Revisiting the expression signature of pks15/1 unveils regulatory patterns controlling phenolphtiocerol and phenolglycolipid production in pathogenic mycobacteria.

One of the most important and exclusive characteristics of mycobacteria is their cell wall. Amongst its constituent components are two related families of glycosylated lipids, diphthioceranates and phthiocerol dimycocerosate (PDIM) and its variant phenolic glycolipids (PGL). PGL have been associated with cell wall impermeability, phagocytosis, defence against nitrosative and oxidative stress and, intriguingly, biofilm formation. In bacteria from the Mycobacterium tuberculosis complex (MTBC), the biosynthetic pathway of the phenolphthiocerol moiety of PGL depends upon the expression of several genes encoding type I polyketide synthases (PKS), namely ppsA-E and pks15/1 which constitute the PDIM + PGL locus, and that are highly conserved in PDIM/PGL-producing strains. Consensus has not been achieved regarding the genetic organization of pks15/1 locus and knowledge is lacking on its transcriptional signature. Here we explore publicly available datasets of transcriptome data (RNA-seq) from more than 100 MTBC experiments in 40 growth conditions to outline the transcriptional structure and signature of pks15/1, using a differential expression approach to infer the regulatory patterns involving these and related genes. We show that pks1 expression is highly correlated with fadD22, Rv2949c, lppX, fadD29 and, also, pks6 and pks12, with the first three putatively integrating into a polycistronic structure. We evidence dynamic transcriptional heterogeneity within the genes involved in phenolphtiocerol and phenolic glycolipid production, most exhibiting up-regulation upon acidic pH and antibiotic exposure and down-regulation under hypoxia, dormancy, and low/high iron concentration. We finally propose a model based on transcriptome data in which σD positively regulates pks1, pks15 and fadD22, while σB and σE factors exert negative regulation at an upper level.

Biosynthesis of cell envelope-associated phenolic glycolipids in Mycobacterium marinum.

Phenolic glycolipids (PGLs) are polyketide synthase-derived glycolipids unique to pathogenic mycobacteria. PGLs are found in several clinically relevant species, including various Mycobacterium tuberculosis strains, Mycobacterium leprae, and several nontuberculous mycobacterial pathogens, such as M. marinum. Multiple lines of investigation implicate PGLs in virulence, thus underscoring the relevance of a deep understanding of PGL biosynthesis. We report mutational and biochemical studies that interrogate the mechanism by which PGL biosynthetic intermediates (p-hydroxyphenylalkanoates) synthesized by the iterative polyketide synthase Pks15/1 are transferred to the noniterative polyketide synthase PpsA for acyl chain extension in M. marinum. Our findings support a model in which the transfer of the intermediates is dependent on a p-hydroxyphenylalkanoyl-AMP ligase (FadD29) acting as an intermediary between the iterative and the noniterative synthase systems. Our results also establish the p-hydroxyphenylalkanoate extension ability of PpsA, the first-acting enzyme of a multisubunit noniterative polyketide synthase system. Notably, this noniterative system is also loaded with fatty acids by a specific fatty acyl-AMP ligase (FadD26) for biosynthesis of phthiocerol dimycocerosates (PDIMs), which are nonglycosylated lipids structurally related to PGLs. To our knowledge, the partially overlapping PGL and PDIM biosynthetic pathways provide the first example of two distinct, pathway-dedicated acyl-AMP ligases loading the same type I polyketide synthase system with two alternate starter units to produce two structurally different families of metabolites. The studies reported here advance our understanding of the biosynthesis of an important group of mycobacterial glycolipids.

Inactivation of tesA reduces cell wall lipid production and increases drug susceptibility in mycobacteria.

Phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs) are structurally related lipids noncovalently bound to the outer cell wall layer of Mycobacterium tuberculosis, Mycobacterium leprae, and several opportunistic mycobacterial human pathogens. PDIMs and PGLs are important effectors of virulence. Elucidation of the biosynthesis of these complex lipids will not only expand our understanding of mycobacterial cell wall biosynthesis, but it may also illuminate potential routes to novel therapeutics against mycobacterial infections. We report the construction of an in-frame deletion mutant of tesA (encoding a type II thioesterase) in the opportunistic human pathogen Mycobacterium marinum and the characterization of this mutant and its corresponding complemented strain control in terms of PDIM and PGL production. The growth and antibiotic susceptibility of these strains were also probed and compared with the parental wild-type strain. We show that deletion of tesA leads to a mutant that produces only traces of PDIMs and PGLs, has a slight growth yield increase and displays a substantial hypersusceptibility to several antibiotics. We also provide a robust model for the three-dimensional structure of M. marinum TesA (TesAmm) and demonstrate that a Ser-to-Ala substitution in the predicted catalytic Ser of TesAmm renders a mutant that recapitulates the phenotype of the tesA deletion mutant. Overall, our studies demonstrate a critical role for tesA in mycobacterial biology, advance our understanding of the biosynthesis of an important group of polyketide synthase-derived mycobacterial lipids, and suggest that drugs aimed at blocking PDIM and/or PGL production might synergize with antibiotic therapy in the control of mycobacterial infections.

Cooperation between a coenzyme A-independent stand-alone initiation module and an iterative type I polyketide synthase during synthesis of mycobacterial phenolic glycolipids.

Several Mycobacterium tuberculosis strains, Mycobacterium leprae, and other mycobacterial pathogens produce a group of small-molecule virulence factors called phenolic glycolipids (PGLs). PGLs play key roles in pathogenicity and host-pathogen interaction. Thus, elucidation of the PGL biosynthetic pathway will not only expand our understanding of natural product biosynthesis, but may also illuminate routes to novel therapeutics to afford alternative lines of defense against mycobacterial infections. In this study, we report an investigation of the enzymatic requirements for the production of long-chain p-hydroxyphenylalkanoate intermediates of PGL biosynthesis. We demonstrate a functional cooperation between a coenzyme A-independent stand-alone didomain initiation module (FadD22) and a 6-domain reducing iterative type I polyketide synthase (Pks15/1) for production of p-hydroxyphenylalkanoate intermediates in in vitro and in vivo FadD22-Pks15/1 reconstituted systems. Our results suggest that Pks15/1 is an iterative type I polyketide synthase with a relaxed control of catalytic cycle iterations, a mechanistic property that explains the origin of a characteristic alkyl chain length variability seen in mycobacterial PGLs. The FadD22-Pks15/1 reconstituted systems lay an initial foundation for future efforts to unveil the mechanism of iterative catalysis control by which the structures of the final products of Pks15/1 are defined, and to scrutinize the functional partnerships of the FadD22-Pks15/1 system with downstream enzymes of the PGL biosynthetic pathway.

Anti-PGL-Tb1 responses as an indicator of the immune restoration syndrome in HIV-TB patients.

A prospective and multi-centre study has allowed us to analyse antibody responses and Mycobacterium tuberculosis clinical isolate genotypes on 24 consecutive HIV-TB co-infected patients treated with Highly Active Antiretroviral Therapy (HAART) who either went on to develop a TB Immune Restoration Syndrome (TB-IRS), or not. Circulating free and immune-complexed antibodies against ManLAM, ESAT-6/CFP10 and PGL-Tb1 in HIV-TB co-infected patients were measured by ELISA at the initiation of anti-TB treatment, at the date of HAART initiation and thereafter. Presence of circulating B cells was also monitored by in vitro antibody production (IVAP) against ESAT-6/CFP10 and PGL-Tb1. Finally, 16 out of 24M. tuberculosis clinical isolates from patients with TB-IRS were genotyped using spoligotyping and MIRUs-VNTR typing. Eleven patients (45.8%) experienced TB-IRS (TB-IRS+). Significantly, lower anti-PGL-Tb1 antibody levels were identified in TB-IRS+ compared to TB-IRS-negative patients prior to TB-IRS development. These very low levels were neither related to CD4 counts nor with complexed antibodies. No difference in antibody levels was observed with the other tested antigens. In addition, no specific strain genotype was associated with TB-IRS. The presence of specific anti-PGL-Tb1 antibodies only in TB-IRS-negative patients represents for the first time an indicator of a potential protective response or a diagnostic biomarker for the detection of non-progression to TB-IRS in HIV-TB co-infected patients starting HAART.

