A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos (original) (raw)
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Cell Wall Deficiency in Mycobacteria: Latency and Persistence
Understanding Tuberculosis - Deciphering the Secret Life of the Bacilli, 2012
Understanding Tuberculosis-Deciphering the Secret Life of the Bacilli 194 The period between 1882 and 1940, after Robert Koch discovered the cause of tuberculosis, was marked by series of papers reporting about the appearance of L-form elements in cultures of mycobacteria, such as filterable forms, branching filaments, syncytial growth, large spheres and "variegated mycelia", all of which characterize mycobacterial growth. Mattman summarized the known data about the ability of M. tuberculosis to convert to cell wall deficient forms and suggested a "L-cycle" for mycobacteria (Mattman et al., 1960; Mattman, 1970, 2001). Fig. 2. Lida Mattman Despite the long history in tuberculosis research, the nature of cell wall deficiency and its association with persistence in life of mycobacteria still remain obscure. Unfortunately, over the last several decades, investigations on these unusual forms of tubercle bacilli have been ignored and neglected. Information about forming of mycobacterial L-forms in vitro (in the laboratory), as well in vivo (within the body) is based mainly on studies concerning their morphological appearance. Two periods in L-form research of mycobacteria should be distinguished: before introduction of chemotherapy against tuberculosis, and after. Observations made in the beginning of 20 th century on mycobacterial pleomorphism and Lform elements provide evidence for existence of L-forms without contact with antimicrobial drugs (Calmette & Valti, 1926; Much, 1931). In the following decades, examinations regarding modification of morphology and L-form transformation by antimicrobials became the starting point of additional information on mycobacterial properties (Dorozhkova &
Journal of Biological Chemistry, 2014
BACKGROUND: Biosynthesis and functions of Mycobacterium marinum lipooligosaccharides (LOS) remain elusive. RESULTS: M. marinum mutants expressing various LOS profiles were generated and used to infect macrophages and amoebae. CONCLUSION: Deep LOS mutants are more efficiently phagocytosed than those lacking only LOS-IV. SIGNIFICANCE: Three novel biosynthetic genes and the effect of the LOS content in modulating uptake by phagocytes are reported. ________________________________________________________________________________ ABSTRACT Mycobacterium marinum is a waterborne pathogen responsible for tuberculosis-like infections in ectotherms and is an occasional opportunistic human pathogen. In the environment, M. marinum also interacts with amoebae, which may serve as a natural reservoir for this microorganism. However, the description of mycobacterial determinants in the early interaction with macrophages or amoebae remains elusive. Lipooligosaccharides (LOS) are cell surface exposed glycolipids capable to modulate the host immune system, suggesting that they may be involved in the early interactions of M. marinum with macrophages. Herein, we addressed whether LOS composition affects the uptake of M. marinum by professional phagocytes. Mutants with various truncated LOS variants were generated, leading to the identification of several previously uncharacterized biosynthetic genes (wbbL2, MMAR_2321 and MMAR_2331). Biochemical and structural approaches allowed resolving the structures of LOS precursors accumulating in this set of mutants. These strains, with structurally defined LOS profiles, were then used to infect both macrophages and Acanthamoebae. An inverse correlation between LOS completeness and uptake of mycobacteria by phagocytes was found, allowing to propose three mutant classes: class I (papA4), devoid of LOS, highly efficiently phagocytosed; class II, accumulating only early LOS intermediates (wbbL2, MMAR_2331), also efficiently phagocytosed but less than class I mutants; class III, lacking LOS-IV (losA, MMAR_2319 and MMAR_2321), phagocytosed similarly to the control strain. These results indicate that phagocytosis is conditioned by the LOS pattern and that the LOS pathway used by M. marinum in macrophages is conserved during infection of amoebae.
Identification of Two Mycobacterium marinum Loci That Affect Interactions with Macrophages
Infection and Immunity, 2004
Mycobacterium marinum is closely related to Mycobacterium tuberculosis, the cause of tuberculosis in humans. M. marinum has become an important model system for the study of the molecular mechanisms involved in causing tuberculosis in humans. Through molecular genetic analysis of the differences between pathogenic and nonpathogenic mycobacteria, we identified two loci that affect the ability of M. marinum to infect macrophages, designated mel 1 and mel 2 . In silico analyses of the 11 putative genes in these loci suggest that mel 1 encodes secreted proteins that include a putative membrane protein and two putative transglutaminases, whereas mel 2 is involved in secondary metabolism or biosynthesis of fatty acids. Interestingly, mel 2 is unique to M. marinum and the M. tuberculosis complex and not present in any other sequenced mycobacterial species. M. marinum mutants with mutations in mel 1 and mel 2 , constructed by allelic exchange, are defective in the ability to infect both murine and fish macrophage cell lines. These data suggest that the genes in mel 1 and mel 2 are important for the ability of M. marinum to infect host cells. on December 2, 2015 by guest http://iai.asm.org/ Downloaded from 6906 EL-ETR ET AL. INFECT. IMMUN. on December 2, 2015 by guest http://iai.asm.org/ Downloaded from FIG. 4. Uptake (A) and adherence (B) of M. marinum (Mm), M. smegmatis (Ms), and M. smegmatis containing individual cosmid clones with THP-1 cells. Data represent the means and standard deviations of assays done in triplicate from a representative experiment. Data are expressed relative to M. smegmatis uptake (A) and adherence (B).
