Both Phthiocerol Dimycocerosates and Phenolic Glycolipids Are Required for Virulence of Mycobacterium marinum

Abstract

c Phthiocerol dimycocerosates (PDIMs) and structurally related phenolic glycolipids (PGLs) are complex cell wall lipids unique to pathogenic mycobacteria. While these lipids have been extensively studied in recent years, there are conflicting reports on some aspects of their biosynthesis and on the role of PDIMs and especially PGLs in virulence of Mycobacterium tuberculosis. This has been complicated by the natural deficiency of PGLs in many clinical strains of M. tuberculosis and the frequent loss of PDIMs in laboratory M. tuberculosis strains. In this study, we isolated seven mutants of Mycobacterium marinum deficient in PDIMs and/or PGLs in which multiple genes of the PDIM/PGL biosynthetic locus were disrupted by transposon insertion. Zebrafish infection experiments showed that M. marinum strains lacking one or both of these lipids were avirulent, suggesting that both PDIMs and PGLs are required for virulence. We also found that these strains were hypersensitive to antibiotics and exhibited increased cell wall permeability. Our studies provide new insights into the biosynthesis of PDIMs/PGLs and may help us to understand the role of PDIMs and PGLs in M. tuberculosis virulence. P athogenic mycobacteria produce two structurally related, methyl-branched fatty acid-containing lipids called phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs). PDIMs and PGLs have long-chain fatty acid backbones consisting of 3-methoxy (or 3-keto, 3-hydroxy), 4-methyl, 9,11-dihydroxy glycols (phthiocerols) and p-glycosylated phenylglycols (glycosyl phenolphthiocerols), respectively, that are diesterified with di-, tri-, and tetramethyl-branched acyl chains (mycocerosates) (reviewed in reference 28). PDIMs have been identified in Mycobacterium tuberculosis, M. africanum, M. bovis, M. leprae, M. marinum, M. ulcerans, M. kansasii, M. haemophilum, M. microti, and M. gastri, all of which are pathogenic for humans or animals. PGLs are produced by the same set of pathogenic mycobacterial species, except that in M. tuberculosis only a subset of clinical isolates produces PGLs.The role of PDIMs in virulence was first suggested by two independent studies using signature-tagged transposon mutagenesis, which identified mutants of M. tuberculosis that were unable to either produce or properly localize PDIMs to the cell wall and demonstrated that these mutants were attenuated in animal models of infection (8,12,33). Since then, circumstantial evidence supporting a role for PDIMs in M. tuberculosis virulence has accumulated. The role of PGLs in M. tuberculosis virulence is less clear and is confounded by the fact that laboratory strains (H37Rv, Erdman) and many clinical isolates, including CDC1551 and MT103, are naturally deficient in PGL production due to a 7-basepair deletion in pks15/1, while some clinical isolates of the East Asian lineage have an intact pks15/1 gene and produce PGLs (31). Mutations of pks15/1 in M. tuberculosis HN878, a strain that produces both PDIMs and PGLs and exhibits a hypervirulent phenotype in infect...

A Nonsense Mutation in Mycobacterium marinum That Is Suppressible by a Novel Mechanism

“…PGLs were visualized by spraying the plate with 1% (wt/vol) ␣-naphthol (Acros Organics, Geel, Belgium) prepared in 95% ethanol and 5% sulfuric acid and heating for 5 min (26).…”

“…PDIM and PGLs are abundant and complex cell wall-associated lipids that play roles in cell impermeability and virulence in pathogenic mycobacteria (23)(24)(25)(26)(27). The spontaneous and frequent loss of PDIM has been widely reported in the literature during routine laboratory manipulation of Mycobacterium tuberculosis, the causative agent of human tuberculosis (20)(21)(22).…”

Phenotypic Profiling of Mycobacterium tuberculosis EspA Point Mutants Reveals that Blockage of ESAT-6 and CFP-10 Secretion In Vitro Does Not Always Correlate with Attenuation of Virulence

“…Aliquots (100 l) were added to 96-well plates with black-walled, clearbottomed wells. Nile red or ethidium bromide was added to cells, and fluorescence was measured as described previously (24).…”

Characterization of phthiocerol and phthiodiolone dimycocerosate esters of M. tuberculosis by multiple-stage linear ion-trap MS

“…In pathogenesis, the role of PDIMs of M. tuberculosis was recognized by the studies that identifi ed that mutants of M. tuberculosis were unable to either produce or properly localize PDIMs to the cell envelopeand that demonstrated that PDIM-defi cient strains were attenuated in animal models of infection ( 8,11,(13)(14)(15)(16)(17). Recently, M. tuberculosis and its close pathogenic relative M. marinum were reported to manipulate macrophage recruitment through coordinated use of membrane PDIM and PGLs to initiate infections ( 18 ).…”