mycobactin and Tuberculosis

The alarming rise in drug-resistant clinical cases of tuberculosis (TB) has necessitated the rapid development of newer chemotherapeutic agents with novel mechanisms of action. The mycobactin biosynthesis pathway, conserved only among the mycolata family of actinobacteria, a group of intracellularly surviving bacterial pathogens that includes

Topics: Antitubercular Agents; Bacterial Proteins; Biosynthetic Pathways; Drug Discovery; Drug Resistance, Bacterial; Humans; Iron; Mycobacterium tuberculosis; Oxazoles; Tuberculosis

Mycobacteria, like other bacteria, archaea and eukaryotic cells, naturally release extracellular vesicles (EVs) to interact with their environment. EVs produced by pathogenic bacteria are involved in many activities including cell-cell communication, immunomodulation, virulence and cell survival. Although EVs released by thick cell wall microorganisms like mycobacteria were recognized only recently, studies of Mycobacterium tuberculosis EVs already point to their important roles in host pathogen interactions, opening exciting new areas of investigation. This minireview will summarize the current understanding of mycobacterial EV biology and roles in pathogenesis and will discuss their potential therapeutic applications.

Topics: Cell Wall; Dendritic Cells; Extracellular Vesicles; Host-Pathogen Interactions; Humans; Immunomodulation; Iron; Macrolides; Macrophages; Mycobacterium tuberculosis; Oxazoles; T-Lymphocytes; Tuberculosis; Virulence

Most mycobacteria are able to make mycobactin for themselves. But, Mycobacterium paratuberculosis, M. avium atypical like wood-pigeon mycobacteria and some strains of M. avium typical for the primary isolation, lack this capacity and require mycobactin for growth in the laboratory.

Topics: Animals; Columbidae; Mycobacterium; Mycobacterium avium; Nontuberculous Mycobacteria; Oxazoles; Paratuberculosis; Species Specificity; Tuberculosis; Tuberculosis, Avian

In this study, we have designed and synthesized pyrazoline analogues that partially mimic the structure of mycobactin, to address the requirement of novel therapeutics to tackle the emerging global challenge of antimicrobial resistance (AMR). Our investigation resulted in the identification of novel lead compounds

Topics: Animals; Antitubercular Agents; Bacterial Proteins; Calcium Channel Blockers; Ion Transport; Iron Chelating Agents; Male; Membrane Transport Proteins; Mycobacterium tuberculosis; Oxazoles; Rats; Rats, Sprague-Dawley; Tissue Distribution; Tuberculosis; Verapamil

Mycobacterium abscessus is an emerging pathogen that critically depends on iron for growth and pathogenesis. The acquisition of iron in Mycobacterium tuberculosis is governed by siderophores called mycobactins, synthesized by the

Topics: Humans; Iron; Mycobacterium abscessus; Mycobacterium tuberculosis; Siderophores; Tuberculosis

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis. Access to iron in host macrophages depends on iron-chelating siderophores called mycobactins and is strongly correlated with Mtb virulence. Here, the crystal structure of an Mtb enzyme involved in mycobactin biosynthesis, MbtN, in complex with its FAD cofactor is presented at 2.30 Å resolution. The polypeptide fold of MbtN conforms to that of the acyl-CoA dehydrogenase (ACAD) family, consistent with its predicted role of introducing a double bond into the acyl chain of mycobactin. Structural comparisons and the presence of an acyl carrier protein, MbtL, in the same gene locus suggest that MbtN acts on an acyl-(acyl carrier protein) rather than an acyl-CoA. A notable feature of the crystal structure is the tubular density projecting from N(5) of FAD. This was interpreted as a covalently bound polyethylene glycol (PEG) fragment and resides in a hydrophobic pocket where the substrate acyl group is likely to bind. The pocket could accommodate an acyl chain of 14-21 C atoms, consistent with the expected length of the mycobactin acyl chain. Supporting this, steady-state kinetics show that MbtN has ACAD activity, preferring acyl chains of at least 16 C atoms. The acyl-binding pocket adopts a different orientation (relative to the FAD) to other structurally characterized ACADs. This difference may be correlated with the apparent ability of MbtN to catalyse the formation of an unusual cis double bond in the mycobactin acyl chain.

