Revisiting the ß-Lactams for Tuberculosis Therapy with a Compound-Compound Synthetic Lethality Approach.

The suboptimal effectiveness of ß-lactam antibiotics against Mycobacterium tuberculosis has hindered the utility of this compound class for tuberculosis treatment. However, the results of treatment with a second-line regimen containing meropenem plus a ß-lactamase inhibitor were found to be encouraging in a case study of extensively drug-resistant tuberculosis (M. C. Payen, S. De Wit, C. Martin, R. Sergysels, et al., Int J Tuberc Lung Dis 16:558-560, 2012, https://doi.org/10.5588/ijtld.11.0414). We hypothesized that the innate resistance of M. tuberculosis to ß-lactams is mediated in part by noncanonical accessory proteins that are not considered the classic targets of ß-lactams and that small-molecule inhibitors of those accessory targets might sensitize M. tuberculosis to ß-lactams. In this study, we screened an NIH small-molecule library for the ability to sensitize M. tuberculosis to meropenem. We identified six hit compounds, belonging to either the N-arylindole or benzothiophene chemotype. Verification studies confirmed the synthetic lethality phenotype for three of the N-arylindoles and one benzothiophene derivative. The latter was demonstrated to be partially bioavailable via oral administration in mice. Structure-activity relationship studies of both structural classes identified analogs with potent antitubercular activity, alone or in combination with meropenem. Transcriptional profiling revealed that oxidoreductases, MmpL family proteins, and a 27-kDa benzoquinone methyltransferase could be the targets of the N-arylindole potentiator. In conclusion, our compound-compound synthetic lethality screening revealed novel small molecules that were capable of potentiating the action of meropenem, presumably via inhibition of the innate resistance conferred by ß-lactam accessory proteins. ß-Lactam compound-compound synthetic lethality may be an alternative approach for drug-resistant tuberculosis.

Silencing of host cell CYBB gene expression by the nuclear effector AnkA of the intracellular pathogen Anaplasma phagocytophilum.

Coevolution of intracellular bacterial pathogens and their host cells resulted in the appearance of effector molecules that when translocated into the host cell modulate its function, facilitating bacterial survival within the hostile host environment. Some of these effectors interact with host chromatin and other nuclear components. In this report, we show that the AnkA protein of Anaplasma phagocytophilum, which is translocated into the host cell nucleus, interacts with gene regulatory regions of host chromatin and is involved in downregulating expression of CYBB (gp91(phox)) and other key host defense genes. AnkA effector protein rapidly accumulated in nuclei of infected cells coincident with changes in CYBB transcription. AnkA interacted with transcriptional regulatory regions of the CYBB locus at sites where transcriptional regulators bind. AnkA binding to DNA occurred at regions with high AT contents. Mutation of AT stretches at these sites abrogated AnkA binding. Histone H3 acetylation decreased dramatically at the CYBB locus during A. phagocytophilum infection, particularly around AnkA binding sites. Transcription of CYBB and other defense genes was significantly decreased in AnkA-transfected HL-60 cells. These data suggest a mechanism by which intracellular pathogens directly regulate host cell gene expression mediated by nuclear effectors and changes in host chromatin structure.

REMap: Operon map of M. tuberculosis based on RNA sequence data.

A map of the transcriptional organization of genes of an organism is a basic tool that is necessary to understand and facilitate a more accurate genetic manipulation of the organism. Operon maps are largely generated by computational prediction programs that rely on gene conservation and genome architecture and may not be physiologically relevant. With the widespread use of RNA sequencing (RNAseq), the prediction of operons based on actual transcriptome sequencing rather than computational genomics alone is much needed. Here, we report a validated operon map of Mycobacterium tuberculosis, developed using RNAseq data from both the exponential and stationary phases of growth. At least 58.4% of M. tuberculosis genes are organized into 749 operons. Our prediction algorithm, REMap (RNA Expression Mapping of operons), considers the many cases of transcription coverage of intergenic regions, and avoids dependencies on functional annotation and arbitrary assumptions about gene structure. As a result, we demonstrate that REMap is able to more accurately predict operons, especially those that contain long intergenic regions or functionally unrelated genes, than previous operon prediction programs. The REMap algorithm is publicly available as a user-friendly tool that can be readily modified to predict operons in other bacteria.

Efficacy of Adjunctive Tofacitinib Therapy in Mouse Models of Tuberculosis.

