Key physiological differences between bacterial and mammalian metabolism provide opportunities for the development of novel antimicrobials. We examined the role of the multifunctional enzyme S-adenosylhomocysteine/Methylthioadenosine (SAH/MTA) nucleosidase (Pfs) in the virulence of S. enterica var Typhimurium ( S. Typhimurium) in mice, using a defined Pfs deletion mutant (i.e. Δ pfs ). Pfs was essential for growth of S. Typhimurium in M9 minimal medium, in tissue cultured cells, and in mice. Studies to resolve which of the three known functions of Pfs were key to murine virulence suggested that downstream production of autoinducer-2, spermidine and methylthioribose were non-essential for Salmonella virulence in a highly sensitive murine model. Mass spectrometry revealed the accumulation of SAH in S. Typhimurium Δ pfs and complementation of the Pfs mutant with the specific SAH hydrolase from Legionella pneumophila reduced SAH levels, fully restored growth ex vivo and the virulence of S. Typhimurium Δ pfs for mice. The data suggest that Pfs may be a legitimate target for antimicrobial development, and that the key role of Pfs in bacterial virulence may be in reducing the toxic accumulation of SAH which, in turn, suppresses an undefined methyltransferase.

Coxiella burnetii is a Gram-negative bacterium which causes Q fever, a complex and life-threatening infection with both acute and chronic presentations. C. burnetii invades a variety of host cell types and replicates within a unique vacuole derived from the host cell lysosome. In order to understand how C. burnetii survives within this intracellular niche, we have investigated the carbon metabolism of both intracellular and axenically cultivated bacteria. Both bacterial populations were shown to assimilate exogenous [ 13 C]glucose or [ 13 C]glutamate, with concomitant labeling of intermediates in glycolysis and gluconeogenesis, and in the TCA cycle. Significantly, the two populations displayed metabolic pathway profiles reflective of the nutrient availabilities within their propagated environments. Disruption of the C. burnetii glucose transporter, CBU0265, by transposon mutagenesis led to a significant decrease in [ 13 C]glucose utilization but did not abolish glucose usage, suggesting that C. burnetii express additional hexose transporters which may be able to compensate for the loss of CBU0265. This was supported by intracellular infection of human cells and in vivo studies in the insect model showing loss of CBU0265 had no impact on intracellular replication or virulence. Using this mutagenesis and [ 13 C]glucose labeling approach, we identified a second glucose transporter, CBU0347, the disruption of which also showed significant decreases in 13 C-label incorporation but did not impact intracellular replication or virulence. Together, these analyses indicate that C. burnetii may use multiple carbon sources in vivo and exhibits greater metabolic flexibility than expected.

All mycobacterial species, including pathogenic Mycobacterium tuberculosis , synthesize an abundant class of phosphatidylinositol mannosides (PIMs) that are essential for normal growth and viability. These glycolipids are important cell-wall and/or plasma-membrane components in their own right and can also be hyperglycosylated to form other wall components, such as lipomannan and lipoarabinomannan. We have investigated the steps involved in the biosynthesis of the major PIM species in a new M. smegmatis cell-free system. A number of apolar and polar PIM intermediates were labelled when this system was continuously labelled or pulse–chase-labelled with GDP-[ 3 H]Man, and the glycan head groups and the acylation states of these species were determined by chemical and enzymic treatments and octyl-Sepharose chromatography respectively. These analyses showed that (1) the major apolar PIM species, acyl-PIM2, can be synthesized by at least two pathways that differ in the timing of the first acylation step, (2) early PIM intermediates containing a single mannose residue can be modified with two fatty acid residues, (3) formation of polar PIM species from acyl-PIM2 is amphomycin-sensitive, indicating that polyprenol phosphate-Man, rather than GDP-Man, is the donor for these reactions, (4) modification of acylated PIM4 with α1-2- or α1-6-linked mannose residues is probably the branch point in the biosyntheses of polar PIM and lipoarabinomannan respectively and (5) GDP strongly inhibits the synthesis of early PIM intermediates and increases the turnover of polyprenol phosphate-Man. These findings are incorporated into a revised pathway for mycobacterial PIM biosynthesis.

Mycobacteria are the causative agents of tuberculosis and several other significant diseases in humans. All species of mycobacteria synthesize abundant cell-wall mannolipids (phosphatidylinositol mannosides, lipoarabinomannan), a cytoplasmic methylmannose polysaccharide and O-mannosylated glycoproteins. To investigate whether these molecules are essential for mycobacterial growth, we have generated a Mycobacterium smegmatis mannose auxotroph by targeted deletion of the gene encoding phosphomannose isomerase (PMI). The PMI deletion mutant displayed a mild hyperseptation phenotype, but grew normally in media containing an exogenous source of mannose. When this mutant was suspended in media without mannose, ongoing synthesis of both the mannolipids and methylmannose polysaccharides was halted and the hyperseptation phenotype became more pronounced. These changes preceded a dramatic loss of viability after 10 h in mannose-free media. Mannose starvation did not lead to detectable changes in cell-wall ultrastructure or permeability to hydrophobic drugs, or to changes in the rate of biosynthesis of other plasma-membrane or wall-associated phospholipids. These results show that mannose metabolism is required for growth of M. smegmatis and that one or more mannose-containing molecules may play a role in regulating septation and cell division in these bacteria.

