Formation of dTDP-Rhamnose Is Essential for Growth of Mycobacteria

Abstract

It was determined that the dTDP-rhamnose synthesis gene, rmlD, could be inactivated in Mycobacterium smegmatis only in the presence of a rescue plasmid carrying functional rmlD. Hence, dTDP-rhamnose biosynthesis is essential for the growth of mycobacteria and the targeting of dTDP-rhamnose synthesis for new tuberculosis drugs is supported.The mycobacterial cell wall consists of a mycolic acid layer tethered to peptidoglycan via the polysaccharide arabinogalactan (3,13,14). Arbinogalactan is attached to peptidoglycan via ␣-L-rhamnopyranosyl-(133)-␣-D-N-acetyglucosaminosyl-1-phosphate (13). This structural arrangement strongly suggests that rhamnosyl residues are essential for the growth and viability of mycobacteria.L-Rhamnosyl residues are not present in humans. They are synthesized in bacteria from dTDP-glucose via the enzymes RmlB, RmlC, and RmlD (5,8,15,17). There is no salvage pathway for the formation of dTDP-L-rhamnose (dTDP-Rha) as there is with GDP-L-fucose (19,20). The dTDP-Rha synthetic enzymes are soluble and have been studied by X-ray crystallography (1, 6, 7). Given these facts, the dTDP-Rha formation enzymes have significant potential as targets for new tuberculosis drugs. Therefore, it is important to directly demonstrate that an enzyme involved in the formation of these enzymes is essential.The final enzyme in the series synthesizing dTDP-Rha is dTDP-6-deoxy-L-lyxo-4-hexulose reductase. Its gene, rmlD, is the first gene of an operon which also contains wbbL and manB (12). This arrangement occurs in all the mycobacterial genomes sequenced. The gene wbbL encodes the rhamnosyl transferase that inserts rhamnose into the cell wall and is also expected to be essential. Thus, a nonpolar rmlD knockout mutation was desired so that complementation would be straightforward.The basic strategy used was to prepare a copy of rmlD interrupted with a kanamycin resistance cassette orientated in the same direction as rmlD with the hope that, after gene replacement, the downstream wbbL and manB genes would be transcribed from the kanamycin resistance promoter. The disrupted rmlD gene was then used to replace wild-type rmlD in the presence of an appropriate rescue plasmid.Constructing the rmlD replacement plasmid (pFP201) and obtaining the first homologous recombination event.A partial genomic DNA library of a mycobacterial lab strain (18) was constructed by isolation of SmaI fragments of approximately 3.5 kb and ligation into pCR-Blunt (Invitrogen, Carlsbad, Calif.). The rmlD gene was located by colony hybridization (9) using Mycobacterium tuberculosis rmlD (10) as a probe. Preliminary sequence data showed that the rmlD DNA sequence was almost identical to that of Mycobacterium smegmatis mc 2 155 (The Institute for Genomic Research website [http: //www.tigr.org/]). Then a 1.6-kb DNA fragment (containing rmlD and 417 bp upstream and 328 bp downstream of it) was cut out by EcoRI and XhoI, filled in with the Klenow fragment, and then inserted into pCR-Blunt to generate pCR-rmlD. By using methods previously described (1...

GacA is essential for Group A Streptococcus and defines a new class of monomeric dTDP‐4‐dehydrorhamnose reductases (RmlD)

“…Insertions for gacA demonstrate that gacA is indeed essential in the GAS strains 5448 (M1T1) and NZ131 (M49) when growing in rich media. These data are in agreement with a previous study conducted on Mycobacterium smegmatis (Ma et al ., 2002), where rmlD was shown to be essential for mycobacterial growth. To validate gacA essentiality in an independent manner, we employed a previously published conditionally lethal approach that takes advantage of a theophylline‐sensitive synthetic riboswitch functional in GAS (Le Breton et al ., 2015).…”

“…The dTDP‐L‐rhamnose biosynthesis is an interesting target for the development of new drugs since (i) the pathway affects either the viability or virulence of many bacteria, including Mycobacterium spp. (Ma et al ., 2002), Pseudomonas spp. (Engels et al ., 1985) and E. faecalis (Teng et al ., 2009) and (ii) the pathway does not exist in humans, reducing the risk of side‐effects by off‐target effects.…”

“…The production of dTDP‐L‐rhamnose is critical for GAC biosynthesis but also more broadly for the viability or virulence of other medically important bacteria including Mycobacterium spp . (Ma et al ., 2002), Pseudomonas spp . (Engels et al ., 1985) and Enterococcus faecalis (Teng et al ., 2005).…”

Identification and Active Expression of the Mycobacterium tuberculosis Gene Encoding 5-Phospho-α-D-ribose-1-diphosphate: Decaprenyl-phosphate 5-Phosphoribosyltransferase, the First Enzyme Committed to Decaprenylphosphoryl-D-arabinose Synthesis

“…The structure of arabinogalactan is conveniently divided into three regions, namely the linker region (2), a D-galactofuranosyl region (3), and an arabinofuranosyl region (3). Enzymes required for arabinogalactan formation have been shown to be essential for mycobacterial growth (4,5). The donor for the arabinofuranosyl residues has been shown to be decaprenylphosphoryl-D-arabinose (6 -8).…”

Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in Pathogenic Fungi Magnaporthe grisea and Botryotinia fuckeliana

“…This product is then converted to dTDP-4-keto-rhamnose by a 3,5-epimerase (rmlC). An enzyme with 4-reductase activity (rmlD) converts the keto-rhamnose to dTDP-Rha (15)(16)(17). Plants have a single protein (Rhm (UDP-L-rhamnose synthase) also annotated as URS) that converts UDP-Glc to UDP-Rha (18,19).…”