The global tuberculosis (TB) epidemic and the spread of multi- and extensively-drug resistant strains of Mycobacterium tuberculosis (M.tb) have been fueled by low adherence to following lengthy treatment protocols, and the rapid spread of HIV (Human Immunodeficiency Virus). Persistence of the infection in immunocompetent individuals follows from the ability of M.tb to subvert host immune responses in favor of survival within macrophages. Alternative host-directed strategies are therefore being currently sought to improve treatment efficacy and duration. In this study, we evaluated tofacitinib, a new oral Janus kinase (JAK) blocker with anti-inflammatory properties, in shortening tuberculosis treatment. BALB/c mice, which are immunocompetent, showed acceleration of M.tb clearance achieving apparent sterilization after 16 weeks of adjunctive tofacitinib therapy at average exposures higher than recommended in humans, while mice receiving standard treatment alone did not achieve clearance until 24 weeks. True sterilization with tofacitinib was not achieved until five months. C3HeB/FeJ mice, which show reduced pro-inflammatory cytokines during M.tb infection, did not show improved clearance with adjunctive tofacitinib therapy, indicating that the nature of granulomatous lesions and host immunity may influence responsiveness to tofacitinib. Our findings suggest that the JAK pathway could be explored further for host-directed therapy in immunocompetent individuals.

Synthetic lethality reveals mechanisms of Mycobacterium tuberculosis resistance to ß-lactams.

UNLABELLED: Most ß-lactam antibiotics are ineffective against Mycobacterium tuberculosis due to the microbe's innate resistance. The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains has prompted interest to repurpose this class of drugs. To identify the genetic determinants of innate ß-lactam resistance, we carried out a synthetic lethality screen on a transposon mutant library for susceptibility to imipenem, a carbapenem ß-lactam antibiotic. Mutations in 74 unique genes demonstrated synthetic lethality. The majority of mutations were in genes associated with cell wall biosynthesis. A second quantitative real-time PCR (qPCR)-based synthetic lethality screen of randomly selected mutants confirmed the role of cell wall biosynthesis in ß-lactam resistance. The global transcriptional response of the bacterium to ß-lactams was investigated, and changes in levels of expression of cell wall biosynthetic genes were identified. Finally, we validated these screens in vivo using the MT1616 transposon mutant, which lacks a functional acyl-transferase gene. Mice infected with the mutant responded to ß-lactam treatment with a 100-fold decrease in bacillary lung burden over 4 weeks, while the numbers of organisms in the lungs of mice infected with wild-type bacilli proliferated. These findings reveal a road map of genes required for ß-lactam resistance and validate synthetic lethality screening as a promising tool for repurposing existing classes of licensed, safe, well-characterized antimicrobials against tuberculosis. IMPORTANCE: The global emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains has threatened public health worldwide, yet the pipeline of new tuberculosis drugs under development remains limited. One strategy to cope with the urgent need for new antituberculosis agents is to repurpose existing, approved antibiotics. The carbapenem class of ß-lactam antibiotics has been proposed as one such class of drugs. Our study identifies molecular determinants of innate resistance to ß-lactam drugs in M. tuberculosis, and we demonstrate that functional loss of one of these genes enables successful treatment of M. tuberculosis with ß-lactams in the mouse model.

STEVOR is a Plasmodium falciparum erythrocyte binding protein that mediates merozoite invasion and rosetting.

Variant surface antigens play an important role in Plasmodium falciparum malaria pathogenesis and in immune evasion by the parasite. Although most work to date has focused on P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1), two other multigene families encoding STEVOR and RIFIN are expressed in invasive merozoites and on the infected erythrocyte surface. However, their role during parasite infection remains to be clarified. Here we report that STEVOR functions as an erythrocyte-binding protein that recognizes Glycophorin C (GPC) on the red blood cell (RBC) surface and that its binding correlates with the level of GPC on the RBC surface. STEVOR expression on the RBC leads to PfEMP1-independent binding of infected RBCs to uninfected RBCs (rosette formation), while antibodies targeting STEVOR in the merozoite can effectively inhibit invasion. Our results suggest a PfEMP1-independent role for STEVOR in enabling infected erythrocytes at the schizont stage to form rosettes and in promoting merozoite invasion.

A screen for non-coding RNA in Mycobacterium tuberculosis reveals a cAMP-responsive RNA that is expressed during infection.

A key to the success of Mycobacterium tuberculosis (Mtb) is the bacteria's ability to survive and thrive in the presence of numerous stresses mounted by the host. Small, non-coding RNAs (sRNAs) have been shown to modulate numerous stress responses in bacteria, yet to date only two studies have screened the Mtb transcriptome to identify sRNA. Their association with oxidative and acid stress has been demonstrated but the cellular function and role of these sRNAs in the pathogenesis of tuberculosis (TB) remain unknown. Here, we have identified an sRNA, ncrMT1302, in a locus involved in cAMP metabolism and demonstrate that expression of ncrMT1302 responds to changes in pH and cAMP concentration. The differential expression of ncrMT1302 observed in wild-type Mtb during growth is abolished in a strain lacking MT1302, an adenylyl cyclase encoding gene. We report that ncrMT1302 is expressed in Mtb residing in the lungs of mice during an active infection.