Gayathri Nagalingam

M. tuberculosis induces potent activation of IDO-1, but this is not essential for the immunological control of infection.

Indoleamine 2,3-dioxygenesae-1 (IDO-1) catalyses the initial, rate-limiting step in tryptophan metabolism, thereby regulating tryptophan availability and the formation of downstream metabolites, including picolinic and quinolinic acid. We found that Mycobacterium tuberculosis infection induced marked upregulation of IDO-1 expression in both human and murine macrophages in vitro and in the lungs of mice following aerosol challenge with M. tuberculosis. The absence of IDO-1 in dendritic cells enhanced the activation of mycobacteria-specific T cells in vitro. Interestingly, IDO-1-deficiency during M. tuberculosis infection in mice was not associated with altered mycobacteria-specific T cell responses in vivo. The bacterial burden of infected organs, pulmonary inflammatory responses, and survival were also comparable in M. tuberculosis-infected IDO-1 deficient and wild type animals. Tryptophan is metabolised into either picolinic acid or quinolinic acid, but only picolinic acid inhibited the growth of M. tuberculosis in vitro. By contrast macrophages infected with pathogenic mycobacteria, produced quinolinic, rather than picolinic acid, which did not reduce M. tuberculosis growth in vitro. Therefore, although M. tuberculosis induces robust expression of IDO-1 and activation of tryptophan metabolism, IDO-1-deficiency fails to impact on the immune control and the outcome of the infection in the mouse model of tuberculosis.

Microparticles from Mycobacteria-Infected Macrophages Promote Inflammation and Cellular Migration

Mycobacterium tuberculosis infection is characterized by a strong inflammatory response whereby a few infected macrophages within the granuloma induce sustained cellular accumulation. The mechanisms coordinating this response are poorly characterized. We hypothesized that microparticles (MPs), which are submicron, plasma membrane-derived vesicles released by cells under both physiological and pathological conditions, are involved in this process. Aerosol infection of mice with M. tuberculosis increased CD45+ MPs in the blood after 4 wk of infection, and in vitro infection of human and murine macrophages with mycobacteria enhanced MP release. MPs derived from mycobacteria-infected macrophages were proinflammatory, and when injected into uninfected mice they induced significant neutrophil, macrophage, and dendritic cell recruitment to the injection site. When incubated with naive macrophages, these MPs enhanced proinflammatory cytokine and chemokine release, and they aided in the disruption of the integrity of a respiratory epithelial cell monolayer, providing a mechanism for the egress of cells to the site of M. tuberculosis infection in the lung. In addition, MPs colocalized with the endocytic recycling marker Rab11a within macrophages, and this association increased when the MPs were isolated from mycobacteria-infected cells. M. tuberculosis–derived MPs also carried mycobacterial Ag and were able to activate M. tuberculosis–specific CD4+ T cells in vivo and in vitro in a dendritic cell–dependent manner. Collectively, these data identify an unrecognized role for MPs in host response against M. tuberculosis by promoting inflammation, intercellular communication, and cell migration.

Total Synthesis of Teixobactin

The first total synthesis of the cyclic depsipeptide natural product teixobactin is described. Synthesis was achieved by solid-phase peptide synthesis, incorporating the unusual l-allo-enduracididine as a suitably protected synthetic cassette and employing a key on-resin esterification and solution-phase macrolactamization. The synthetic natural product was shown to possess potent antibacterial activity against a range of Gram-positive pathogenic bacteria, including a virulent strain of Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus (MRSA).

New tuberculosis drug leads from naturally occurring compounds.

Tuberculosis (TB) continues to be a significant cause of mortality and morbidity worldwide. An estimated 2 billion individuals are infected with Mycobacterium tuberculosis and annually there are approximately 10 million new cases of clinical TB and 1.5 million deaths. Currently available drugs and vaccines have had no significant impact on TB control. In addition, the emergence of drug resistant TB is considered a public health crisis, with some strains now resistant to all available drugs. Unfortunately, the growing burden of antibiotic resistance is coupled with decreased effort in the development of new antibiotics. Natural sources are attractive starting points in the search for anti-tubercular drugs because they are extremely rich in chemical diversity and have privileged antimicrobial activity. This review will discuss recent advances in the development of TB drug leads from natural products, with a particular focus on anti-mycobacterial compounds in late-stage preclinical and clinical development.

