David E. Minnikin

Polar Lipid Composition in the Classification of Nocardia and Related Bacteria

Strains representing the mycolic acid-containing taxa Nocardia, Mycobacterium, Gordona, Corynebacterium, Bacterionema, and the “rhodochrous” complex were analysed for polar lipids by two-dimensional, thin-layer chromatography; diphosphatidylglycerol and phosphatidylinositol were found in all strains and phosphatidylethanolamine was absent only in extracts of Corynebacterium and Bacterionema. Mono- and diacyl phosphatidylinositol dimannosides were present in all Nocardia, Mycobacterium, Corynebacterium, and rhodochrous strains and in the single strain of Gordona aurantiaca examined; Bacterionema and strains of the other Gordona species had only a monoacyl phosphatidylinositol dimannoside. Phosphatidylglycerol was present in substantial amounts in extracts of two strains of Bacterionema matruchotii and in reduced proportions in strains of several other species. Unidentified glycolipids were detected in the lipids of a majority of the organisms investigated.

Intracellular lipophilic inclusions of mycobacteria in vitro and in sputum

Although most mycobacterial lipids are thought to be associated with the cell envelope, the authors previously observed substantial deposits of intracellular lipophilic material. A Nile-red-based cytological assay was used to determine factors which affect the presence and natural history of intracellular lipophilic inclusions (ILIs) in Mycobacterium smegmatis. Development of ILIs was associated with stationary-phase cultures in broth and with aged (6 days) colonies on agar. Using variants of Youmans defined medium, the frequency and size of ILIs was observed to be minimal in carbon-poor medium. ILIs were observed to form within 15 min after provision of fatty acids to the medium and after a period of several days in nitrogen-poor medium. Analysis of the non-polar lipid extracts of ILI-rich and -poor preparations indicated that the triacylglycerols (TAGs) were a major component of the inclusions. The acyl substituents of the TAGs varied according to whether they were formed in Middlebrook 7H9 broth, in low-nitrogen Youmans broth or rapidly after oleic acid supplementation of Youmans broth. These studies support a storage function for TAGs in mycobacteria in addition to their previously suggested occurrence as components of the cell envelope. To assess a possible role for ILIs in Mycobacterium tuberculosis, a combined acid-fast (Auramine)/Nile red assay was applied to heavily positive sputum samples from patients with tuberculosis. Strong intracellular Nile red signals were obtained from acid-fast cells, indicating that ILI occur in M. tuberculosis in vivo. This may reflect a distinct physiological state of these cells, which it has not been possible to reproduce in vitro. These findings indicate that the uptake of long-chain fatty acids and TAG biosynthetic and degradative pathways are important aspects of mycobacterial lipid metabolism, meriting further investigation.

A Type II Pathway for Fatty Acid Biosynthesis Presents Drug Targets in Plasmodium falciparum

ABSTRACT It has long been held that the malaria parasite, Plasmodium sp., is incapable of de novo fatty acid synthesis. This view has recently been overturned with the emergence of data for the presence of a fatty acid biosynthetic pathway in the relict plastid of P. falciparum (known as the apicoplast). This pathway represents the type II pathway common to plant chloroplasts and bacteria but distinct from the type I pathway of animals including humans. Specific inhibitors of the type II pathway, thiolactomycin and triclosan, have been reported to target this Plasmodium pathway. Here we report further inhibitors of the plastid-based pathway that inhibit Plasmodium parasites. These include several analogues of thiolactomycin, two with sixfold-greater efficacy than thiolactomycin. We also report that parasites respond very rapidly to such inhibitors and that the greatest sensitivity is seen in ring-stage parasites. This study substantiates the importance of fatty acid synthesis for blood-stage parasite survival and shows that this pathway provides scope for the development of novel antimalarial drugs.

Mycolic acid biosynthesis and enzymic characterization of the beta-ketoacyl-ACP synthase A-condensing enzyme from Mycobacterium tuberculosis.

Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a beta-ketoacyl-ACP synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA.

