Characterization of Two Novel Inhibitors of the Mycobacterium tuberculosis Cytochrome bc1 Complex.

Drug-resistant tuberculosis is a global health care threat calling for novel effective treatment options. Here, we report on two novel cytochrome bc1 inhibitors (MJ-22 and B6) targeting the Mycobacterium tuberculosis respiratory chain with excellent intracellular activities in human macrophages. Both hit compounds revealed very low mutation frequencies and distinct cross-resistance patterns with other advanced cytochrome bc1 inhibitors.

Low prevalence of DHFR and DHPS mutations in Pneumocystis jirovecii strains obtained from a German cohort.

BACKGROUND: Pneumocystis pneumonia (PCP) is an opportunistic and potentially life-threatening infection of immunocompromised individuals. A combination of trimethoprim-sulfamethoxazole is widely used for prophylaxis and treatment of PCP. Polymorphisms in the drug targets, the dihydropteroate synthase (DHPS) or the dihydrofolate reductase (DHFR) are presumably a reason for treatment failure. METHODS: We retrospectively examined the prevalence of DHPS and DHFR mutations in Pneumocystis jirovecii isolates obtained from HIV-infected and non-HIV-infected PCP patients. DHFR and DHPS genes were amplified using semi-nested PCR followed by sequencing. Obtained data were correlated with clinical findings. RESULTS: Sequencing of the DHPS gene was achieved in 81 out of 128 isolates (63%), the DHFR-gene was successfully sequenced in 96 isolates (75%). The vast majority of DHFR and DHPS sequences were either wild-type or showed synonymous single nucleotide polymorphisms. Only one sample contained a double mutation at DHPS codon 55 and codon 57 which was associated with treatment failure in some studies. No linkage of treatment failure to a DHFR or DHPS genotype was observed. In our cohort, 35 of 95 Patients (37%) were HIV-positive and 60 (63%) were HIV-negative. The overall mortality rate was 24% with a much higher rate among non-HIV patients. CONCLUSION: DHPS and DHFR mutations exist but are infrequent in our cohort. The contribution of gene polymorphisms to treatment failure needs further research. In immunocompromised HIV-negative patients PCP is associated with high mortality rates. Prophylactic treatment is warranted in this patient subset.

The cysteine desulfurase IscS of Mycobacterium tuberculosis is involved in iron-sulfur cluster biogenesis and oxidative stress defence.

The complex multiprotein systems for the assembly of protein-bound iron-sulfur (Fe-S) clusters are well defined in Gram-negative model organisms. However, little is known about Fe-S cluster biogenesis in other bacterial species. The ISC (iron-sulfur cluster) operon of Mycobacterium tuberculosis lacks several genes known to be essential for the function of this system in other organisms. However, the cysteine desulfurase IscSMtb (Rv number Rv3025c; Mtb denotes M. tuberculosis) is conserved in this important pathogen. The present study demonstrates that deleting iscSMtb renders the cells microaerophilic and hypersensitive to oxidative stress. Moreover, the ∆iscSMtb mutant shows impaired Fe-S cluster-dependent enzyme activity, clearly indicating that IscSMtb is associated with Fe-S cluster assembly. An extensive interaction network of IscSMtb with Fe-S proteins was identified, suggesting a novel mechanism of sulfur transfer by direct interaction with apoproteins. Interestingly, the highly homologous IscS of Escherichia coli failed to complement the ∆iscSMtb mutant and showed a less diverse protein-interaction profile. To identify a structural basis for these observations we determined the crystal structure of IscSMtb, which mirrors adaptations made in response to an ISC operon devoid of IscU-like Fe-S cluster scaffold proteins. We conclude that in M. tuberculosis IscS has been redesigned during evolution to compensate for the deletion of large parts of the ISC operon.

Discovery of dual-active ethionamide boosters inhibiting the Mycobacterium tuberculosis ESX-1 secretion system.

Drug-resistant Mycobacterium tuberculosis (Mtb) remains a major public health concern requiring complementary approaches to standard anti-tuberculous regimens. Anti-virulence molecules or compounds that enhance the activity of antimicrobial prodrugs are promising alternatives to conventional antibiotics. Exploiting host cell-based drug discovery, we identified an oxadiazole compound (S3) that blocks the ESX-1 secretion system, a major virulence factor of Mtb. S3-treated mycobacteria showed impaired intracellular growth and a reduced ability to lyse macrophages. RNA sequencing experiments of drug-exposed bacteria revealed strong upregulation of a distinct set of genes including ethA, encoding a monooxygenase activating the anti-tuberculous prodrug ethionamide. Accordingly, we found a strong ethionamide boosting effect in S3-treated Mtb. Extensive structure-activity relationship experiments revealed that anti-virulence and ethionamide-boosting activity can be uncoupled by chemical modification of the primary hit molecule. To conclude, this series of dual-active oxadiazole compounds targets Mtb via two distinct mechanisms of action.

