Search

Tuberculosis (TB) kills an estimated 1–2 million people per year and is the leading cause of death in people with HIV (PWH). The CD4+ T helper (Th) populations play significant roles in protective immunity to Mycobacterium tuberculosis (Mtb), and are also important host cells for HIV proliferation and persistence. Emerging evidence in PWH suggests that Th17 and Th22 cells may be preferentially depleted during HIV infection, an outcome that could pose a risk for Mtb containment during co-infection. We employed our humanized mouse model of co-infection to assess pulmonary and peripheral changes in Th17 and Th22 frequency and function due to infection with HIV, Mtb, or both. As determined with multi-plex ELISA, co-infection with HIV suppressed the cytokine response to Mtb including IL-17A, IL-22, IL-6, and IL-1β. Multi-parameter flow cytometric analysis revealed that Mtb and HIV/Mtb infection led to increased Th17+ cells in lung and decreased Th17+ cells in spleen. Following HIV or HIV/Mtb infection, Th22+ cells were significantly decreased in both lung and spleen. Importantly, HIV preferentially infected Th17 cells compared to Th22 and other Th subpopulations. Greater HIV viral load was also observed in the lung, but not spleen, of animals with Mtb/HIV co-infection compared to those with HIV mono-infection. Overall, these results suggest that HIV may compromise Th22 immunity, and exploit Th17+ cells to promote viral pathogenesis, in the setting of Mtb and HIV co-infection. Y.B. Martinez-Martinez is supported by Conacyt-I2T2 in Mexico, Contex, and the James W. McLaughlin Fellowship Fund. Grant support was provided by NIH R01AI147948 award to J. Endsley

Mycobacterium tuberculosis and HIV remain top global public health concern. Complexities associated with Mtb/HIV coinfections, including in those virally suppressed with anti-retroviral therapy (ART), present significant challenges to efforts to reduce both infections. The pattern of cytokines produced by T helper (Th) lymphocytes plays a central role in susceptibility, relapse and reinfection to TB. To understand how HIV and ART status affects recovery of Th cell effector function, we conducted a prospective study of HIV+ or HIV- subjects from Nairobi, Kenya. Blood samples were taken at start of TB treatment and 5 months after. Cytokine bead array was used to assess human Th cell-derived cytokines isolated from plasma from Nil, TB1 and Mitogen QuantiFERON tubes, including; IL-4, IL-9, IL-10, IL-17A, IL-17F, IL-22, IFN-γ and TNF-α. Intracellular cytokine staining (ICS) of cryopreserved peripheral blood mononuclear cells was also performed to determine changes in baseline numbers of Th1, Th2, Th17, Th22, IL-10+ Th cells, as well as HIV+ Th cells, across treatment groups. Results show reduced plasma IL-9, IFNg and IL-22 activation by mitogen in TB+HIV+ subjects that is independent of ART status, while IL-17A is moderately reduced in TB+HIV+ART- subjects. Cytokine recall to TB1 antigen was observed to be similar across all treatment groups, with the exception that a moderate reduction of IFNg and IL-17A was observed in the TB-HIV+ group. In subjects compliant for ART, ISC analysis revealed greater numbers of HIV+ cells in TB+HIV+, compared to TB-HIV+, groups. These preliminary results identify defects in effector cytokines produced by Th cells that may persist after ART and contribute to poorer TB treatment outcomes in subjects with HIV. Supported by NIH, NIAID, 1R01AI147948

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is responsible for approximately 10 million new infections and 1.5 million deaths each year. Pattern recognition receptors (PRRs) expressed on the surface of macrophages play an important role in orchestrating the immune response to pathogens. Amongst these, the C-type lectin receptors (CLRs) are an important PRR system that is increasingly understood to mediate innate immunity to mycobacteria. We recently described a new anti-bacterial and anti-inflammatory role for the macrophage galactose-type lectin-1 (MGL-1) in a murine model of experimental TB. MGL is a type II CLR that is known to recognize galactose and N-acetylgalactosamine monosaccharides. We observed that exposure to Mtb activated expression of both murine MGL orthologues, MGL-1 and MGL-2. The specific pathogen-associated molecular patterns that activate MGL orthologue transcription, however, have yet to be determined. Here we demonstrate that expression of MGL-1 and MGL-2 are differentially regulated by cytokines, TLR agonists, and mycobacterial ligands. MGL-1 expression is activated by dexamethasone, IL-4, IL-10, and the mycobacterial cell wall glycolipid Trehalose 6,6′-dimycolate. In contrast, MGL-2 expression is activated by TLR2/6 and TLR2/1 agonists and total lipid extracts of mycobacteria. Use of a MyD88 inhibitor further showed that molecular regulation of MGL-1 and MGL-2 transcription, following exposure of macrophages to mycobacteria, is TLR pathway-dependent. These results identify overlapping and unique host immune pathways that differentially activate MGL orthologues following exposure to Mtb and could be targeted through immune modulation.

