Kuni Takayama

Pathway to Synthesis and Processing of Mycolic Acids in Mycobacterium tuberculosis

SUMMARY Mycobacterium tuberculosis is known to synthesize α-, methoxy-, and keto-mycolic acids. We propose a detailed pathway to the biosynthesis of all mycolic acids in M. tuberculosis. Fatty acid synthetase I provides C20-S-coenzyme A to the fatty acid synthetase II system (FAS-IIA). Modules of FAS-IIA and FAS-IIB introduce cis unsaturation at two locations on a growing meroacid chain to yield three different forms of cis,cis-diunsaturated fatty acids (intermediates to α-, methoxy-, and keto-meroacids). These are methylated, and the mature meroacids and carboxylated C26-S-acyl carrier protein enter into the final Claisen-type condensation with polyketide synthase-13 (Pks13) to yield mycolyl-S-Pks13. We list candidate genes in the genome encoding the proposed dehydrase and isomerase in the FAS-IIA and FAS-IIB modules. We propose that the processing of mycolic acids begins by transfer of mycolic acids from mycolyl-S-Pks13 to d-mannopyranosyl-1-phosphoheptaprenol to yield 6-O-mycolyl-β-d-mannopyranosyl-1-phosphoheptaprenol and then to trehalose 6-phosphate to yield phosphorylated trehalose monomycolate (TMM-P). Phosphatase releases the phosphate group to yield TMM, which is immediately transported outside the cell by the ABC transporter. Antigen 85 then catalyzes the transfer of a mycolyl group from TMM to the cell wall arabinogalactan and to other TMMs to produce arabinogalactan-mycolate and trehalose dimycolate, respectively. We list candidate genes in the genome that encode the proposed mycolyltransferases I and II, phosphatase, and ABC transporter. The enzymes within this total pathway are targets for new drug discovery.

Studies on the electron transfer system: LXVII. Polyacrylamide gel electrophoresis of the mitochondrial electron transfer complexes

Abstract A new method is described for the fractionation of the hydrophobic proteins of the mitochondrial electron transfer chain. The four constituent complexes of the chain, complex I (DPNH-coenzyme Q reductase), Complex II (succinic-coenzyme Q reductase), Complex III (reduced coenzyme Q-cytochrome c reductase), and Complex IV (cytochrome c ; O 2 oxidoreductase E.C.1.9.3.1) and also succinic dehydrogenase (succinic; (acceptor) oxidoreductase, E.C. 1.3.99.1), after extraction with acetone, were completely soluble in a solvent system consisting of phenol-acetic acid-water (2:1:1, w/v/v). Zone electrophoresis of the solubilized mitochondrial complexes and of succinic dehydrogenase on polyacrylamide gel (7.5% in acrylamide, 35% in acetic acid and 5 M in urea) revealed, in every case, many protein bands with a specific and reproducible pattern for each enzyme complex. These bands include not only the proteins with known prosthetic groups but also other uncharacterized proteins. Bands were identified corresponding to (1) the flavoprotein of Complex II, (2) the flavine protein of the soluble succinic dehydrogenase, and (3) the cytochrome c 1 of Complex III. Bands were tentatively identified corresponding to the nonheme iron protein, to cytochrome b of Complex III, and to cytochrome a of Complex IV. The results support the concept that the electron transfer chain is composed of a series of proteins of small molecular weight.

Effect of Isoniazid on the In Vivo Mycolic Acid Synthesis, Cell Growth, and Viability of Mycobacterium tuberculosis

When an actively growing culture of the H37Ra strain of Mycobacterium tuberculosis was exposed to isoniazid at a concentration of 0.5 μg/ml, the cells began to lose their ability to synthesize mycolic acids immediately. After 60 min, the cells had completely lost this ability. The synthesis of the three mycolate components—α-mycolate, methoxymycolate, and β-mycolate—was inhibited. The viability of the isoniazid-treated cells was unaffected up to about 60 min of exposure, after which time there was a gradual decline in the viability to about 18% after 180 min. Correspondingly, growth of the drug-treated cells slowed down and stopped after 24 hr. The inhibition of the synthesis of mycolic acids was reversible if the drug was removed before the loss of viability set in. Incubation of the viable cells in the absence of the drug for 24 hr restored the mycolate synthesis. These results strongly suggest that the inhibition of the synthesis of the mycolic acids is closely associated with the primary mechanism of action of isoniazid on the tubercle bacilli. The sequence of events which leads to the loss of viability of cells exposed to isoniazid is described.