Location of double bonds and cyclopropane rings in fatty acids by mass spectrometry

Abstract Electron-impact mass spectrometric procedures for locating the position of double bonds and cyclopropane rings in long-chain fatty acids are reviewed. Since unsaturation is not located directly by mass spectrometry, the properties of suitable derivatives are summarized. Epoxides are readily prepared from double bonds and on opening of the ring with various reagents useful derivatives are obtained, the most promising to date being hydroxymethoxy esters whose trimethylsilyl ethers give good mass spectra. Trimethylsilyl ethers of vicinal diols, prepared by direct hydroxylation, are recommended for the analysis of polyunsaturated fatty acid esters using combined gas chromatography-mass spectrometry. Oxymercuration-demercuration techniques are very convenient and one particular procedure can specifically locate unsaturation up to five carbons distant from the carboxyl group. An alternative approach enables the location of double bonds and cyclopropane rings in fatty acids by direct mass spectrometry of pyrrolidides. Cyclopropane rings can be positively located in fatty acid esters by mass spectrometry of isomeric ketones or methoxy derivatives prepared by chromium trioxide oxidation on poron trifluoride-catalysed methoxylation, respectively. A variety of other procedures are also considered and some guidelines are given for choosing a method to suit a particular unsaturated acid.

Analogues of thiolactomycin: potential drugs with enhanced anti-mycobacterial activity.

Analogues of the antibiotic thiolactomycin (TLM) have been synthesized and have been shown to have enhanced activity against whole cells of Mycobacterium tuberculosis H37Rv and against mycolic acid biosynthesis in cell extracts of Mycobacterium smegmatis. TLM has a methyl-branched butadienyl side chain attached at position 5 on a thiolactone ring, namely 4-hydroxy-3,5-dimethyl-5H-thiophen-2-one. Various combinations of strong bases were explored to create a reactive anion at position 5 on the thiolactone ring which could react with halides to produce 5-substituted derivatives; the best reagent was two equivalents of lithium-bis-(trimethylsilyl)amide in tetrahydrofuran. The analogue with a 5-tetrahydrogeranyl substituent showed the best biological activity with an MIC(90) for M. tuberculosis of 29 micro M and 92% mycolate inhibition in extracts of M. smegmatis, as compared to 125 micro M and 54%, respectively, for TLM; other related C(10) and C(15) isoprenoid derivatives had similar biological activity. These isoprenoid-based derivatives did not inhibit type II fatty acid synthase from M. smegmatis, but compounds with iso-butyl and iso-butenyl side chains did show some inhibitory activity against this enzyme. These short-chain derivatives did not inhibit mycolate synthesis or have significant antibiotic activity. Treatment of the thiolactone with a weaker base, sodium hydride in tetrahydrofuran, gave 3-alkyl-3,5-dimethyl-thiophene-2,4-dione analogues, which had no effect on fatty acid or mycolate synthesis. However, the geranyl derivative had an MIC(99) of 60 micro M for M. tuberculosis, one quarter that (240 micro M) of TLM, demonstrating its excellent antibiotic potential against an unknown cellular target.

Classification of thermophilic streptomycetes, including the description of Streptomyces thermoalcalitolerans sp. nov.

A polyphasic taxonomic study was undertaken to clarify relationships within and between representative thermophilic alkalitolerant streptomycetes isolated from soil and appropriate marker strains. The resultant data, notably those from DNA-DNA relatedness studies, support the taxonomic integrity of the validly described species Streptomyces thermodiastaticus, Streptomyces thermoviolaceus and Streptomyces thermovulgaris. However, the genotypic and phenotypic data clearly show that Streptomyces thermonitrificans Desai and Dhala 1967 and S. thermovulgaris (Henssen 1957) Goodfellow et al. 1987 represent a single species. On the basis of priority, S. thermonitrificans is a later subjective synonym of S. thermovulgaris. Similarly, 10 out of the 11 representative thermophilic alkalitolerant isolates had a combination of properties consistent with their classification as S. thermovulgaris. The remaining thermophilic alkalitolerant isolate, Streptomyces strain TA56, merited species status. The name Streptomyces thermoalcalitolerans sp. nov. is proposed for this strain. A neutrophilic thermophilic isolate, Streptomyces strain NAR85, was identified as S. thermodiastaticus.