Insights into the function of the WhiB-like protein of mycobacteriophage TM4--a transcriptional inhibitor of WhiB2.

WhiB-like proteins of actinomycetes are known to co-ordinate iron-sulfur (Fe-S) clusters and are believed to have regulatory functions in many essential bacterial processes. The systematic determination of the genome sequences of mycobacteriophages has revealed the presence of several whiB-like genes in these viruses. Here we focussed on the WhiB-like protein of mycobacteriophage TM4, WhiBTM4. We provide evidence that this viral protein is capable of co-ordinating a Fe-S cluster. The UV-visible absorption spectra obtained from freshly purified and reconstituted WhiBTM4 were consistent with the presence of an oxygen sensitive [2Fe-2S] cluster. Expression of WhiBTM4 in the mycobacterial host led to hindered septation resembling a WhiB2 knockout phenotype whereas basal expression of WhiBTM4 led to superinfection exclusion. The quantification of mRNA-levels during phage infection showed that whiBTM4 is a highly transcribed early phage gene and a dominant negative regulator of WhiB2. Strikingly, both apo-WhiB2 of Mycobacterium tuberculosis and apo-WhiBTM4 were capable of binding to the conserved promoter region upstream of the whiB2 gene indicating that WhiB2 regulates its own synthesis which is inhibited in the presence of WhiBTM4. Thus, we provide substantial evidence supporting the hypothesis of viral and bacterial WhiB proteins being important Fe-S containing transcriptional regulators with DNA-binding capability.

The cytotoxic early protein 77 of mycobacteriophage L5 interacts with MSMEG_3532, an L-serine dehydratase of Mycobacterium smegmatis.

Mycobacteriophage L5 is a temperate phage infecting a broad range of mycobacterial species. Upon induction of lytic growth, L5 rapidly switches off host protein synthesis. We have recently identified the mycobacteriophage L5 early protein gp77 as a host shut-off protein that acts growth inhibitory in the mycobacterial host when expressed through the corresponding phage promoter. Here we present data showing that this purified phage protein of unknown function specifically binds to protein MSMEG_3532 when incubated with cell lysates of Mycobacterium smegmatis. This interaction was confirmed by pull-down assays using purified MSMEG_3532 as bait which co-purified with gp77. The amino acid sequence of MSMEG_3532 is nearly identical to that of threonine dehydratases, serine dehydratases and an L-threo-3-hydroxyaspartate dehydratase. An enzymatic assay identified this host protein as a pyridoxal-5'-phosphate-dependent L-serine dehydratase (SdhA) which converts L-serine to pyruvate. This is the first biochemical characterization of a SdhA derived from mycobacteria. Though the addition of purified gp77 to the established in vitro assay had no influence on SdhA activity at a saturating L-serine concentration, the specific interaction of phage protein and dehydratase in vivo may well have a role in altering the amino acid pool or the products of amino acid metabolism in favour of phage maturation.

Comprehensive Host Cell-Based Screening Assays for Identification of Anti-Virulence Drugs Targeting Pseudomonas aeruginosa and Salmonella Typhimurium.

The prevalence of bacterial pathogens being resistant to antibiotic treatment is increasing worldwide, leading to a severe global health challenge. Simultaneously, the development and approval of new antibiotics stagnated in the past decades, leading to an urgent need for novel approaches to avoid the spread of untreatable bacterial infections in the future. We developed a highly comprehensive screening platform based on quantification of pathogen driven host-cell death to detect new anti-virulence drugs targeting Pseudomonas aeruginosa (Pa) and Salmonella enterica serovar Typhimurium (ST), both known for their emerging antibiotic resistance. By screening over 10,000 small molecules we could identify several substances showing promising effects on Pa and ST pathogenicity in our in vitro infection model. Importantly, we could detect compounds potently inhibiting bacteria induced killing of host cells and one novel comipound with impact on the function of the type 3 secretion system (T3SS) of ST. Thus, we provide proof of concept data of rapid and feasible medium- to high-throughput drug screening assays targeting virulence mechanisms of two major Gram-negative pathogens.

Corticosteroids inhibit Mycobacterium tuberculosis-induced necrotic host cell death by abrogating mitochondrial membrane permeability transition.

Corticosteroids are host-directed drugs with proven beneficial effect on survival of tuberculosis (TB) patients, but their precise mechanisms of action in this disease remain largely unknown. Here we show that corticosteroids such as dexamethasone inhibit necrotic cell death of cells infected with Mycobacterium tuberculosis (Mtb) by facilitating mitogen-activated protein kinase phosphatase 1 (MKP-1)-dependent dephosphorylation of p38 MAPK. Characterization of infected mixed lineage kinase domain-like (MLKL) and tumor necrosis factor receptor 1 (TNFR1) knockout cells show that the underlying mechanism is independent from TNFα-signaling and necroptosis. Our results link corticosteroid function and p38 MAPK inhibition to abrogation of necrotic cell death mediated by mitochondrial membrane permeability transition, and open new avenues for research on novel host-directed therapies (HDT).