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.

Beyond rational-biosensor-guided isolation of 100 independently evolved bacterial strain variants and comparative analysis of their genomes.

BACKGROUND: In contrast to modern rational metabolic engineering, classical strain development strongly relies on random mutagenesis and screening for the desired production phenotype. Nowadays, with the availability of biosensor-based FACS screening strategies, these random approaches are coming back into fashion. In this study, we employ this technology in combination with comparative genome analyses to identify novel mutations contributing to product formation in the genome of a Corynebacterium glutamicum L-histidine producer. Since all known genetic targets contributing to L-histidine production have been already rationally engineered in this strain, identification of novel beneficial mutations can be regarded as challenging, as they might not be intuitively linkable to L-histidine biosynthesis. RESULTS: In order to identify 100 improved strain variants that had each arisen independently, we performed > 600 chemical mutagenesis experiments, > 200 biosensor-based FACS screenings, isolated > 50,000 variants with increased fluorescence, and characterized > 4500 variants with regard to biomass formation and L-histidine production. Based on comparative genome analyses of these 100 variants accumulating 10-80% more L-histidine, we discovered several beneficial mutations. Combination of selected genetic modifications allowed for the construction of a strain variant characterized by a doubled L-histidine titer (29 mM) and product yield (0.13 C-mol C-mol-1) in comparison to the starting variant. CONCLUSIONS: This study may serve as a blueprint for the identification of novel beneficial mutations in microbial producers in a more systematic manner. This way, also previously unexplored genes or genes with previously unknown contribution to the respective production phenotype can be identified. We believe that this technology has a great potential to push industrial production strains towards maximum performance.

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.

A Reinvestigation of the Role of the Sorbic Acid Tail on the Antibacterial and Anti-Tuberculosis Properties of Moiramide B.

Due to worldwide increasing resistances, there is a considerable need for antibacterial compounds with modes of action not yet realized in commercial antibiotics. One such promising structure is the acetyl-CoA carboxylase (ACC) inhibitor moiramide B which shows strong antibacterial activity against gram-positive bacteria such as Bacillus subtilis and weaker activities against gram-negative bacteria. However, the narrow structure-activity relationship of the pseudopeptide unit of moiramide B represents a formidable challenge for any optimization strategy. In contrast, the lipophilic fatty acid tail is considered an unspecific vehicle responsible only for the transport of moiramide into the bacterial cell. Here we show that the sorbic acid unit, in fact, is highly relevant for ACC inhibition. A hitherto undescribed sub-pocket at the end of the sorbic acid channel binds strongly aromatic rings and allows the development of moiramide derivatives with altered antibacterial profiles including anti-tubercular activity.

Identification of novel scaffolds targeting Mycobacterium tuberculosis.

Drug resistance in Mycobacterium tuberculosis is relentlessly progressing while only a handful of novel drug candidates are developed. Here we describe a GFP-based high-throughput screening of 386,496 diverse compounds to identify putative tuberculosis drug candidates. In an exploratory analysis of the model organism M. bovis BCG and M. smegmatis and the subsequent screening of the main library, we identified 6354 compounds with anti-mycobacterial activity. These hit compounds were predominantly selective for mycobacteria while dozens had activity in the low µM range. We tested toxicity against the human monocyte/macrophage cell line THP-1 and elaborated activity against M. tuberculosis growing in liquid broth, under unique conditions such as non-replicating persistence or inhibition of M. tuberculosis residing in macrophages. Finally, spontaneous compound-resistant M. tuberculosis mutants were selected and subsequently analyzed by whole genome sequencing. In addition to compounds targeting the well-described proteins Pks13 and MmpL3, we identified two novel scaffolds targeting the bifunctional guanosine pentaphosphate synthetase/ polyribonucleotide nucleotidyltransferase GpsI, or interacting with the aminopeptidase PepB, a probable pro-drug activator. KEY MESSAGES: A newly identified scaffold targets the bifunctional enzyme GpsI. The aminopeptidase PepB is interacting with a second novel scaffold. Phenotypic screenings regularly identify novel compounds targeting Pks13 and MmpL3.

Increased drug permeability of a stiffened mycobacterial outer membrane in cells lacking MFS transporter Rv1410 and lipoprotein LprG.

The major facilitator superfamily transporter Rv1410 and the lipoprotein LprG (Rv1411) are encoded by a conserved two-gene operon and contribute to virulence in Mycobacterium tuberculosis. Rv1410 was originally postulated to function as a drug efflux pump, but recent studies suggested that Rv1410 and LprG work in concert to insert triacylglycerides and lipoarabinomannans into the outer membrane. Here, we conducted microscopic analyses of Mycobacterium smegmatis lacking the operon and observed a cell separation defect, while surface rigidity measured by atomic force microscopy was found to be increased. Whereas Rv1410 expressed in Lactococcus lactis did not confer drug resistance, deletion of the operon in Mycobacterium abscessus and M. smegmatis resulted in increased susceptibility toward vancomycin, novobiocin and rifampicin. A homology model of Rv1410 revealed a periplasmic loop as well as a highly conserved aspartate, which were found to be essential for the operon's function. Interestingly, influx of the fluorescent dyes BCECF-AM and calcein-AM in de-energized M. smegmatis cells was faster in the deletion mutant. Our results unambiguously show that elevated drug susceptibility in the deletion mutant is caused by increased drug influx through a defective mycobacterial cell envelope and not by drug efflux mediated by Rv1410.

Molecular Mechanisms of Intrinsic Streptomycin Resistance in Mycobacterium abscessus.