Nontoxic Metal-Cyclam Complexes, a New Class of Compounds with Potency against Drug-Resistant Mycobacterium tuberculosis.

Tuberculosis (TB) accounted for 1.5 million deaths in 2014, and new classes of anti-TB drugs are required. We report a class of functionalized 1,8-disubstituted cyclam derivatives that display low micromolar activity against pathogenic mycobacteria. These compounds inhibit intracellular growth of Mycobacterium tuberculosis, are nontoxic to human cell lines, and are active against multidrug-resistant M. tuberculosis strains, indicating a distinct mode of action. These compounds warrant further appraisal as novel agents to control TB in humans.

Inhalation of Respirable Crystalline Rifapentine Particles Induces Pulmonary Inflammation.

Rifapentine is an anti-tuberculosis (anti-TB) drug with a prolonged half-life, but oral delivery results in low concentrations in the lungs because of its high binding (98%) to plasma proteins. We have shown that inhalation of crystalline rifapentine overcomes the limitations of oral delivery by significantly enhancing and prolonging the drug concentration in the lungs. The delivery of crystalline particles to the lungs may promote inflammation. This in vivo study characterizes the inflammatory response caused by pulmonary deposition of the rifapentine particles. The rifapentine powder was delivered to BALB/c mice by intratracheal insufflation at a dose of 20 mg/kg. The inflammatory response in the lungs and bronchoalveolar lavage (BAL) was examined at 12 h, 24 h, and 7 days post-treatment by flow cytometry and histopathology. At 12 and 24 h post-treatment, there was a significant influx of neutrophils into the lungs, and this returned to normal by day 7. A significant recruitment of macrophages occurred in the BAL at 24 h. Consistent with these findings, histopathological analysis demonstrated pulmonary vascular congestion and significant macrophage recruitment at 12 and 24 h post-treatment. In conclusion, the pulmonary delivery of crystalline rifapentine caused a transient neutrophil-associated inflammatory response in the lungs that resolved over 7 days. This observation may limit pulmonary delivery of rifapentine to once a week at a dose of 20 mg/kg or less. The effectiveness of weekly dosing with inhalable rifapentine will be assessed in murine Mycobacterium tuberculosis infection.

Mycobacterium tuberculosis components expressed during chronic infection of the lung contribute to long-term control of pulmonary tuberculosis in mice

Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide, yet current control strategies, including the existing BCG vaccine, have had little impact on disease control. The tubercle bacillus modifies protein expression to adapt to chronic infection of the host, and this can potentially be exploited to develop novel therapeutics. We identified the gene encoding the first step of the Mycobacterium tuberculosis sulphur assimilation pathway, cysD, as highly induced during chronic infection in the mouse lung, suggesting therapies based on CysD could be used to target infection. Vaccination with the composite vaccine CysVac2, a fusion of CysD and the immunogenic Ag85B of M. tuberculosis, resulted in the generation of multifunctional CD4+ T cells (interferon (IFN)-γ+TNF+IL-2+IL-17+) in the lung both pre- and post-aerosol challenge with M. tuberculosis. CysVac2 conferred significant protection against pulmonary M. tuberculosis challenge and was particularly effective at controlling late-stage infection, a property not shared by BCG. CysVac2 delivered as a booster following BCG vaccination afforded greater protection against M. tuberculosis challenge than BCG alone. The antigenic components of CysVac2 were conserved amongst M. tuberculosis strains, and protective efficacy afforded by CysVac2 was observed across varying murine MHC haplotypes. Strikingly, administration of CysVac2 to mice previously infected with M. tuberculosis reduced bacterial load and immunopathology in the lung compared with BCG-vaccinated mice. These results indicate that CysVac2 warrants further investigation to assess its potential to control pulmonary TB in humans.

