Phosphatidylinositol Is an Essential Phospholipid of Mycobacteria

Jackson, Mary; Crick, Dean C.; Brennan, Patrick J.

doi:10.1074/jbc.m004658200

Cited by 207 publications

(216 citation statements)

References 42 publications

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“…After PgsA1 (MSMEG_2933, Rv2612c in M. tuberculosis H37Rv), PimA (MSMEG_2935, Rv2610c in M. tuberculosis H37Rv), and the acyltransferase MSMEG_2934 (Rv2611c in M. tuberculosis H37Rv), PimBЈ (Rv2188c in M. tuberculosis H37Rv) is now the fourth enzyme of the PIM pathway found to be essential in M. smegmatis and/or M. tuberculosis (5,13,27). 5 Although this finding implies that PI, PIM 1 , and PIM 2 are essential phospho(glyco)lipids, it is at present difficult to distinguish which of their roles as metabolic end products or as precursors for more mannosylated molecules (LM, LAM, and biosynthetic intermediates) specifically accounts for their essentiality.…”

Section: Fatty Acyl Chains ([M ϫ H]mentioning

confidence: 99%

New Insights into the Early Steps of Phosphatidylinositol Mannoside Biosynthesis in Mycobacteria

Guerin

Kaur

Somashekar

et al. 2009

Journal of Biological Chemistry

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103

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Phosphatidyl-myo-inositol mannosides (PIMs) are key glycolipids of the mycobacterial cell envelope. They are considered not only essential structural components of the cell but also important molecules implicated in host-pathogen interactions. Although their chemical structures are well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still incomplete. Here we show for the first time that although both mannosyltransferases PimA and PimB (MSMEG_4253) recognize phosphatidyl-myo-inositol (PI) as a lipid acceptor, PimA specifically catalyzes the transfer of a Manp residue to the 2-position of the myo-inositol ring of PI, whereas PimB exclusively transfers to the 6-position. Moreover, whereas PimB can catalyze the transfer of a Manp residue onto the PI-monomannoside (PIM 1 ) product of PimA, PimA is unable in vitro to transfer Manp onto the PIM 1 product of PimB. Further assays using membranes from Mycobacterium smegmatis and purified PimA and PimB indicated that the acylation of the Manp residue transferred by PimA preferentially occurs after the second Manp residue has been added by PimB. Importantly, genetic evidence is provided that pimB is an essential gene of M. smegmatis. Altogether, our results support a model wherein Ac 1 PIM 2 , a major form of PIMs produced by mycobacteria, arises from the consecutive action of PimA, followed by PimB, and finally the acyltransferase MSMEG_2934. The essentiality of these three enzymes emphasizes the interest of novel anti-tuberculosis drugs targeting the initial steps of PIM biosynthesis. PIMs3 are unique mannolipids found in abundant quantities in the inner and outer membranes of the cell envelope of Mycobacterium spp. and a few other actinomycetes. 4 They are based on a phosphatidyl-myo-inositol (PI) lipid anchor carrying one to six Manp residues and up to four acyl chains (for review see Refs. 1, 2). Based on a conserved mannosyl-PI anchor, they are also thought to be the precursors of the two major mycobacterial lipoglycans, lipomannan (LM) and lipoarabinomannan (LAM) (1, 2). PIMs, LM, and LAM are considered not only essential structural components of the mycobacterial cell envelope (3-6), but also important molecules implicated in host-pathogen interactions in the course of tuberculosis and leprosy (1).Although the chemical structure of PIMs is now well established, knowledge of the enzymes and sequential events leading to their biosynthesis is still fragmentary. According to the currently accepted model, the biosynthetic pathway is initiated by the transfer of two Manp residues and a fatty acyl chain to PI in the cytoplasmic leaflet of the plasma membrane. Based on genetic and biochemical evidence, Korduláková et al. (5) identified PimA (MSMEG_2935 in Mycobacterium smegmatis mc 2 155) as the enzyme that catalyzes the first mannosylation step of the pathway transferring a Manp residue most likely to the 2-position of the myo-inositol (myo-Ins) ring of PI. In contrast, the identity of PimBЈ, the enzyme responsible for the tran...