The phenolic glycolipid of Mycobacterium tuberculosis differentially modulates the early host cytokine response but does not in itself confer hypervirulence.

Mycobacterium tuberculosis possesses a diversity of potential virulence factors including complex branched lipids such as the phenolic glycolipid PGL-tb. PGL-tb expression by the clinical M. tuberculosis isolate HN878 has been associated with a less efficient Th1 response and increased virulence in mice and rabbits. It has been suggested that the W-Beijing family is the only group of M. tuberculosis strains with an intact pks1-15 gene, required for the synthesis of PGL-tb and capable of producing PGL-tb. We have found that some strains with an intact pks1-15 do not produce PGL-tb while others may produce a variant of PGL-tb. We examined the early host cytokine response to infection with these strains in vitro to better understand the effect of PGL-tb synthesis on immune responses. In addition, we generated a PGL-tb-producing H37Rv in order to determine the effect of PGL-tb production on the host immune response during infection by a strain normally devoid of PGL-tb synthesis. We observed that PGL-tb production by clinical M. tuberculosis isolates affected cytokine production differently depending on the background of the strain. Importantly, while ectopic PGL-tb production by H37Rv suppressed the induction of several pro- and anti-inflammatory cytokines in vitro in human monocytes, it did not lead to increased virulence in infected mice and rabbits. Collectively, our data indicate that, while PGL-tb may play a role in the immunogenicity and/or virulence of M. tuberculosis, it probably acts in concert with other bacterial factors which seem to be dependent on the background of the strain.

Mycobacterial phenolic glycolipid virulence factor biosynthesis: mechanism and small-molecule inhibition of polyketide chain initiation.

Phenolic glycolipids (PGLs) are polyketide-derived virulence factors produced by Mycobacterium tuberculosis, M. leprae, and other mycobacterial pathogens. We have combined bioinformatic, genetic, biochemical, and chemical biology approaches to illuminate the mechanism of chain initiation required for assembly of the p-hydroxyphenyl-polyketide moiety of PGLs. Our studies have led to the identification of a stand-alone, didomain initiation module, FadD22, comprised of a p-hydroxybenzoic acid adenylation domain and an aroyl carrier protein domain. FadD22 forms an acyl-S-enzyme covalent intermediate in the p-hydroxyphenyl-polyketide chain assembly line. We also used this information to develop a small-molecule inhibitor of PGL biosynthesis. Overall, these studies provide insights into the biosynthesis of an important group of small-molecule mycobacterial virulence factors and support the feasibility of targeting PGL biosynthesis to develop new drugs to treat mycobacterial infections.

Production and characterization of peptide mimotopes of phenolic glycolipid-I of Mycobacterium leprae.

Phenolic glycolipid-I (PGL-I), a Mycobacterium leprae-specific antigen, has been widely used for the serodiagnosis of leprosy and has been implicated in the pathogenesis of leprosy. In an effort to produce an alternate antigen of PGL-I, the mimotope peptides of PGL-I, W(T/R)LGPY(V/M), were obtained using a monoclonal antibody, III603.8, specific to PGL-I by a phage library. The biotin-labeled predominant mimotope peptide of PGLP1, WTLGPYV, bound to III603.8 in a dose-dependent manner in an immunoassay. However, PGLP1 did not bind to anti-PGL-I antibodies in the serum samples from leprosy patients that were reactive to PGL-I. Although the mimotope peptide of WTLGPYV was not effective as an alternate antigen of PGL-I for the serodiagnosis of leprosy, but it would be of interest to know how the mimotope peptides mimic the role of PGL-I antigen in the pathogenesis of M. leprae infection.

Immunohistological analysis of in situ expression of mycobacterial antigens in skin lesions of leprosy patients across the histopathological spectrum. Association of Mycobacterial lipoarabinomannan (LAM) and Mycobacterium leprae phenolic glycolipid-I (PGL-I) with leprosy reactions.