Mycobacterium tuberculosis is a global pathogen of huge importance which can adapt to several host niche environments in which carbon source availability is likely to vary. We developed and ran a phenotypic screen using butyrate as the sole carbon source to be more reflective of the host lung environment. We screened a library of ∼87,000 small compounds and identified compounds which demonstrated good antitubercular activity against M. tuberculosis grown with butyrate but not with glucose as the carbon source. Among the hits, we identified an oxadiazole series (six compounds) which had specific activity against M. tuberculosis but which lacked cytotoxicity against mammalian cells.

There is an urgent need for new treatments effective against Mycobacterium tuberculosis, the causative agent of tuberculosis. The 8‐hydroxyquinoline series is a privileged scaffold with anticancer, antifungal, and antibacterial activities. We conducted a structure–activity relationship study of the series regarding its antitubercular activity using 26 analogs. The 8‐hydroxyquinolines showed good activity against M. tuberculosis, with minimum inhibitory concentrations (MIC90) of 4 log kill at 10× MIC over 14 days. The majority of the compounds demonstrated cytotoxicity (IC50 of

Arenaviruses are a significant cause of hemorrhagic fever, an often-fatal disease for which there is no approved antiviral therapy. Lassa fever in particular generates high morbidity and mortality in West Africa, where the disease is endemic, and a recent outbreak in Nigeria was larger and more geographically diverse than usual. We are developing LHF-535, a small-molecule viral entry inhibitor that targets the arenavirus envelope glycoprotein, as a therapeutic candidate for Lassa fever and other hemorrhagic fevers of arenavirus origin. Using a lentiviral pseudotype infectivity assay, we determined that LHF-535 had sub-nanomolar potency against the viral envelope glycoproteins from all Lassa virus lineages, with the exception of the glycoprotein from the LP strain from lineage I, which was 100-fold less sensitive than that of other strains. This reduced sensitivity was mediated by a unique amino acid substitution, V434I, in the transmembrane domain of the envelope glycoprotein GP2 subunit. This position corresponds to the attenuation determinant of Candid#1, a live-attenuated Junín virus vaccine strain used to prevent Argentine hemorrhagic fever. Using a virus-yield reduction assay, we determined that LHF-535 potently inhibited Junín virus, but not Candid#1, and the Candid#1 attenuation determinant, F427I, regulated this difference in sensitivity. We also demonstrated that a daily oral dose of LHF-535 at 10 mg/kg protected mice from a lethal dose of Tacaribe virus. Serial passage of Tacaribe virus in LHF-535-treated Vero cells yielded viruses that were resistant to LHF-535, and the majority of drug-resistant viruses exhibited attenuated pathogenesis. These findings provide a framework for the clinical development of LHF-535 as a broad-spectrum inhibitor of arenavirus entry and provide an important context for monitoring the emergence of drug-resistant viruses., Author summary Lassa fever is a viral hemorrhagic fever disease that is transmitted to humans primarily through contact with the urine or feces of infected rodents. The disease is endemic in West Africa, and an unusually large outbreak occurred in Nigeria in early 2018. The case fatality rate was 25% among confirmed cases, underscoring the need for an effective antiviral therapy. Here, we evaluated the small-molecule drug LHF-535, which targets the arenavirus envelope glycoprotein, for broad-spectrum activity against Lassa viruses of different lineages and related arenaviruses that cause hemorrhagic fever diseases in South America. We also selected for LHF-535-resistant viruses and characterized their genotype and phenotype. Using a combination of surrogate systems and wild-type viruses, we determined that all tested Lassa virus strains and New World hemorrhagic fever arenaviruses were sensitive to LHF-535. Sensitivity to the drug was modulated by specific amino acid changes in the viral envelope glycoprotein, and the majority of emerging drug-resistant viruses were attenuated for virulence. Similarly, the live-attenuated vaccine strain for Argentine hemorrhagic fever was also resistant to LHF-535. These findings indicate that LHF-535 targets a viral virulence determinant, the mutation of which may result in the emergence of drug-resistant viruses, but with reduced capacity for virulence.

There is an urgent need for the development of shorter, simpler and more tolerable drugs to treat antibiotic tolerant populations ofMycobacterium tuberculosis. We previously identified a series of phenylhydrazones (PHY) active againstM. tuberculosis. We selected six representative compounds for further analysis. All compounds were active against non-replicatingM. tuberculosis, with two compounds demonstrating greater activity under hypoxic conditions than aerobic culture. Compounds had bactericidal activity with MBC/MIC of

The general secretion (Sec) pathway is a conserved essential pathway in bacteria and is the primary route of protein export across the cytoplasmic membrane. During protein export, the signal peptidase LepB catalyzes the cleavage of the signal peptide and subsequent release of mature proteins into the extracellular space. We developed a target-based whole cell assay to screen for potential inhibitors of LepB, the sole signal peptidase in Mycobacterium tuberculosis, using a strain engineered to underexpress LepB (LepB-UE). We screened 72,000 compounds against both the Lep-UE and wild-type (wt) strains. We identified the phenylhydrazone (PHY) series as having higher activity against the LepB-UE strain. We conducted a limited structure–activity relationship determination around a representative PHY compound with differential activity (MICs of 3.0 μM against the LepB-UE strain and 18 μM against the wt); several analogues were less potent against the LepB overexpressing strain. A number of chemical modifications around the hydrazone moiety resulted in improved potency. Inhibition of LepB activity was observed for a number of compounds in a biochemical assay using cell membrane fraction derived from M. tuberculosis. Compounds did not increase cell permeability, dissipate membrane potential, or inhibit an unrelated mycobacterial enzyme, suggesting a specific mode of action related to the LepB secretory mechanism.