Inhibition by Ethambutol of Mycolic Acid Transfer into the Cell Wall of Mycobacterium smegmatis

Ethambutol simultaneously inhibited the transfer (presumably via mycolyl acetyl trehalose) of mycolic acids into the cell wall and stimulated the synthesis of trehalose dimycolates of Mycobacterium smegmatis. Structural similarities of the drug and mycolyl acetyl trehalose suggested that competitive inhibition was involved.

Adjuvant activity of non-ionic block copolymers. V. Modulation of antibody isotype by lipopolysaccharides, lipid A and precursors

Non-ionic block copolymers and lipopolysaccharides are both effective immunological adjuvants which are thought to act via distinct mechanisms. We hypothesized that they might produce synergistic effects when used together. We prepared a series of lipopolysaccharide (LPS) preparations ranging from the smallest precursor, lipid X through complete LPS with O-polysaccharide chains. Three preparations with reduced toxicity, monophosphoryl lipid A, partially hydrolysed Ra-LPS and LPS of Rhodopseudomonas sphaeroides were also utilized. All LPS preparations except the smallest were effective adjuvants for inducing early antibody responses to trinitrophenyl-conjugated hen egg albumin (TNP-HEA) when injected in squalane-in-water emulsions with copolymer L141. Only the larger LPS preparations induced sustained antibody responses. By itself, emulsions of copolymer L141 induced a predominant IgG1 antibody isotype response with lesser amounts of IgG2a and IgG2b. Surprisingly, all of the LPS preparations tested increased the proportion of IgG2 isotypes even though some had little effect on overall titres. The detoxified Ra-LPS (Ra-detox) was the most effective preparation for both increasing antibody titres and inducing the desirable IgG2a and IgG2b isotypes. These results demonstrate that the combination of LPS and block polymer adjuvants can produce synergistic effects without unacceptable toxicities.

Identification of a novel α(1→6) mannopyranosyltransferase MptB from Corynebacterium glutamicum by deletion of a conserved gene, NCgl1505, affords a lipomannan-and lipoarabinomannan-deficient mutant

Mycobacterium tuberculosis and Corynebacterium glutamicum share a similar cell wall structure and orthologous enzymes involved in cell wall assembly. Herein, we have studied C. glutamicum NCgl1505, the orthologue of putative glycosyltransferases Rv1459c from M. tuberculosis and MSMEG3120 from Mycobacterium smegmatis. Deletion of NCgl1505 resulted in the absence of lipomannan (Cg‐LM‐A), lipoarabinomannan (Cg‐LAM) and a multi‐mannosylated polymer (Cg‐LM‐B) based on a 1,2‐di‐O‐C16/C18:1‐(α‐D‐glucopyranosyluronic acid)‐(1→3)‐glycerol (GlcAGroAc2) anchor, while syntheses of triacylated‐phosphatidyl‐myo‐inositol dimannoside (Ac1PIM2) and Man1GlcAGroAc2 were still abundant in whole cells. Cell‐free incubation of C. glutamicum membranes with GDP‐[14C]Man established that C. glutamicum synthesized a novel α(1→6)‐linked linear form of Cg‐LM‐A and Cg‐LM‐B from Ac1PIM2 and Man1GlcAGroAc2 respectively. Furthermore, deletion of NCgl1505 also led to the absence of in vitro synthesized linear Cg‐LM‐A and Cg‐LM‐B, demonstrating that NCgl1505 was involved in core α(1→6) mannan biosynthesis of Cg‐LM‐A and Cg‐LM‐B, extending Ac1PI[14C]M2 and [14C]Man1GlcAGroAc2 primers respectively. Use of the acceptor α‐D‐Manp‐(1→6)‐α‐D‐Manp‐O‐C8 in an in vitro cell‐free assay confirmed NCgl1505 as an α(1→6) mannopyranosyltransferase, now termed MptB. While Rv1459c and MSMEG3120 demonstrated similar in vitroα(1→6) mannopyranosyltransferase activity, deletion of the Rv1459c homologue in M. smegmatis did not result in loss of mycobacterial LM/LAM, indicating a functional redundancy for this enzyme in mycobacteria.