Topics: Amino Acid Sequence; Binding Sites; Crystallography, X-Ray; Enoyl-(Acyl-Carrier Protein) Reductase (NADPH, B-Specific); Flavin-Adenine Dinucleotide; Humans; Models, Molecular; Molecular Sequence Data; Mycobacterium tuberculosis; Oxazoles; Protein Conformation; Sequence Alignment; Tuberculosis

Improved biomarkers are needed for tuberculosis. To develop tests based on products secreted by tubercle bacilli that are strictly associated with viability, we evaluated 3 bacterial-derived, species-specific, small molecules as biomarkers: 2 mycobactin siderophores and tuberculosinyladenosine. Using liquid chromatography-tandem mass spectrometry, we demonstrated the presence of 1 or both mycobactins and/or tuberculosinyladenosine in serum and whole lung tissues from infected mice and sputum, cerebrospinal fluid (CSF), or lymph nodes from infected patients but not uninfected controls. Detection of the target molecules distinguished host infection status in 100% of mice with both serum and lung as the target sample. In human subjects, we evaluated detection of the bacterial small molecules (BSMs) in multiple body compartments in 3 patient cohorts corresponding to different forms of tuberculosis. We detected at least 1 of the 3 molecules in 90%, 71%, and 40% of tuberculosis patients' sputum, CSF, and lymph node samples, respectively. In paucibacillary forms of human tuberculosis, which are difficult to diagnose even with culture, detection of 1 or more BSM was rapid and compared favorably to polymerase chain reaction-based detection. Secreted BSMs, detectable in serum, warrant further investigation as a means for diagnosis and therapeutic monitoring in patients with tuberculosis.

Topics: Adenosine; Animals; Bacterial Typing Techniques; Biomarkers; Chromatography, Liquid; Humans; Lung; Mice; Mycobacterium tuberculosis; Oxazoles; Sputum; Tandem Mass Spectrometry; Tuberculosis

Iron is an essential nutrient for most bacterial pathogens, but is restricted by the host immune system. Mycobacterium tuberculosis (Mtb) utilizes two classes of small molecules, mycobactins and carboxymycobactins, to capture iron from the human host. Here, we show that an Mtb mutant lacking the mmpS4 and mmpS5 genes did not grow under low iron conditions. A cytoplasmic iron reporter indicated that the double mutant experienced iron starvation even under high-iron conditions. Loss of mmpS4 and mmpS5 did not change uptake of carboxymycobactin by Mtb. Thin layer chromatography showed that the ΔmmpS4/S5 mutant was strongly impaired in biosynthesis and secretion of siderophores. Pull-down experiments with purified proteins demonstrated that MmpS4 binds to a periplasmic loop of the associated transporter protein MmpL4. This interaction was corroborated by genetic experiments. While MmpS5 interacted only with MmpL5, MmpS4 interacted with both MmpL4 and MmpL5. These results identified MmpS4/MmpL4 and MmpS5/MmpL5 as siderophore export systems in Mtb and revealed that the MmpL proteins transport small molecules other than lipids. MmpS4 and MmpS5 resemble periplasmic adapter proteins of tripartite efflux pumps of Gram-negative bacteria, however, they are not only required for export but also for efficient siderophore synthesis. Membrane association of MbtG suggests a link between siderophore synthesis and transport. The structure of the soluble domain of MmpS4 (residues 52-140) was solved by NMR and indicates that mycobacterial MmpS proteins constitute a novel class of transport accessory proteins. The bacterial burden of the mmpS4/S5 deletion mutant in mouse lungs was lower by 10,000-fold and none of the infected mice died within 180 days compared to wild-type Mtb. This is the strongest attenuation observed so far for Mtb mutants lacking genes involved in iron utilization. In conclusion, this study identified the first components of novel siderophore export systems which are essential for virulence of Mtb.

Topics: Animals; Bacterial Outer Membrane Proteins; Bacterial Proteins; Carrier Proteins; Chromatography, Thin Layer; Female; Host-Pathogen Interactions; Iron; Lung; Mice; Mice, Inbred BALB C; Mycobacterium tuberculosis; Oxazoles; Protein Structure, Tertiary; Receptors, Cell Surface; Sequence Deletion; Siderophores; Spleen; Survival Rate; Tuberculosis; Virulence

Although the antimalarial agent artemisinin itself is not active against tuberculosis, conjugation to a mycobacterial-specific siderophore (microbial iron chelator) analogue induces significant and selective antituberculosis activity, including activity against multi- and extensively drug-resistant strains of Mycobacterium tuberculosis. The conjugate also retains potent antimalarial activity. Physicochemical and whole-cell studies indicated that ferric-to-ferrous reduction of the iron complex of the conjugate initiates the expected bactericidal Fenton-type radical chemistry on the artemisinin component. Thus, this "Trojan horse" approach demonstrates that new pathogen-selective therapeutic agents in which the iron component of the delivery vehicle also participates in triggering the antibiotic activity can be generated. The result is that one appropriate conjugate has potent and selective activity against two of the most deadly diseases in the world.