The presence of mycobacterial antigens in leprosy skin lesions was studied by immunohistological methods using monoclonal antibodies (MAbs) to Mycobacterium leprae-specific phenolic glycolipid I (PGL-I) and to cross-reactive mycobacterial antigens of 36 kd, 65 kd, and lipoarabinomannan (LAM). The staining patterns with MAb to 36 kd and 65 kd were heterogeneous and were also seen in the lesions of other skin diseases. The in situ staining of PGL-I and LAM was seen only in leprosy. Both antigens were abundantly present in infiltrating macrophages in the lesions of untreated multibacillary (MB) patients, whereas only PGL-I was occasionally seen in scattered macrophages in untreated paucibacillary lesions. During treatment, clearance of PGL-I from granulomas in MB lesions occurred before that of LAM, although the former persisted in scattered macrophages in some treated patients. This persistence of PGL-I in the lesions paralleled high serum anti-PGL-I antibody titers but was not indicative for the presence of viable bacilli in the lesions. Interestingly, we also observed a differential expression pattern of PGL-I and LAM in the lesions of MB patients with reactions during the course of the disease as compared with those without reactions. In conclusion, the in situ expression pattern of PGL-I and LAM in MB patients may assist in early diagnosis of reactions versus relapse.

Competency of human-derived Mycobacterium leprae to use palmitic acid in the synthesis of phenolic glycolipid-I and phthiocerol dimycocerosate and to release CO2 in axenic culture.

Insufficient numbers of viable Mycobacterium leprae have hampered metabolic studies using human-derived M. leprae. In this study, sufficient numbers of M. leprae were obtained from an untreated lepromatous patient to titrate the effects of pH on the metabolism of 14C-palmitic acid by M. leprae. Catabolic metabolism (oxidation of 14C-palmitic acid and release of 14CO2) was maximal when M. leprae were incubated at 33 degrees C and suspended in Middlebrook 7H9, ADC supplemented medium that had been buffered to maintain a pH of 4.8. Anabolic metabolism (synthesis of 14C-phenolic glycolipid-I and its precursor, 14C-phthiocerol dimycocerosate) was maximal when the pH was maintained at 6.8.

Comparison of DNA fragment patterns between the phenolic glycolipid-Tb producers and non-producers of Mycobacterium tuberculosis.

Differences in ability to produce the specific phenolic glycolipid-Tb (PGL-Tb) antigen among Mycobacterium tuberculosis strains have been reported. One of the explanations would be the genotypic variation between the strains. In this study, we compared the DNA fragment patterns after digestion of DNA with various restriction enzymes between the PGL-Tb producing and non-producing strains of M. tuberculosis. Three clinical isolates of M. tuberculosis producing the PGL-Tb antigen detectable by thin-layer chromatography, and M. tuberculosis H37Rv and M. bovis BCG not producing the antigen were grown in Sauton medium. The chromosomal DNA was digested with the restriction endonucleases, Eco RI, Sau3A I, BamH I, Xho I, Sma I, Pst I, Hinc II, and Bst EII. Most of the restriction enzymes used gave no clear DNA bands or no DNA fragment common just to the PGL-Tb producing strains. When DNAs were digested with Bst EII, however, there was a 13 kb DNA fragment common to the PGL-Tb producing isolates of M. tuberculosis and not present in the H37Rv strain and M. bovis BCG. This study thus suggests that there might be differences in DNA fragment patterns between the PLG-Tb producing and non-producing strains of M. tuberculosis.

In vitro effects of antimicrobial agents on Mycobacterium leprae in mouse peritoneal macrophages.

