Mycobacterial cell wall components

Barry C.E. Mdluli K. (1996) Drug sensitivity and environmental adaptation of mycobacterial cell wall components. Trends Microbiol, 4, 275-281. 

Adipex-P phentermine. acyuvant peptide (muramyl dipeptide MDP) is a Af-acetylmuramyl dipeptide, identified as the minimum structural constituent of the mycobacterial cell wall component of Freund s complete adjuvant, which is necessary for adjuvant activity. It and many of its analogues have been investigated as adjuvants in the immunization of animals, as (immunostimulant) immunomodulators. It also has some pyrogenic activity. 

C19H32N4O11 492.482 Identified as the minimum structural constit. of the mycobacterial cell wall component of Freund s complete adjuvant which is necessary for adjuvant activity. 

The carbohydrate-rich bacterial cell wall presents a structural challenge of a very different nature to the case studies above. Although the available sample amount for analysis is less of a problem in comparison to the situation with bioactive mammalian or helminth glycoproteins, the extremely diverse range of glycoconjugates, as well as novel saccharide residues and non-saccharide substituents that may be present, exerts a high premium on detailed structural characterization. Studies of mycobacterial cell wall components (Figure 16) exemplify recent trends in mass spectrometric analysis in this area of biopolymer research. 

The antimicrobial action of ethambutol, like that of isoniazid, is specific for mycobacteria, suggesting a target in the unique components of the mycobacterial cell wall. Cells treated with ethambutol accumulate an isoprenoid intermediate, decaprenyl-arabinose which is the source ofarabinose in the arabinogalactan polymer. This suggests that ethambutol blocks assembly of the arabinogalactan through inhibition of an arabinosyl transferase enzyme. 

Fractionation of mycobacteria resulted in the identification of two cellular immunostimulatory components, namely TDM and MDPs. Both are normally found in association with the mycobacterial cell wall. TDM is composed of a molecule of trehalose (a disaccharide consisting of two molecules of a-D-glucose linked via an a 1-1 glycosidic bond), linked to two molecules of my-colic acid (a long-chain aliphatic hydrocarbon-based acid) found almost exclusively in association with mycobacteria. TDM, although retaining its adjuvanticity, is relatively non-toxic. 

Cyclopropyl fatty acids with C12, Cjg and C21 have been formed in many gram-negative and gram-positive bacteria [198]. The mycolic acid 142 (Cso) is a major component of the mycobacterial cell wall of the human strain of M. tuber-culosiSyEq. (57) [199]. 

Isoniazid (INH) is a synthetic derivative of isonico-tinic acid. It has bactericidal activity against both intra- and extra-cellular mycobacteria. It also displays anti-bacterial activity in caseous lesions, but only in proliferating cells. Losing genes which code for catalase and peroxidase is the major mechanism through which resistance occurs. Single mutations can rapidly result in such resistance if isoniazid is used alone. Its mechanism of action is presumably based on inhibition of the synthesis of mycolic acids, unique and essential components of the mycobacterial cell wall. 

Isoniazid possibly exerts its action by inhibiting the synthesis of mycolic acid which is an essential component of mycobacterial cell wall. It is also postulated that the ability of isoniazid to suppress the formation of DNA and RNA and also inhibition of various oxidative mechanisms may be responsible for its action. 

Susceptible strains of Mycobacterium tuberculosis and other mycobacteria are inhibited in vitro by ethambutol, 1-5 mcg/mL. Ethambutol inhibits mycobacterial arabinosyl transferases, which are encoded by the embCAB operon. Arabinosyl transferases are involved in the polymerization reaction of arabinoglycan, an essential component of the mycobacterial cell wall. Resistance to ethambutol is due to mutations resulting in overexpression of emb gene products or within the embB structural gene. 

Isoniazid Inhibits synthesis of mycolic acids, an essential component of mycobacterial cell walls Bactericidal activity against susceptible strains of M tuberculosis First-line agent for tuberculosis treatment of latent infection less active against other mycobacteria Oral, IV hepatic clearance (half-life 1 h) reduces levels of phenytoin Toxicity Flepatotoxic, peripheral neuropathy (give pyridoxine to prevent)... 

Ethambutol Inhibits mycobacterial arabinosyl transferases, which are involved in the polymerization reaction of arabinoglycan an essential component of the mycobacterial cell wall Bacteriostatic activity against susceptible mycobacteria Given as four-drug initial combination therapy for tuberculosis until drug sensitivities are known also used for atypical mycobacterial infections Oral t mixed clearance (half-life 4 h) dose must be reduced in renal failure Toxicity Retrobulbar neuritis... 

Bacterial DNA, long a component of the earlier whole cell vaccines, has been shown to have an immunostimulatory effect on immune cells and is a potent inducer of cytokines such as IL-1, IL-6, and IL-12. Monophosphoryl A, a component of mycobacterial cell walls, reacts with receptors on antigen producing cells and generates a Thl response due to the production of IL-2 and IFN-y. 