We conducted an evaluation of the phenoxyalkylbenzimidazole series based on the exemplar 2-ethyl-1-(3-phenoxypropyl)-1H-benzo[d]imidazole for its antitubercular activity. Four segments of the molecule were examined systematically to define a structure-activity relationship with respect to biological activity. Compounds had submicromolar activity against Mycobacterium tuberculosis; the most potent compound had a minimum inhibitory concentration (MIC) of 52 nM and was not cytotoxic against eukaryotic cells (selectivity index = 523). Compounds were selective for M. tuberculosis over other bacterial species, including the closely related Mycobacterium smegmatis. Compounds had a bacteriostatic effect against aerobically grown, replicating M. tuberculosis, but were bactericidal against nonreplicating bacteria. Representative compounds had moderate to high permeability in MDCK cells, but were rapidly metabolized in rodents and human liver microsomes, suggesting the possibility of rapid in vivo hepatic clearance mediated by oxidative metabolism. These results indicate that the readily synthesized phenoxyalkylbenzimidazoles are a promising class of potent and selective antitubercular agents, if the metabolic liability can be solved.

This is the first report of 5-styryl-oxathiazol-2-ones as inhibitors of the Mycobacterium tuberculosis (Mtb) proteasome. As part of the study, the structure–activity relationship of oxathiazolones as Mtb proteasome inhibitors has been investigated. Furthermore, the prepared compounds displayed a good selectivity profile for Mtb compared to the human proteasome. The 5-styryl-oxathiazol-2-one inhibitors identified showed little activity against replicating Mtb, but were rapidly bactericidal against nonreplicating bacteria. (E)-5-(4-Chlorostyryl)-1,3,4-oxathiazol-2-one) was most effective, reducing the colony-forming units (CFU)/mL below the detection limit in only seven days at all concentrations tested. The results suggest that this new class of Mtb proteasome inhibitors has the potential to be further developed into novel antitubercular agents for synergistic combination therapies with existing drugs.

We demonstrated that the 3-substituted benzothiophene-1,1-dioxide class of compounds are effective inhibitors of Mycobacterium tuberculosis growth under aerobic conditions. We examined substitution at the C-3 position of the benzothiophene-1,1-dioxide series systematically to delineate structure–activity relationships influencing potency and cytotoxicity. Compounds were tested for inhibitory activity against virulent M. tuberculosis and eukaryotic cells. The tetrazole substituent was most potent, with a minimum inhibitory concentration (MIC) of 2.6 µM. However, cytotoxicity was noted with even more potency (Vero cell TC50 = 0.1 µM). Oxadiazoles had good anti-tubercular activity (MICs of 3–8 µM), but imidazoles, thiadiazoles and thiazoles had little activity. Cytotoxicity did not track with anti-tubercular activity, suggesting different targets or mode of action between bacterial and eukaryotic cells. However, we were unable to derive analogs without cytotoxicity; all compounds synthesized were cytotoxic (TC50 of 0.1–5 µM). We conclude that cytotoxicity is a liability in this series precluding it from further development. However, the series has potent anti-tubercular activity and future efforts towards identifying the mode of action could result in the identification of novel drug targets.

Background Fluorescent proteins are used widely as reporter genes in many organisms. We previously codon-optimized mCherry for Mycobacterium tuberculosis and generated expression constructs with high level expression in mycobacteria with multiple uses in vitro and in vivo. However, little is known about the expression of fluorescent proteins in mycobacteria and the translational start codon for mCherry has not been experimentally determined. Results We determined the translational start site for functional (fluorescent) mCherry in mycobacteria. Several potential translational start codons were identified; introduction of downstream stop codons by mutagenesis was used to determine which start codon was utilized in the bacterial cells. Fluorescent protein was expressed from a construct which would allow translation of a protein of 226 amino acids or a protein of 235 amino acids. No fluorescence was seen when a construct which could give rise to a protein of 219 amino acids was used. Similar results were obtained in mycobacteria and in Escherichia coli. Western blotting confirmed that mCherry was expressed from the constructs encoding 235 or 226 amino acids, but not from the plasmid encoding 219 amino acids. N-terminal sequencing and mass determination confirmed that the mature protein was 226 amino acids and commenced with the amino acid sequence AIIKE. Conclusion We conclude that mCherry is expressed in M. tuberculosis as a smaller protein than expected lacking the GFP-derived N-terminal sequence designed to allow efficient fusions.