Search

Mycobacterium tuberculosis (Mtb) is one of the most successful human pathogens. Several cytokines are known to increase virulence of bacterial pathogens, leading us to investigate whether Interferon-γ (IFN-γ), a central regulator of the immune defense against Mtb, has a direct effect on the bacteria. We found that recombinant and T-cell derived IFN-γ rapidly induced a dose-dependent increase in the oxygen consumption rate (OCR) of Mtb, consistent with increased bacterial respiration. This was not observed in attenuated Bacillus Calmette–Guérin (BCG), and did not occur for other cytokines tested, including TNF-α. IFN-γ binds to the cell surface of intact Mtb, but not BCG. Mass spectrometry identified mycobacterial membrane protein large 10 (MmpL10) as the transmembrane binding partner of IFN-γ, supported by molecular modelling studies. IFN-γ binding and the OCR response was absent in Mtb Δmmpl10 strain and restored by complementation with wildtype mmpl10. RNA-sequencing and RT-PCR of Mtb exposed to IFN-γ revealed a distinct transcriptional profile, including genes involved in virulence. In a 3D granuloma model, IFN-γ promoted Mtb growth, which was lost in the Mtb Δmmpl10 strain and restored by complementation, supporting the involvement of MmpL10 in the response to IFN-γ. Finally, IFN-γ addition resulted in sterilization of Mtb cultures treated with isoniazid, indicating clearance of phenotypically resistant bacteria that persist in the presence of drug alone. Together our data are the first description of a mechanism allowing Mtb to respond to host immune activation that may be important in the immunopathogenesis of TB and have use in novel eradication strategies. Mycobacterium tuberculosis senses IFN-γ via the membrane protein MmpL10, which results in stimulation of bacterial respiration and virulence gene expression, worsens infection and paradoxically prevents the formation of isoniazid tolerant persisters. [ABSTRACT FROM AUTHOR]

In order to sustain a persistent infection, Mycobacterium tuberculosis (Mtb) must adapt to a changing environment that is shaped by the developing immune response. This necessity to adapt is evident in the flexibility of many aspects of Mtb metabolism, including a respiratory chain that consists of two distinct terminal cytochrome oxidase complexes. Under the conditions tested thus far, the bc1/aa3 complex appears to play a dominant role, while the alternative bd oxidase is largely redundant. However, presence of two terminal oxidases in this obligate pathogen implies that respiratory requirements might change during infection. We report that the cytochrome bd oxidase is specifically required for resisting the adaptive immune response. While the bd oxidase was dispensable for growth in resting macrophages and the establishment of infection in mice, this complex was necessary for optimal fitness after the initiation of adaptive immunity. This requirement was dependent on lymphocyte-derived interferon gamma (IFNγ), but did not involve nitrogen and oxygen radicals that are known to inhibit respiration in other contexts. Instead, we found that ΔcydA mutants were hypersusceptible to the low pH encountered in IFNγ-activated macrophages. Unlike wild type Mtb, cytochrome bd-deficient bacteria were unable to sustain a maximal oxygen consumption rate (OCR) at low pH, indicating that the remaining cytochrome bc1/aa3 complex is preferentially inhibited under acidic conditions. Consistent with this model, the potency of the cytochrome bc1/aa3 inhibitor, Q203, is dramatically enhanced at low pH. This work identifies a critical interaction between host immunity and pathogen respiration that influences both the progression of the infection and the efficacy of potential new TB drugs. Author summary: Tuberculosis, caused by Mycobacterium tuberculosis (Mtb) is a serious global health problem that is responsible for over one million deaths annually, more than any other single infectious agent. In the host, Mtb can adapt to a wide variety of immunological and environmental pressures which is integral to its success as a pathogen. Accordingly, the respiratory capacity of Mtb is flexible. The electron transport chain of Mtb has two terminal oxidases, the cytochrome bc1/aa3 super complex and cytochrome bd, that contribute to the proton motive force and subsequent production of energy in the form of ATP. The bc1/aa3 super complex is required for optimal growth during infection but the role of cytochrome bd is unclear. Here we report that the cytochrome bd oxidase is required for resisting the adaptive immune response, in particular, acidification of the phagosome induced by lymphocyte-derived IFNγ. We found that the cytochrome bd oxidase is specifically required under acidic conditions, where the bc1/aa3 complex is preferentially inhibited. Additionally, we show that acidic conditions increased the potency of Q203, a cytochrome bc1/aa3 inhibitor and candidate tuberculosis therapy. This work defines a new link between the host immune response and the respiratory requirements of Mtb that affects the potency of a potential new therapeutic. [ABSTRACT FROM AUTHOR]

The quinolone decoquinate is coadministered with feed for treatment of parasites which cause coccidiosis in poultry. However, from a drug-development perspective, the biological activity is often not adequately exploited due to poor physicochemical properties. Here we convert decoquinate into N-alkyl quinolone amides that, in contrast to decoquinate, are active against the tuberculosis bacterium with MIC90 values ranging from 1.4 to 3.64 µM, and quinoline O-carbamates active against apicomplexan parasites that cause malaria, toxoplasmosis, and neosporosis with IC50 values of 0.32–1.5 nM for the best derivative. Uniquely for the TB-active amides, disruption of cell wall homoeostasis is identified as one target. With IC50 values against fetal lung fibroblast cells of 40 to >100 μM, the derivatives are selective for the pathogens. Structures of the most active derivatives are determined by NMR spectroscopy and X-ray crystallography. Analogues lacking the decyl side chain of decoquinate are inactive. Decoquinate is a drug used in veterinary practice, which displays antimalarial activity in vitro but has poor bioavailability. Here, the authors convert decoquinate into more soluble amide and carbamate derivatives and assess their efficacy against tuberculosis bacteria and apicomplexan parasites. [ABSTRACT FROM AUTHOR]