Autophagy promotes efficient T cell responses to restrict high-dose Mycobacterium tuberculosis infection in mice.

Although autophagy sequesters Mycobacterium tuberculosis (Mtb) in in vitro cultured macrophages, loss of autophagy in macrophages in vivo does not result in susceptibility to a standard low-dose Mtb infection until late during infection, leaving open questions regarding the protective role of autophagy during Mtb infection. Here we report that loss of autophagy in lung macrophages and dendritic cells results in acute susceptibility of mice to high-dose Mtb infection, a model mimicking active tuberculosis. Rather than observing a role for autophagy in controlling Mtb replication in macrophages, we find that autophagy suppresses macrophage responses to Mtb that otherwise result in accumulation of myeloid-derived suppressor cells and subsequent defects in T cell responses. Our finding that the pathogen-plus-susceptibility gene interaction is dependent on dose has important implications both for understanding how Mtb infections in humans lead to a spectrum of outcomes and for the potential use of autophagy modulators in clinical medicine.

Discovery, Synthesis, and Optimization of 1,2,4-Triazolyl Pyridines Targeting Mycobacterium tuberculosis.

The rise in multidrug resistant tuberculosis cases underscores the urgent need to develop new treatment strategies for tuberculosis. Herein, we report the discovery and synthesis of a new series of compounds containing a 3-thio-1,2,4-triazole moiety that show inhibition of Mycobacterium tuberculosis (Mtb) growth and survival. Structure-activity relationship studies led us to identify several potent analogs displaying low micromolar to nanomolar inhibitory activity, specifically against Mtb. The potent analogs demonstrated no cytotoxicity in mammalian cells at over 100 times the effective concentration required in Mtb and were bactericidal against Mtb during infection of macrophages. In the exploratory ADME investigations, we observed suboptimal ADME characteristics, which prompted us to identify potential metabolic liabilities for further optimization. Our preliminary investigations into the mechanism of action suggest that this series is not engaging the promiscuous targets that arise from many phenotypic screens. We selected for resistant mutants with the nanomolar potent nitro-containing compound 20 and identified resistant isolates with mutations in genes required for coenzyme F420 biosynthesis and the nitroreductase Ddn. This suggests that the aromatic nitro-1,2,4-triazolyl pyridines are activated by F420-dependent Ddn activity, similar to the nitro-containing TB drug pretomanid. We were able to circumvent the requirement for F420-dependent Ddn activity using compounds that contained non-nitro groups, identifying a key feature to be modified to avoid this predominant resistance mechanism. These studies provide the foundation for the development of a new class of 1,2,4-triazole compounds for the treatment of tuberculosis.

Type I IFN signaling in the absence of IRGM1 promotes M. tuberculosis replication in immune cells by suppressing T cell responses.

Polymorphisms in the IRGM gene are associated with susceptibility to tuberculosis in humans. A murine ortholog of Irgm, Irgm1, is also essential for controlling Mycobacterium tuberculosis (Mtb) infection in mice. Multiple processes have been associated with IRGM1 activity that could impact the host response to Mtb infection, including roles in autophagy-mediated pathogen clearance and expansion of activated T cells. However, what IRGM1-mediated pathway is necessary to control Mtb infection in vivo and the mechanistic basis for this control remains unknown. We dissected the contribution of IRGM1 to immune control of Mtb pathogenesis in vivo and found that Irgm1 deletion leads to higher levels of IRGM3-dependent type I interferon signaling. The increased type I interferon signaling precludes T cell expansion during Mtb infection. The absence of Mtb-specific T cell expansion in Irgm1-/- mice results in uncontrolled Mtb infection in neutrophils and alveolar macrophages, which directly contributes to susceptibility to infection. Together, our studies reveal that IRGM1 is required to promote T cell-mediated control of Mtb infection in neutrophils, which is essential for the survival of Mtb-infected mice. These studies also uncover new ways type I interferon signaling can impact TH1 immune responses.

Design, Synthesis, and Evaluation of Novel Δ2-Thiazolino 2-Pyridone Derivatives That Potentiate Isoniazid Activity in an Isoniazid-Resistant Mycobacterium tuberculosis Mutant.

Mycobacterium tuberculosis (Mtb) drug resistance poses an alarming threat to global tuberculosis control. We previously reported that C10, a ring-fused thiazolo-2-pyridone, inhibits Mtb respiration, blocks biofilm formation, and restores the activity of the antibiotic isoniazid (INH) in INH-resistant Mtb isolates. This discovery revealed a new strategy to address INH resistance. Expanding upon this strategy, we identified C10 analogues with improved potency and drug-like properties. By exploring three heterocycle spacers (oxadiazole, 1,2,3-triazole, and isoxazole) on the ring-fused thiazolo-2-pyridone scaffold, we identified two novel isoxazoles, 17h and 17j. 17h and 17j inhibited Mtb respiration and biofilm formation more potently with a broader therapeutic window, were better potentiators of INH-mediated inhibition of an INH-resistant Mtb mutant, and more effectively inhibited intracellular Mtb replication than C10. The (-)17j enantiomer showed further enhanced activity compared to its enantiomer and the 17j racemic mixture. Our potent second-generation C10 analogues offer promise for therapeutic development against drug-resistant Mtb.

A promising bioconjugate vaccine against hypervirulent Klebsiella pneumoniae.

Hypervirulent Klebsiella pneumoniae (hvKp) is globally disseminating as a community-acquired pathogen causing life-threatening infections in healthy individuals. The fact that a dose as little as 50 bacteria is lethal to mice illustrates the dramatic increase of virulence associated with hvKp strains compared with classical K. pneumoniae (cKp) strains, which require lethal doses greater than 107 bacteria. Until recently, these virulent strains were mostly antibiotic-susceptible. However, multidrug-resistant (MDR) hvKp strains have been emerging, spawning a new generation of hypervirulent "superbugs." The mechanisms of hypervirulence are not fully defined, but overproduction of capsular polysaccharide significantly impedes host clearance, resulting in increased pathogenicity of hvKp strains. While there are more than 80 serotypes of K. pneumoniae, the K1 and K2 serotypes cause the vast majority of hypervirulent infections. Therefore, a glycoconjugate vaccine targeting these 2 serotypes could significantly reduce hvKp infection. Conventionally, glycoconjugate vaccines are manufactured using intricate chemical methodologies to covalently attach purified polysaccharides to carrier proteins, which is widely considered to be technically challenging. Here we report on the recombinant production and analytical characterization of bioconjugate vaccines, enzymatically produced in glycoengineered Escherichia coli cells, against the 2 predominant hypervirulent K. pneumoniae serotypes, K1 and K2. The K. pneumoniae bioconjugates are immunogenic and efficacious, protecting mice against lethal infection from 2 hvKp strains, NTUH K-2044 and ATCC 43816. This preclinical study constitutes a key step toward preventing further global dissemination of hypervirulent MDR hvKp strains.