Topics: Antitubercular Agents; Artemisinins; Drug Design; Iron Chelating Agents; Malaria; Molecular Structure; Mycobacterium tuberculosis; Oxazoles; Tuberculosis

Iron availability affects the course of tuberculosis infection, and the ability to acquire this metal is known to be essential for replication of Mycobacterium tuberculosis in human macrophages. M. tuberculosis overcomes iron deficiency by producing siderophores. The relevance of siderophore synthesis for iron acquisition by M. tuberculosis has been demonstrated, but the molecules involved in iron uptake are currently unknown. We have identified two genes (irtA and irtB) encoding an ABC transporter similar to the YbtPQ system involved in iron transport in Yersinia pestis. Inactivation of the irtAB system decreases the ability of M. tuberculosis to survive iron-deficient conditions. IrtA and -B do not participate in siderophore synthesis or secretion but are required for efficient utilization of iron from Fe-carboxymycobactin, as well as replication of M. tuberculosis in human macrophages and in mouse lungs. We postulate that IrtAB is a transporter of Fe-carboxymycobactin. The irtAB genes are located in a chromosomal region previously shown to contain genes regulated by iron and the major iron regulator IdeR. Taken together, our results and previous observations made by other groups regarding two other genes in this region indicate that this gene cluster is dedicated to siderophore synthesis and transport in M. tuberculosis.

Topics: Animals; ATP-Binding Cassette Transporters; Bacterial Proteins; Iron; Mice; Mice, Inbred C57BL; Mycobacterium tuberculosis; Oxazoles; Tuberculosis; Virulence

The causative agent of Johne's disease is Mycobacterium avium subsp. paratuberculosis. This is a chronic, debilitating gastrointestinal disorder that affects ruminants and is responsible for significant economic loss. The specimen processing method that combines C(18)-carboxypropylbetaine (CB-18) treatment and lytic enzyme decontamination has been shown to improve the diagnosis of mycobacterioses. This processing method was applied to the isolation of M. avium subsp. paratuberculosis from ruminant tissue samples. The BACTEC 12B liquid culture system was used but was supplemented with 1% egg yolk emulsion, 4 microg of mycobactin J, and 0.5% pyruvate (12B/EMP) for use in conjunction with this method. The final concentration of antibiotics used was 10 microg of vancomycin, 30 microg of amphotericin B, and 20 microg of nalidixic acid (VAN) per ml. A 7H10-based solid medium was also used that included mycobactin J, pyruvate, and VAN but excluded the egg yolk emulsion (7H10/MPV). Several M. avium subsp. paratuberculosis isolates were examined during the evaluation of this processing method. It was observed that treatment with lytic enzymes stimulated the growth of M. avium subsp. paratuberculosis; however, the growth of one isolate was markedly inhibited due to the presence of vancomycin. Subsequently, the vancomycin concentration in the VAN formulation was reduced to 2 microg/ml. A blinded panel of 60 previously characterized tissue samples from bovine and bison were then processed and analyzed by smear and culture. Historically, 31 and 37 specimens were classified as positive by histology and culture, respectively. The overall sensitivity and specificity of smear relative to culture following CB-18 processing were 97.6 and 89.5%, respectively. The 12B/EMP/VAN liquid culture system recovered M. avium subsp. paratuberculosis from 39 specimens, whereas 7H10/MPV and Herrold's egg yolk media recovered M. avium subsp. paratuberculosis from 26 and 16 specimens, respectively. The average times to positive were 7.4 +/- 8.3, 29.9 +/- 2.6, and 24 +/- 0 days, respectively. The contamination rates were 4.8, 22.6, and 20.0%, respectively.

Topics: Animals; Bacteriological Techniques; Betaine; Bison; DNA, Bacterial; Ileum; Indicators and Reagents; Kinetics; Muscle, Smooth; Mycobacterium avium; Mycobacterium Infections; Oxazoles; Polymerase Chain Reaction; Ruminants; Specimen Handling; Time Factors; Tuberculosis

The pathogenicity of 40 strains of Mycobacterium avium, M. paratuberculosis, M. intracellulare and M. lepraemurium was investigated in chickens, rabbits, guinea-pigs, mice and calves. Mycobactin dependence and serological type were also determined. There was no evidence that mycobactin dependence was related to pathogenicity. Antigenic similarities were demonstrated between M. avium and M. paratuberculosis, and one isolate had the pathogenic characteristics of both species.

Topics: Animals; Cattle; Chickens; Guinea Pigs; Mice; Mice, Inbred C57BL; Mycobacterium; Mycobacterium avium; Mycobacterium lepraemurium; Oxazoles; Rabbits; Tuberculosis; Tuberculosis, Avian