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Section: Fatty Acyl Chains ([M ϫ H]mentioning

confidence: 99%

New Insights into the Early Steps of Phosphatidylinositol Mannoside Biosynthesis in Mycobacteria

Guerin

Kaur

Somashekar

et al. 2009

Journal of Biological Chemistry

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103

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“…This cooccurrence profile is not in contradiction with the existence of many microbial genomes that contain ips and imp genes but do not contain dipA-dipB genes. For example, in Mycobacterium tuberculosis and other actinobacteria IPS plays an essential role in the production of the major inositol-containing thiol and cell wall lipoglycans (23).…”

Section: Prediction Of Novel Dip Pathway Genes By Comparative Genomicsmentioning

confidence: 99%

Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di- myo -inositol-phosphate

Rodionov

Kurnasov

Stec

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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Di-myo-inositol 1,1 -phosphate (DIP) is a major osmoprotecting metabolite in a number of hyperthermophilic species of archaea and bacteria. Although the DIP biosynthesis pathway was previously proposed, genes encoding only two of the four required enzymes, inositol-1-phosphate synthase and inositol monophosphatase, were identified. In this study we used a comparative genomic analysis to predict two additional genes of this pathway (termed dipA and dipB) that remained missing. In Thermotoga maritima both candidate genes (in an originally misannotated locus TM1418) form an operon with the inositol-1-phosphate synthase encoding gene (TM1419). A predicted inositol-monophosphate cytidylyltransferase activity was directly confirmed for the purified product of T. maritima gene dipA cloned and expressed in Escherichia coli. The entire DIP pathway was reconstituted in E. coli by cloning of the TM1418 -TM1419 operon in pBAD expression vector and confirmed to function in the crude lysate. 31 P NMR and MS analysis revealed that DIP synthesis proceeds via a phosphorylated DIP intermediate, P-DIP, which is generated by the dipBencoded enzyme, now termed P-DIP synthase. This previously unknown intermediate is apparently converted to the final product, DIP, by an inositol monophosphatase-like phosphatase. These findings allowed us to revise the previously proposed DIP pathway. The genomic survey confirmed its presence in the species known to use DIP for osmoprotection. Among several newly identified species with a postulated DIP pathway, Aeropyrum pernix was directly proven to produce this osmolyte.comparative genomics ͉ di-myo-inositol-1,3-phosphate biosynthesis ͉ Thermotoga maritima T he most common mechanism of osmoadaptation in microorganisms involves the accumulation of specific organic osmolytes, so-called compatible solutes, amino acids, sugars, and polyols, that can be taken up from the environment or synthesized de novo (1-3). In thermophiles and hyperthermophiles, compatible solutes are generally different from those found in mesophiles (4), and many of them additionally contribute to thermoprotection.Di-myo-inositol 1,1Ј-phosphate (DIP) is one of the major compatible solutes in a number of thermophilic archaea of the genera Pyrococcus, Methanococcus, Thermococcus, and Archaeoglobus and bacteria belonging to the genera Aquifex, Rubrobacter, and Thermotoga, including Thermotoga maritima and Thermotoga neapolitana (4-7). DIP levels are highly increased at supraoptimal growth temperatures in both Methanococcus igneus and Thermotogales, suggesting that this solute also plays a thermoprotective role (6,8).Based on the study in M. igneus (9) and the reported stereochemistry of DIP (10), the pathway of DIP biosynthesis was originally proposed to occur in four steps: (i) synthesis of L-myoinositol-1-phosphate (L-I-1-P) from glucose-6-phosphate by NAD ϩ -dependent L-I-1-P synthase [inositol-1-phosphate synthase (IPS)]; (ii) hydrolysis of some of the L-I-1-P by inositol monophosphatase (IMP); (iii) coupling of the L-I-1-P with ...