Cellular Microbiology, 2020
Mycobacterium marinum is a model organism for pathogenic Mycobacterium species, including Mycobacterium tuberculosis, the causative agent of tuberculosis. These pathogens enter phagocytes and replicate within the Mycobacterium-containing vacuole (MCV), possibly followed by vacuole exit and growth in the host cell cytosol. Mycobacteria release siderophores called mycobactins to scavenge iron, an essential, yet poorly soluble and available micro-nutrient. To investigate the role of M. marinum mycobactins, we purified by organic solvent extraction and identified by mass spectrometry the lipid-bound mycobactin (MBT) and the water-soluble variant carboxymycobactin (cMBT). Moreover, we generated by specialized phage transduction a defined M. marinum ΔmbtB deletion mutant predicted to be defective for mycobactin production. The M. marinum ΔmbtB mutant strain showed a severe growth defect in broth and phagocytes, which was partially complemented by supplying the mbtB gene on a plasmid. Furthermore, purified Fe-MBT or Fe-cMBT improved the growth of wild-type as well as ΔmbtB mutant bacteria on minimal plates, but only Fe-cMBT promoted the growth of wild-type M. marinum during phagocyte infection. Finally, the intracellular growth of M. marinum ΔmbtB in Acanthamoeba castellanii amoebae was restored by co-infection with wild-type bacteria. Our study identifies and characterizes the M. marinum MBT and cMBT siderophores and reveals the requirement of mycobactins for extraand intracellular growth of the pathogen.
2020
Phthiocerol dimycocerosates (PDIMs) are a class of mycobacterial lipids that promote virulence in Mycobacterium tuberculosis and Mycobacterium marinum. It has recently been shown that PDIMs work in concert with the M. tuberculosis Type VII secretion system ESX-1 to permeabilize the phagosomal membranes of infected macrophages. As the zebrafish-M. marinum model of infection has revealed the critical role of PDIM at the host-pathogen interface, we set to determine if PDIMs contributed to phagosomal permeabilization in M. marinum. Using an ΔmmpL7 mutant defective in PDIM transport, we find the PDIM-ESX-1 interaction to be conserved in an M. marinum macrophage infection model. However, we find PDIM and ESX-1 mutants differ in their degree of defect, with the PDIM mutant retaining more membrane damaging activity. Using an in vitro hemolysis assay— a common surrogate for cytolytic activity, we find that PDIM and ESX-1 differ in their contributions: the ESX-1 mutant loses hemolytic activit...
Comparative pathogenesis of Mycobacterium marinum and Mycobacterium tuberculosis
Cellular Microbiology, 2008
A thorough understanding of Mycobacterium tuberculosis pathogenesis in humans has been elusive in part because of imperfect surrogate laboratory hosts, each with its own idiosyncrasies. Mycobacterium marinum is the closest genetic relative of the M. tuberculosis complex and is a natural pathogen of ectotherms. In this review, we present evidence that the similar genetic programmes of M. marinum and M. tuberculosis and the corresponding host immune responses reveal a conserved skeleton of Mycobacterium host-pathogen interactions. While both species have made niche-specific refinements, an essential framework has persisted. We highlight genetic comparisons of the two organisms and studies of M. marinum in the developing zebrafish. By pairing M. marinum with the simplified immune system of zebrafish embryos, many of the defining mechanisms of mycobacterial pathogenesis can be distilled and investigated in a tractable host/pathogen pair.
Journal of Biological …, 1997
Surface-exposed unusual lipids containing phthiocerol and phenolphthiocerol are found only in the cell wall of slow-growing pathogenic mycobacteria and are thought to play important roles in host-pathogen interaction. The enzymology and molecular genetics of biosynthesis of phthiocerol and phenolphthiocerol are unknown. We postulate the domain organization of a set of multifunctional enzymes and a cluster of genes (pps) that would encode these enzymes for the biosynthesis of phthiocerol and phenolphthiocerol. A cosmid containing the postulated pps gene cluster was identified by screening a genomic library of Mycobacterium bovis BCG with the postulated homologous domains from mycocerosic acid synthase and fatty acid synthase genes as probes. Homologous cosmids were also identified in the genomic libraries of Mycobacterium tuberculosis and Mycobacterium leprae. M. bovis BCG was transformed with a pps disruption construct, made from the BCG cosmid by introducing the hygromycin resistance gene as the positive-selectable marker and the sacB gene as the counter-selectable marker. Gene disruption by homologous recombination with double crossover was confirmed by polymerase chain reaction and Southern hybridization. Chromatographic analysis showed that the phenolphthiocerol derivative, mycoside B, and phthiocerol dimycocerosates were not produced by the gene knockout mutants. This result confirms the identity of the pps genes. With the identification of the pps gene clusters in both M. tuberculosis and M. leprae, it should be possible to test the postulated roles of these unique lipids in tuberculosis and leprosy.
Dissecting the mycobacterial cell envelope and defining the composition of the native mycomembrane
Scientific reports, 2017
The mycobacterial envelope is unique, containing the so-called mycomembrane (MM) composed of very-long chain fatty acids, mycolic acids (MA). Presently, the molecular composition of the MM remains unproven, due to the diversity of methods used for determining its composition. The plasma membranes (PM) and the native MM-containing cell walls (MMCW) of two rapid-growing mycobacterial species, Mycobacterium aurum and M. smegmatis, were isolated from their cell lysates by differential ultracentrifugation. Transmission electron microscopy and biochemical analyses demonstrated that the two membranes were virtually pure. Bottom-up quantitative proteomics study indicated a different distribution of more than 2,100 proteins between the PM and MMCW. Among these, the mannosyltransferase PimB, galactofuranosyltransferase GlfT2, Cytochrome p450 and ABC transporter YjfF, were most abundant in the PM, which also contain lipoglycans, phospholipids, including phosphatidylinositol mannosides, and onl...