Free glycosylphosphatidylinositols (GPIs) are an important class of membrane lipids in many pathogenic protozoa. In this study, we have investigated the subcellular distribution and intracellular trafficking of an abundant class of free GPIs [termed glycosylinositolphospholipids (GIPLs)] in Leishmania mexicana promastigotes. The intracellular transport of the GIPLs and the major GPI-anchored glycoprotein gp63 was measured by following the incorporation of these molecules into sphingolipid-rich, detergent-resistant membranes (DRMs) in the plasma membrane. In metabolic-labelling experiments, mature GIPLs and gp63 were transported to DRMs in the plasma membrane with a t 1/2 of 70 and 40min, respectively. Probably, GIPL transport to the DRMs involves a vesicular mechanism, as transport of both the GIPLs and gp63 was inhibited similarly at 10°C. All GIPL intermediates were quantitatively recovered in Triton X-100-soluble membranes and were largely orientated on the cytoplasmic face of the endoplasmic reticulum, as shown by their sensitivity to exogenous phosphatidylinositol-specific phospho-lipase C. On the contrary, a significant proportion of the mature GIPLs (≈50% of iM4) were accessible to membrane-impermeable probes on the surface of live promastigotes. These results suggest that the GIPLs are flipped across intracellular or plasma membranes during surface transport and that a significant fraction may populate the cytoplasmic leaflet of the plasma membrane. Finally, treatment of L. mexicana promastigotes with myriocin, an inhibitor of sphingolipid biosynthesis, demonstrated that ongoing sphingolipid biosynthesis is not required for the plasma-membrane transport of either gp63 or the GIPLs and that DRMs persist even when cellular levels of the major sphingolipid are depleted by 70%.

The variant-specific surface proteins (VSPs) of the ancient protist Giardia duodenalis (syn.: Giardia intestinalis , Giardia lamblia ) are cysteine- and threonine-rich polypeptides that can vary considerably in sequence and size. In the present study, we have purified a VSP (VSP4A1, formerly called CRISP-90) from a cloned Giardia isolate, derived from a sheep, by Triton X-114 phase partitioning and anion-exchange chromatography. Analysis of the purified VSP4A1 showed that this protein is post-translationally modified with both glycans and lipid. The glycans of VSP4A1 were detected and partially characterized by (1) compositional analysis, which indicated the presence of GlcNAc and Glc (0.5 and 1.0 mol/mol of protein respectively), and (2) the specific labelling of VSP4A1 with galactosyltransferase/UDP-[ 3 H]Gal. The glycans were released by β-elimination, suggesting that they are O-linked to the protein. Bio-Gel P4 chromatography of the released galactosylated glycans and further compositional analysis suggested that the major glycan on the VSP is a trisaccharide with Glc at the reducing terminus. These and other results indicate the absence of any N-linked glycans on the VSP and suggest instead that it is elaborated with a novel type of short O-linked glycan. Compositional analysis and radiolabelling experiments also indicated that VSP4A1 is modified with covalently linked palmitate (1 mol/mol of protein). Hydroxylamine treatment at neutral pH of [ 3 H]palmitate-labelled VSP4A1 indicated that the acyl chain may be attached by a thioester linkage. A likely location for the lipid modification appears to be in the region of the C-terminal domain where it may facilitate association of the protein with the plasma membrane.

The monosaccharide D -arabinopyranose has only been found in glycoconjugates of the trypanosomatid parasites Leishmania major, Endotrypanum schaudinni and Crithidia fasciculata. The donor molecule for the relevant arabinosyltransferases is known to be GDP-α- D -Ara p in L. major and C. fasciculata, and the latter organism is being used to study the biosynthesis of GDP-α- D -Ara p. In this study, we describe the structure of the terminal product of arabinose metabolism in C. fasciculata, namely lipoarabinogalactan. This molecule was purified by hydrophobic-interaction chromatography and studied by a variety of techniques, including gas chromatography–mass spectrometry, electrospray mass spectrometry and chemical and enzymic digestions. These data show that lipoarabinogalactan contains a previously described D -arabino- D -galactan polysaccharide component covalently attached to a glycosylphosphatidylinositol type of membrane anchor that is similar to, but not identical with, that found in the lipophosphoglycans of the Leishmania .