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“…Allelic replacement was confirmed by Southern hybridization as described in ref. 32. The blots were hybridized with a 2.5-kb 32 P-labeled DNA probe generated by digestion of the PCR fragment subcloned in the pBS(KS Ϫ ) vector as described above.…”

Section: Construction Of M Smegmatis ⌬Msmeg4250 and Cloning Of Rv2181mentioning

confidence: 99%

“…32. MSMEG4250 and its flanking regions, including two natural PstI sites, were amplified by PCR with the forward 5Ј-AACCTCGGCATCCCGGTG-3Ј and reverse 5Ј-GCAAGACGGGCCTGGCC-3Ј primers.…”

Section: Construction Of M Smegmatis ⌬Msmeg4250 and Cloning Of Rv2181mentioning

confidence: 99%

Biosynthesis of mycobacterial lipoarabinomannan: Role of a branching mannosyltransferase

Kaur

Berg

Dinadayala

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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110

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M ycobacterial diseases, such as tuberculosis and leprosy, remain serious human public health problems. One critical feature that contributes to the particular pathogenicity and physiology of mycobacteria is the unique, highly hydrophobic and impermeable cell wall (1). Beyond the cytoplasmic membrane, the cell wall of Mycobacterium tuberculosis consists of a core of mycolic acids, arabinogalactan and peptidoglycan, providing the template for insertion of products such as the phthiocerol-containing lipids, the trehalose mycolates, phosphatidylinositol mannosides (PIMs), and their more glycosylated derivatives, lipomannan (LM) and lipoarabinomannan (LAM) (2), all of which contribute to the particular physiology and disease induction capacity of Mycobacterium species. LAM, in its various forms, including those with mannose (Man) ''caps'' (ManLAM), has been implicated in many of the key aspects of the pathogenesis of tuberculosis and leprosy, such as induction of phagocytosis, phagosomal alteration and inhibition of fusion with lysosomes, and induction of innate, humoral, and acquired T cell-mediated immunity (3, 4). However, all of these studies (often conflicting) were conducted with the isolated molecule. Mutants devoid of LAM are crucial to fully resolve its role in disease pathogenesis and bacterial physiology.Although structurally well defined (5), the underlying enzymology and genetics of LAM biosynthesis are unknown. It has been believed, although not empirically demonstrated, that both LM and LAM have their origins in the PIMs, because all contain a phosphatidylinositol (PI) moiety (5, 6). The first step in PIM synthesis involves the transfer of a Manp residue to the 2 position of the myo-inositol ring of PI to form PIM 1 , catalyzed by PimA (7,8). Biosynthesis proceeds by means of the sequential addition of Manp residues to PIM 1 or its acylated counterpart (AcPIM 1 ), § catalyzed in part by PimB and PimC, to produce PIM species having from two (PIM 2 ) to three (PIM 3 ) Manp residues (9, 10). The Manp units at position 6 of the inositol of PIM 3 are further elongated with Manp to generate higher PIMs (PIM 4 , PIM 5 , and PIM 6 ) (11, 12). However, the mannosyltransferases (ManTs) involved in these biosynthetic steps have not been identified. PIM 6 is likely a ''dead-end'' product, because it contains 2-linked Manp, a combination not found in the mannan core of LM͞ LAM (13); PIM 4 is the likely precursor for the subsequent extension of the mannan chain, giving rise to ''linear LM'' (12), which is further mannosylated at the C-2 positions, resulting in mature, branched LM. LAM itself retains the PI end and mannan backbone of LM; the Araf (arabinofuranose) residues originate in decaprenyl-P-arabinofuranose (C 50 -P-Araf ), and addition is partially catalyzed by EmbC (14).The only well characterized glycosyltransferases (GTs) implicated in PIM͞LM͞LAM biosynthesis are the three initial ManTs, PimA, PimB, and PimC, and EmbC (8-10, 14). Apparently, the ManTs that use GDP-Man as sugar donors occur on the cytoplas...

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Phosphatidylinositol Is an Essential Phospholipid of Mycobacteria

Cited by 207 publications

References 42 publications

New Insights into the Early Steps of Phosphatidylinositol Mannoside Biosynthesis in Mycobacteria

New Insights into the Early Steps of Phosphatidylinositol Mannoside Biosynthesis in Mycobacteria

Genomic identification and in vitro reconstitution of a complete biosynthetic pathway for the osmolyte di- myo -inositol-phosphate

Biosynthesis of mycobacterial lipoarabinomannan: Role of a branching mannosyltransferase

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