Protective efficacy of recombinant BCG over-expressing protective, stage-specific antigens of Mycobacterium tuberculosis

Tuberculosis (TB) remains a major cause of mortality and morbidity worldwide, yet current control strategies, including the existing BCG vaccine, have had little impact on disease control. CysVac2, a fusion protein comprising stage-specific Mycobacterium tuberculosis antigens, provided superior protective efficacy against chronic M. tuberculosis infection in mice, compared to BCG. To determine if the delivery of CysVac2 in the context of BCG could improve BCG-induced immunity and protection, we generated a recombinant strain of BCG overexpressing CysVac2 (rBCG:CysVac2). Expression of CysVac2 in BCG was facilitated by the M. tuberculosis hspX promoter, which is highly induced inside phagocytic cells and induces strong cellular immune responses to antigens expressed under its regulation. Intradermal vaccination with rBCG:CysVac2 resulted in increased monocyte/macrophage recruitment and enhanced antigen-specific CD4+ T cell priming compared to parental BCG, indicating CysVac2 overexpression had a marked effect on rBCG induced-immunity. Further, rBCG:CysVac2 was a more potent inducer of antigen-specific multifunctional CD4+ T cells (CD4+IFN-γ+TNF+IL-2+) than BCG after vaccination of mice. This improved immunogenicity however did not influence protective efficacy, with both BCG and rBCG:CysVac2 affording comparable level of protection aerosol infection with M. tuberculosis. Boosting either BCG or rBCG:CysVac2 with the CysVac2 fusion protein resulted in a similar improvement in protective efficacy. These results demonstrate that the expression of protective antigens in BCG can augment antigen-specific immunity after vaccination but does not alter protection against infection, further highlighting the challenge of developing effective vaccines to control TB.

Total Synthesis of Ecumicin

The first total synthesis of the potent anti-mycobacterial cyclic depsipeptide natural product ecumicin is described. Synthesis was achieved via a solid-phase strategy, incorporating the synthetic non-proteinogenic amino acids N-methyl-4-methoxy-l-tryptophan and threo-β-hydroxy-l-phenylalanine into the growing linear peptide chain. The synthesis employed key on-resin esterification and dimethylation steps as well as a final macrolactamization between the unusual N-methyl-4-methoxy-l-tryptophan unit and a bulky N-methyl-l-valine residue. The synthetic natural product possessed potent antimycobacterial activity against the virulent H37Rv strain of Mycobacterium tuberculosis (MIC90 = 312 nM).

Delta inulin-based adjuvants promote the generation of polyfunctional CD4 + T cell responses and protection against Mycobacterium tuberculosis infection

There is an urgent need for the rational design of safe and effective vaccines to protect against chronic bacterial pathogens such as Mycobacterium tuberculosis. Advax™ is a novel adjuvant based on delta inulin microparticles that enhances immunity with a minimal inflammatory profile and has entered human trials to protect against viral pathogens. In this report we determined if Advax displays broad applicability against important human pathogens by assessing protective immunity against infection with M. tuberculosis. The fusion protein CysVac2, comprising the M. tuberculosis antigens Ag85B (Rv1886c) and CysD (Rv1285) formulated with Advax provided significant protection in the lungs of M. tuberculosis-infected mice. Protection was associated with the generation of CysVac2-specific multifunctional CD4+ T cells (IFN-γ+TNF+IL-2+). Addition to Advax of the TLR9 agonist, CpG oligonucleotide (AdvaxCpG), improved both the immunogenicity and protective efficacy of CysVac2. Immunisation with CysVac2/AdvaxCpG resulted in heightened release of the chemoattractants, CXCL1, CCL3, and TNF, and rapid influx of monocytes and neutrophils to the site of vaccination, with pronounced early priming of CysVac2-specific CD4+ T cells. As delta inulin adjuvants have shown an excellent safety and tolerability profile in humans, CysVac2/AdvaxCpG is a strong candidate for further preclinical evaluation for progression to human trials.