Mycobacterium marinum MMAR_2380, a predicted transmembrane acyltransferase, is essential for the presence of the mannose cap on lipoarabinomannan

Lipoarabinomannan (LAM) is a major glycolipid in the mycobacterial cell envelope. LAM consists of a mannosylphosphatidylinositol (MPI) anchor, a mannan core and a branched arabinan domain. The termini of the arabinan branches can become substituted with one to three α(1→2)-linked mannosyl residues, the mannose cap, producing ManLAM. ManLAM has been associated with a range of different immunomodulatory properties of Mycobacterium tuberculosis during infection of the host. In some of these effects, the presence of the mannose cap on ManLAM appears to be crucial for its activity. So far, in the biosynthesis of the mannose cap on ManLAM, two enzymes have been reported to be involved: a mannosyltransferase that adds the first mannosyl residue of the mannose caps to the arabinan domain of LAM, and another mannosyltransferase that elongates the mannose cap up to three mannosyl residues. Here, we report that a third gene is involved, MMAR_2380, which is the Mycobacterium marinum orthologue of Rv1565c. MMAR_2380 encodes a predicted transmembrane acyltransferase. In M. marinum ΔMMAR_2380, the LAM arabinan domain is still intact, but the mutant LAM lacks the mannose cap. Additional effects of mutation of MMAR_2380 on LAM were observed: a higher degree of branching of both the arabinan domain and the mannan core, and a decreased incorporation of [1,2-14C]acetate into the acyl chains in mutant LAM as compared with the wild-type form. This latter effect was also observed for related lipoglycans, i.e. lipomannan (LM) and phosphatidylinositol mannosides (PIMs). Furthermore, the mutant strain showed increased aggregation in liquid cultures as compared with the wild-type strain. All phenotypic traits of M. marinum ΔMMAR_2380, the deficiency in the mannose cap on LAM and changes at the cell surface, could be reversed by complementing the mutant strain with MMAR_2380. Strikingly, membrane preparations of the mutant strain still showed enzymic activity for the arabinan mannose-capping mannosyltransferase similar to that of the wild-type strain. Although the exact function of MMAR_2380 remains unknown, we show that the protein is essential for the presence of a mannose cap on LAM

Mycobacterium tuberculosis β-lactamase variant reduces sensitivity to ampicillin/avibactam in a zebrafish-Mycobacterium marinum model of tuberculosis

Abstract The β-lactamase of Mycobacterium tuberculosis, BlaC, hydrolyzes β-lactam antibiotics, hindering the use of these antibiotics for the treatment of tuberculosis. Inhibitors, such as avibactam, can reversibly inhibit the enzyme, allowing for the development of combination therapies using both antibiotic and inhibitor. However, laboratory evolution studies using Escherichia coli resulted in the discovery of single amino acid variants of BlaC that reduce the sensitivity for inhibitors or show higher catalytic efficiency against antibiotics. Here, we tested these BlaC variants under more physiological conditions using the M. marinum infection model of zebrafish, which recapitulates hallmark features of tuberculosis, including the intracellular persistence of mycobacteria in macrophages and the induction of granuloma formation. To this end, the M. tuberculosis blaC gene was integrated into the chromosome of a blaC frameshift mutant of M. marinum. Subsequently, the resulting strains were used to infect zebrafish embryos in order to test the combinatorial effect of ampicillin and avibactam. The results show that embryos infected with an M. marinum strain producing BlaC show lower infection levels after treatment than untreated embryos. Additionally, BlaC K234R showed higher infection levels after treatment than those infected with bacteria producing the wild-type enzyme, demonstrating that the zebrafish host is less sensitive to the combinatorial therapy of β-lactam antibiotic and inhibitor. These findings are of interest for future development of combination therapies to treat tuberculosis

Surface Labeling with Adhesion Protein FimH Improves Binding of Immunotherapeutic Agent Salmonella Ty21a to the Bladder Epithelium