Nitric oxide synthesis and TNF-α secretion in RAW 264.7 macrophages: Mode of action of a fermented papaya preparation

Macrophage inducible nitric oxide synthase is able to generate massive amounts of nitric oxide (NO) which contributes to the host immune defense against viruses and bacteria. Monocyte-macrophages stimulated with the bacterial wall component lipopolysaccharide (LPS) and cytokines such as interferon-gamma (IFN-gamma) express the inducible form of nitric oxide synthase (iNOS). Furthermore, tumor necrosis factor-alpha (TNF-alpha) is one of the central regulatory cytokines in macrophage antimicrobial activity and synergizes with IFN-gamma in the induction of NO synthesis. Because of its pivotal role in both antimicrobial and tumoricidal activities of macrophages, a significant effort has focused on developing therapeutic agents that regulate NO production. In the present study fermented papaya preparation (FPP) is shown to exert both immunomodulatory and antioxidant activity in the macrophage cell line RAW 264.7. Interestingly, a low and a high molecular weight fraction (LMF and HMF, respectively) of FPP exhibited different activity patterns. FPP fractions alone did not affect NO production. However in the presence of IFN-gamma, both LMF and HMF significantly increased iNOS activity and nitrite as well as nitrate accumulation. NO radical formation measured in real-time by electron paramagnetic resonance spectroscopy was higher in the presence of LMF and IFN-gamma. On the contrary, iNOS mRNA levels were enhanced further with HMF than with LMF. Moreover, LMF displayed a stronger superoxide anion scavenging activity than HMF. In the presence of IFN-gamma, both FPP fractions stimulated TNF-alpha secretion. However in non-stimulated macrophages, TNF-alpha secretion was enhanced by HMF only. Since water-soluble FPP fractions contained no lipid A, present data indicate that FPP is a macrophage activator which augments nitric oxide synthesis and TNF-alpha secretion independently of lipopolysaccharides.

Isoniazid Inhibition of the Synthesis of Monounsaturated Long-Chain Fatty Acids in Mycobacterium tuberculosis H37Ra

Isoniazid inhibited C24 and C26 monounsaturated fatty acid synthesis in Mycobacterium tuberculosis H37Ra. Time courses of this inhibition and that of mycolic acid synthesis were similar.

Scanning electron microscopy of the H37Ra strain of Mycobacterium tuberculosis exposed to isoniazid.

The morphology of cells of the H37Ra strain of Mycobacterium tuberculosis exposed to 0.5 μg of isonicotinic acid hydrazide (isoniazid) per ml was examined by scanning electron microscopy (SEM). Cells that were exposed to isoniazid for 3 h showed no detectable change, whereas cells exposed to the drug for 24 h exhibited diverse morphological features. From our examination of these SEM pictures, we have reconstructed the probable sequence of morphological changes to be as follows: (i) the wrinkling of the cell surface was ascribed as the earliest observable change, (ii) the cell surface then became very rough and ragged, (iii) eventually the cytoplasmic material was extruded from the cell, (iv) this event produced a collapsed cell, (v) the cells began to fragment, (vi) the fragmented cells then coalesced to form an amorphous mass of cell debris. Images

Application of fast atom bombardment mass spectrometry and nuclear magnetic resonance on the structural analysis of purified lipid A

Abstract A method is described for the determination of the complete structure of lipid A obtained from the lipopolysaccharides of Salmonella strains which can now be applied A samples obtained from other gram-negative bacteria. The lipopolysaccharides were treated under mild acid conditions to yield a crude monophosphoryl lipid A (MLA) mixture which was then fractionated on a silicic column to yield the structural analogs. Each of the purified MLA analogs was methylated with diazomethane and further fractionated by reverse-phase high performance liquid chromatography to yield a higly purified dimethyl MLA. Such a sample was analyzed by chemical means and by modern spectroscopic methods. The molecular size of dimethyl MLA and fatty acid distribution in the reduciong and distal glucosamines were determined bu utilizing positive ion fast atom bombardment mass spectrometry. The location of all of the ester-linked fatty acids and the single phosphate group as well as the anomeric configuration of the two glucosamines were determined by utilizing proton-nuclear magnetic resonance spectroscopy. Chemical degradation studies on MLA and dimethyl MLA using triethylamine also contributed to determining the location of the ester-linked fatty acids.