Variation of polar lipid composition of Bacillus subtilis (Marburg) with different growth conditions

A previous study of the polar lipids of Bacillus subtilis (Marburg) has shown that at least five phospholipids in addition to glycolipid may be present [ 11. The report shows that by manipulation of growth conditions the lipid composition of this organism can be varied and greatly simplified. In batch cultures, under conditions of apparent phosphate starvation, a close interrelation of phospholipids and phosphate-free polar lipids is observed, the latter increasing in proportion as growth progresses. Certain chemostat cultures (magnesium limitation) are found to contain only one acidic phospholipid, phosphatidylglycerol (PC), and one neutral polar lipid, diglucosyldiglyceride (DG). In phosphate-limited cultures the proportions of phospholipids are reduced and phosphate-free polar lipids are observed in increased proportions; one of these lipids is DG, the other is an acidic peptidolipid. vessel. Lipids were extracted from lyophilized cells with chloroform-methanol (2: 1, v/v) and investigated by two-dimensional thin-layer chromatography [4] (fig. 2 for details). Qualitative identifications of lipids was made by use of specific spray reagents [2], and densitometry [6] gave an approximate measure of the relative proportions of the lipids. The lipids of B. subtilis (Marburg) grown in batch culture included the phospholipids reported in previous studies [I] i.e. diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), PG and traces of its lysine ester. DG was identified in all cultures, and in some extracts an additional phosphorous-free lipid (X) was found (see fig. 1). Preliminary investigations

Stimulation of mycolic acid biosynthesis by incorporation of cis-tetracos-5-enoic acid in a cell-wall preparation from Mycobacterium smegmatis

Mycolic acids are high molecular weight hydroxy fatty acids which are a covalently linked part of the cell wall structure of all mycobacteria and their biosynthetic pathways offer potential drug targets. Three good candidates, cis-tetracos-5-enoic acid and R or S trans-6-methyl-tetracos-4-enoic acids, for the key initial intermediates where mycolic acid biosynthesis might diverge from other metabolic pathways, were tested as possible substrates. A cell-wall preparation from Mycobacterium smegmatis, capable of mycolic acid synthesis, was developed to investigate the possible incorporation of these, and other 16 to 24 carbon acids into mycolic acids. The wall preparations were extracted with hexane and suspended in hexane/water (7:1, v/v), and in this low-water assay, only one of these acids, cis-tetracos-5-enoic acid, stimulated the incorporation of radioactive label from [1-14C]acetate into alpha- and alpha-mycolic acids. The extraction method used did, however, abolish some enzyme activity and mycolic acid biosynthesis was not completely restored by cis-tetracos-5-enoate. The two methyl-branched acids did not enhance the amount of label in epoxymycolic acids. An initial key intermediate in the synthesis of alpha- and alpha-mycolic acids has therefore been positively identified for the first time; intermediates in the initial stages of the biosynthesis of oxygenated mycolic acids such as epoxymycolates remain to be defined.

Identification and structural characterization of an unusual mycobacterial monomeromycolyl-diacylglycerol

Systematic thin layer chromatographic (TLC) analysis of apolar lipids in Mycobacterium kansasii revealed the presence of a previously uncharacterized novel component. The product was ubiquitously found in a panel of M. kansasii clinical isolates, as well as other pathogenic and non‐pathogenic mycobacterial species. TLC analysis of [14C]‐acetate‐ or [14C]‐glycerol‐labelled M. kansasii cultures tentatively assigned the novel product as an unusual triacylglycerol‐related lipid. Subsequent purification, followed by structural determination using 1H‐nuclear magnetic resonance (NMR) and electrospray mass spectrometry (ES/MS), led to the identification of this product as a monomeromycolyl‐diacylglycerol (MMDAG). Treatment of M. kansasii with either isoniazid (INH), a well‐known type II fatty acid synthase (FAS‐II) and mycolic acid biosynthesis inhibitor, or tetrahydrolipstatin (THL), a drug approved for treating obesity, correlated with a reduced incorporation of [14C]‐acetate into both mycolic acids and MMDAG. Addition of INH or THL to the cultures induced major morphological changes and, surprisingly, resulted in an increased number of lipid storage bodies, as determined by electron microscopy. The potent antimycobacterial activity of THL was confirmed against a variety of mycobacterial species, including INH‐susceptible and ‐resistant Mycobacterium tuberculosis strains. Therefore, THL and other β‐lactones may be promising drugs for the development of new antitubercular therapy.