confer resistance. In Escherichia coli, fosfomycin covalently binds to a cysteine normally involved in the enzymic activity, while protein alignments predict an aspartate at this position in the M. tuberculosis MurA. In the present study, it is demonstrated that the wild-type M. tuberculosis MurA is indeed resistant to fosfomycin, and that it becomes sensitive following replacement of the aspartate residue in position 117 by a cysteine. In addition, the study illustrates the use of an inducible expression system in mycobacteria to allow functional characterization of an M. tuberculosis enzyme that is unstable during constitutive expression.

A recombinant expression system was developed to analyse sequence determinants involved in O-glycosylation of proteins in mycobacteria. By expressing peptide sequences corresponding to known glycosylation sites within a chimeric lipoprotein construct, amino acids flanking modified threonine residues were found to have an important influence on glycosylation. The expression system was used to screen mycobacterial sequences selected using a neural network (NetOglyc) trained on eukaryotic O-glycoproteins. Evidence of glycosylation was obtained for eight of 11 proteins tested. The results suggest that sites involved in O-glycosylation of mycobacterial and eukaryotic proteins share similar structural features.z 2000 Federation of European Biochemical Societies.

Porphyromonas gingivalis, a black-pigmenting anaerobe implicated in the aetiology of periodontal disease, contains two loci, rgpA and rgpB, encoding the extracellular Arg-X specific proteases (RGPs, Arg-gingipains), and kgp, which encodes a Lys-X specific protease (KGP, Lys-gingipain). The rgpA and kgp genes encode polyproteins comprising pro-peptide and catalytic domain with large N-and C-terminal extensions which require proteolytic processing at several Arg and Lys residues to generate mature enzymes. The product of rgpB contains only a pro-peptide and the catalytic domain which requires processing at an Arg residue to generate active enzyme. An rgpA rgpB double mutant (E8) of P. gingivalis was constructed to study the role of RGPs in the processing of KGP. A kgp mutant (K1A) was also studied to investigate the role of KGP in the generation of RGPs. E8 was stable in the absence of the antibiotics tetracycline and clindamycin (selection markers for rgpA and rgpB, respectively) and exhibited the same pigmentation, colony morphology and identical growth rates to the parent W50 strain in the absence of antibiotics, in both complex and chemically defined media. The KGP activity of E8, grown in the absence of tetracycline, in whole cultures and in culture supernatants (up to 6 d) was identical to levels in W50. However, in the presence of tetracycline in the growth medium, the level of KGP was reduced to 50 % of levels present in whole cultures of W50. Since tetracycline had no effect on RGP or KGP activity when incorporated into assay buffer, this effect is most likely to be on the synthesis of Kgp polypeptide. K1A was also stable in the absence of antibiotics but was unable to pigment, and remained straw-coloured throughout growth. RGP activity in whole cultures of K1A was identical to levels in W50, but RGP activity in 6 d culture supernatants was reduced to 50 % of levels present in W50. Thus, although KGP is not required for generation of RGP activity from RgpA and RgpB polypeptides, its absence affects the release/transport of RGP into culture supernatant.