BACKGROUND: Bladder cancer is the ninth most common cancer in men. 70% of these tumors are classified as non-muscle invasive bladder cancer and those patients receive 6 intravesical instillations with Mycobacterium bovis BCG after transurethral resection. However, 30% of patients show recurrences after treatment and experience severe side effects that often lead to therapy discontinuation. Recently, another vaccine strain, Salmonella enterica typhi Ty21a, demonstrated promising antitumor activity in vivo. Here we focus on increasing bacterial retention in the bladder in order to reduce the number of instillations required and improve antitumor activity. OBJECTIVE: To increase the binding of Ty21a to the bladder wall by surface labeling of the bacteria with adhesion protein FimH and to study its effect in a bladder cancer mouse model. METHODS: Binding of Ty21a with surface-labeled FimH to the bladder wall was analyzed in vitro and in vivo. The antitumor effect of a single instillation of Ty21a+FimH in treatment was determined in a survival experiment. RESULTS: FimH-labeled Ty21a showed significant (p < 0.0001) improved binding to mouse and human cell lines in vitro. Furthermore, FimH labeled bacteria showed ∼5x more binding to the bladder than controls in vivo. Enhanced binding to the bladder via FimH labeling induced a modest improvement in median but not in overall mice survival. CONCLUSIONS: FimH labeling of Ty21a significantly improved binding to bladder tumor cells in vitro and the bladder wall in vivo. The improved binding leads to a modest increase in median survival in a single bladder cancer mouse study

Structure and Function of RNase AS, a Polyadenylate-Specific Exoribonuclease Affecting Mycobacterial Virulence In Vivo

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Inorganic phosphate limitation modulates capsular polysaccharide composition in mycobacteria

Mycobacterium tuberculosis is protected by an unusual and highly impermeable cell envelope that is critically important for the successful colonization of the host. The outermost surface of this cell envelope is formed by capsular polysaccharides that play an important role in modulating the initial interactions once the bacillus enters the body. Although the bioenzymatic steps involved in the production of the capsular polysaccharides are emerging, information regarding the ability of the bacterium to modulate the composition of the capsule is still unknown. Here, we study the mechanisms involved in regulation of mycobacterial capsule biosynthesis using a high throughput screen for gene products involved in capsular α-glucan production. Utilizing this approach we identified a group of mutants that all carried mutations in the ATP-binding cassette phosphate transport locus pst. These mutants collectively exhibited a strong overproduction of capsular polysaccharides, including α-glucan and arabinomannan, suggestive of a role for inorganic phosphate (P(i)) metabolism in modulating capsular polysaccharide production. These findings were corroborated by the observation that growth under low P(i) conditions as well as chemical activation of the stringent response induces capsule production in a number of mycobacterial species. This induction is, in part, dependent on σ factor E. Finally, we show that Mycobacterium marinum, a model organism for M. tuberculosis, encounters P(i) stress during infection, which shows the relevance of our findings in vivo

Role of Phosphatidylinositol Mannosides in the Interaction between Mycobacteria and DC-SIGN▿ †

The C-type lectin dendritic cell (DC)-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is the major receptor on DCs for mycobacteria of the Mycobacterium tuberculosis complex. Recently, we have shown that although the mannose caps of the mycobacterial surface glycolipid lipoarabinomannan (ManLAM) are essential for the binding to DC-SIGN, genetic removal of these caps did not diminish the interaction of whole mycobacteria with DC-SIGN and DCs. Here we investigated the role of the structurally related glycolipids phosphatidylinositol mannosides (PIMs) as possible ligands for DC-SIGN. In a binding assay with both synthetic and natural PIMs, DC-SIGN exhibited a high affinity for hexamannosylated PIM6, which contains terminal α(1→2)-linked mannosyl residues identical to the mannose cap on ManLAM, but not for di- and tetramannosylated PIM2 and PIM4, respectively. To determine the role of PIM6 in the binding of whole mycobacteria to DC-SIGN, a mutant strain of M. bovis bacillus Calmette-Guérin deficient in the production of PIM6 (ΔpimE) was created, as well as a double knockout deficient in the production of both PIM6 and the mannose caps on LAM (ΔpimE ΔcapA). Compared to the wild-type strain, both mutant strains bound similarly well to DC-SIGN and DCs. Furthermore, the wild-type and mutant strains induced comparable levels of interleukin-10 and interleukin-12p40 when used to stimulate DCs. Hence, we conclude that, like ManLAM, PIM6 represents a bona fide DC-SIGN ligand but that other, as-yet-unknown, ligands dominate in the interaction between mycobacteria and DCs