Other monosaccharide components (of bacterial polysaccharides) that are structurally related to D-ribose include D-riburonic acid,232 identified in the exocellular polysaccharide produced by a strain of Rhizobium meliloti, and D-arabinose, frequently present as the furanose, in polysaccharides of mycobacterial cell-wall.233,234 L-Xylose235,236 should probably be included in the group, as it may be derived from D-arabinose through epimerization at C-4. Biosynthesis of these monosaccharides was not investigated. 

When crude endotoxin from the heptose-less mutant of Salmonella typhimurium is combined with trehalose dimycolate from mycobacteria in oil droplets and injected directly into established tumors (line 10 hepatocellular carcinoma) in syngeneic guinea pigs, rapid regression of the tumors occurs and over 90% of the animals are cured. The three required components for activity in this tumor model are (a) the endotoxin (b) the mycobacterial adjuvant, trehalose dimycolate and (c) a compound satisfying the minimal structural requirement (muramyl dipeptide) for adjuvant activity by bacterial cell wall materials. The mycobacterial cell wall skeleton is able to replace the latter two components. 

The component(s) of the mycobacterial cell wall responsible for conferring intrinsic resistance to antibiotics are not yet known with any certainty. However, it has been shown [91-93] that inhibitors of arabinogalactan synthesis increase mycobacterial sensitivity to antibiotics. When M. avium is treated with inhibitors of mycolic acid biosynthesis there are significant alterations in outer cell wall layers and the cells show an increased antibiotic susceptibility [93], Thus, arabinogalactan and mycolic acids are components of the wall associated with intrinsic resistance of mycobacteria to chemotherapeutic drugs and alteration of these structural building blocks leads to increased intracellular penetration of antibiotics [88,94,95],... 

Retrosynthesis of octaarabinose 417. A component of lipoarabinomannan in mycobacterial cell wall... 

The basic structure of the mycobacterial cell wall is shown in O Fig. 1 (O Sect. 1). The major component of the cell wall is the mycolyl-arabinogalactan-peptidoglycan (mAGP) complex [21]. This consists of an inner peptidoglycan layer attached through a linker unit to an ara-binogalactan polysaccharide, which is in turn esterified to mycolic acids. The mycolic acids are branched hydroxy fatty acids that form the inner leaflet of the lipid bilayer. The outer leaflet... 

Isoniazid interferes with mycolic acid synthesis by inhibiting an enoyl reductase (InhA) which forms part of the fatty acid synthase system in mycobacteria. Mycolic acids are produced by a diversion of the normal fatty acid synthetic pathway in which short-chain (16 carbon) and long-chain (24 carbon) fatty acids are produced by addition of 7 or 11 malonate extension units from malonyl coenzyme A to acetyl coenzyme A. InhA inserts a double bond into the extending fatty acid chain at the 24 carbon stage. The long-chain fatty acids are further extended and condensed to produce the 60-90 carbon (3-hydroxymycolic acids which are important components of the mycobacterial cell wall. Isoniazid is converted inside the mycobacteria to a free radical species by a catalase peroxidase enzyme, KatG. The active free radicals then attack and inhibit the enoyl reductase, InhA, by covalent attachment to the active site. 

Mechanisms Ethambutol inhibits arabinosyl transferases (encoded by the emhCAB operon) involved in the synthesis of arabinogalactan, a component of mycobacterial cell walls. Resistance occurs rapidly via mutations in the emb gene if the drug is used alone. 

The mycobacterial cell wall contains three main components. Firstly, there is a skeleton composed of arabinogalactan mycolate covalently linked through a phosphodiester bond to peptidoglycan. There are also peptides which are removed by proteinolysis and free lipids which are easily extracted by solvents. 

M. tuberculosis lacks a regularly structured, unique surface polysaccharide that could be used as a vaccine target. The major component of the mycobacterial cell wall is a complex peptidoglycan with several covalently linked molecules as for example arabinogalactan and lipoar-abinomannan (LAM). 

Figure 16. A chemical model of the mycobacterial cell wall. The two major components are the mycolyl arabinogalactan-peptidoglycan complex and the lipoarabinomannan/lipomannan. The amount and exact chemical nature of the surface glycolipids are species- and/or strain-specific.

The cell walls of mycobacteria contain three structures peptidoglycan, an arabinogalactan polysaccharide and long chain hydroxy fatty acids (mycolic acids) which are all covalently linked. Additional non-covalently attached lipid components found in the wall include glycolipids, various phospholipids and waxes. The lipid-rich nature of the mycobacterial wall is responsible for the characteristic acid-fastness on staining and serves as a penetration barrier to many antibiotics. Isoniazid and ethambutol have long been known as specific antimycobacterial agents but their mechanisms of action have only recently become more clearly understood. 

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