Mycobacterium leprae synthesizes large quantities of a specific phthiocerol-containing phenolic glycolipid in vivo. We have shown earlier that viable M. leprae readily incorporates radiolabeled palmitic acid into phenolic glycolipid I when residing in cultured macrophages in vitro and that this process is inhibited by the antileprosy drug rifampin. In the present paper we report that application of this observation to the rapid evaluation of over 25 antimicrobial agents for potential antileprosy activity in vitro. All the known antileprosy drugs rifampin, dapsone, clofazimine, and ethionamide inhibited phenolic glycolipid I synthesis. Rifabutin, a spiropiperidyl derivative of rifamycin, also reported to be active in the mouse model, was very effective. Interestingly, the macrolides erythromycin, clarithromycin, and roxithromycin were also found to be active in this system, while D-cycloserine and other cell wall synthesis inhibitors showed no effect. Many of the compounds found to be active in this system have been reported to be effective in vivo in mice. This correlation lends support to the feasibility of using phenolic glycolipid I synthesis for the rapid evaluation of new drugs against leprosy.

Biosynthesis of phenolic glycolipids in M. microti.

Mycobacterium microti readily incorporates radioactive propionate into phenolic glycolipids and phthiocerol dimycocerosates. This process is inhibited by 2- and 3-fluoropropionic acids at concentrations which do not affect overall growth. Incorporation is also inhibited by N-(phosphonomethyl) glycine, an inhibitor of the synthesis of aromatic units, but only at high concentrations which also inhibit bacterial growth.

Inhibition of phenolic glycolipid-I synthesis in extracellular Mycobacterium leprae as an indicator of antimicrobial activity.

The effects of 22 antimicrobial agents on the incorporation of [U14C] palmitic acid ([U14C] PA) into the unique phenolic glycolipid-I (PGL-I) antigen of Mycobacterium leprae were studied. Nude-mouse-propagated M. leprae were incubated in a modified Dubos medium in the presence of antimicrobial agents for 4 days. [U14C] PA was then added and incubation was continued for 8 days. The antileprosy agents dapsone, rifampin, and clofazimine (2 micrograms/ml each) caused a significant reduction in [U14C] PA incorporation into PGL-I. Among other agents, the most active were erythromycin, chloramphenicol, and cerulenin. Low concentrations of ethionamide, tetracycline, and minocycline stimulated label incorporation. This system may prove useful in the evaluation of antileprosy agents.

Biophysical optima for metabolism of Mycobacterium leprae.

The metabolic response of freshly harvested, nude-mouse-derived Mycobacterium leprae to biophysical parameters was studied to facilitate an understanding of axenic culture requirements. Quantitation of intracellular ATP and the rate of [U-14C]palmitic acid incorporation into phenolic glycolipid I (PGL-I) were used as metabolic indicators after axenic incubation in modified Dubos medium under various biophysical conditions. PGL-I synthesis was optimal at 33 degrees C, whereas ATP was optimally maintained at less than or equal to 33 degrees C. Both metabolic indices showed sharp reductions at 37 degrees C. After 5 days of incubation, PGL-I synthesis and ATP maintenance showed pH optima of 5.1 to 5.6, with the higher value appearing optimal for ATP maintenance after extended incubation. Metabolic activity was negatively affected by strong reducing agents, and ATP maintenance was optimal when the gaseous environment was maintained at 2.5 to 10% oxygen. The results may partially explain the failure to cultivate the leprosy bacillus in vitro.

Metabolism of Mycobacterium leprae in macrophages.

The incorporation of 14C-labeled palmitic acid ( [U-14C]PA) into the phenolic glycolipid-I (PGL-I) fraction of Mycobacterium leprae was studied in a murine macrophage system in vitro. Peritoneal macrophages from Swiss Webster mice were infected with fresh viable or Formalin-killed M. leprae harvested from infected footpads of nu/nu mice, and [U-14C]PA was added to the culture medium. Labeled glycolipid synthesized by live M. leprae was fractionated on a Florisil-silicic acid column and identified as PGL-I by using thin-layer chromatography and localization on a polysulfone membrane with an anti-PGL-I monoclonal antibody. Increased incorporation of [U-14C]PA into the PGL-I fraction was observed in macrophages infected with only live M. leprae. Treatment of the infected macrophages with rifampin caused a significant reduction in the incorporation of palmitic acid into PGL-I. These preliminary studies suggest that PGL-I synthesis can be used to quantitate the metabolism of M. leprae in macrophages in vitro.