Streptomycin, the first drug used for the treatment of tuberculosis, shows limited activity against the highly resistant pathogen Mycobacterium abscessus We recently identified two aminoglycoside-acetylating genes [aac(2') and eis2] which, however, do not affect susceptibility to streptomycin. This suggests the existence of a discrete mechanism of streptomycin resistance. M. abscessus BLASTP analysis identified MAB_2385 as a close homologue of the 3″-O-phosphotransferase [APH(3″)] from the opportunistic pathogen Mycobacterium fortuitum as a putative streptomycin resistance determinant. Heterologous expression of MAB_2385 in Mycobacterium smegmatis increased the streptomycin MIC, while the gene deletion mutant M. abscessus ΔMAB_2385 showed increased streptomycin susceptibility. The MICs of other aminoglycosides were not altered in M. abscessus ΔMAB_2385. This demonstrates that MAB_2385 encodes a specific and prime innate streptomycin resistance determinant in M. abscessus We further explored the feasibility of applying rpsL-based streptomycin counterselection to generate gene deletion mutants in M. abscessus Spontaneous streptomycin-resistant mutants of M. abscessus ΔMAB_2385 were selected, and we demonstrated that the wild-type rpsL is dominant over the mutated rpsLK43R in merodiploid strains. In a proof of concept study, we exploited this phenotype for construction of a targeted deletion mutant, thereby establishing an rpsL-based counterselection method in M. abscessus.

Lipoprotein Glycosylation by Protein-O-Mannosyltransferase (MAB_1122c) Contributes to Low Cell Envelope Permeability and Antibiotic Resistance of Mycobacterium abscessus.

Lipoproteins are important components of the mycobacterial cell envelope due to their function in cell wall homeostasis and bacterial virulence. They are post-translationally modified with lipid- and glycosyl-residues in various species and interference with acylation or glycosylation leads to reduced growth and attenuated virulence in Mycobacterium tuberculosis. Lipoproteins are also expressed in the emerging and highly drug resistant pathogen Mycobacterium abscessus which frequently affects the lungs of patients with chronic pulmonary disease or cystic fibrosis. We investigated post-translational modification, acylation and glycosylation, of heterologously expressed (M. tuberculosis LppX and Mpt83) and endogenous (SodC) lipoproteins at the molecular level in M. abscessus and identified MAB_1122c as protein O-mannosyltransferase (Pmt). Both, heterologous and endogenous lipoproteins carried a characteristic lipid anchor with palmitic acid (C16), palmitoleic acid (C16:1), oleic acid (C18), or tuberculostearic acid (C19) modifications. Multiple hexose-moieties were detected in the N-terminal region of the model lipoproteins expressed in M. abscessus. Conservation of lipoprotein glycosylation in M. tuberculosis and M. abscessus was revealed and points toward the existence of an O-glycosylation motif or other regulatory mechanisms regarding this post-translational modification. Deletion of MAB_1122c prevented glycosylation and affected susceptibility to specific antibiotics which are large or target peptidoglycan synthesis and to lysozyme. Cell envelope permeability of M. abscessus Δpmt was increased and mutant bacteria showed reduced survival inside macrophages. The results provide a link between post-translational modification of lipoproteins and the permeability of the mycobacterial cell envelope which stresses the importance of lipoproteins as components of this complex structure.

Elucidation of Mycobacterium abscessus aminoglycoside and capreomycin resistance by targeted deletion of three putative resistance genes.

Objectives: Mycobacterium abscessus is innately resistant to a variety of drugs thereby limiting therapeutic options. Bacterial resistance to aminoglycosides (AGs) is conferred mainly by AG-modifying enzymes, which often have overlapping activities. Several putative AG-modifying enzymes are encoded in the genome of M. abscessus . The aim of this study was to investigate the molecular basis underlying AG resistance in M. abscessus . Methods: M. abscessus deletion mutants deficient in one of three genes potentially involved in AG resistance, aac(2 ' ) , eis1 and eis2 , were generated by targeted gene inactivation, as were combinatorial double and triple deletion mutants. MICs were determined to study susceptibility to a variety of AG drugs and to capreomycin. Results: Deletion of aac(2 ' ) increased susceptibility of M. abscessus to kanamycin B, tobramycin, dibekacin and gentamicin C. Deletion of eis2 increased susceptibility to capreomycin, hygromycin B, amikacin and kanamycin B. Deletion of eis1 did not affect drug susceptibility. Equally low MICs of apramycin, arbekacin, isepamicin and kanamycin A for WT and mutant strains indicate that these drugs are not inactivated by either AAC(2 ' ) or Eis enzymes. Conclusions: M. abscessus expresses two distinct AG resistance determinants, AAC(2 ' ) and Eis2, which confer clinically relevant drug resistance.

Mycobacterium tuberculosis EsxO (Rv2346c) promotes bacillary survival by inducing oxidative stress mediated genomic instability in macrophages.

Mycobacterium tuberculosis (Mtb) survives inside the macrophages by modulating the host immune responses in its favor. The 6-kDa early secretory antigenic target (ESAT-6; esxA) of Mtb is known as a potent virulence and T-cell antigenic determinant. At least 23 such ESAT-6 family proteins are encoded in the genome of Mtb; however, the function of many of them is still unknown. We herein report that ectopic expression of Mtb Rv2346c (esxO), a member of ESAT-6 family proteins, in non-pathogenic Mycobacterium smegmatis strain (MsmRv2346c) aids host cell invasion and intracellular bacillary persistence. Further mechanistic studies revealed that MsmRv2346c infection abated macrophage immunity by inducing host cell death and genomic instability as evident from the appearance of several DNA damage markers. We further report that the induction of genomic instability in infected cells was due to increase in the hosts oxidative stress responses. MsmRv2346c infection was also found to induce autophagy and modulate the immune function of macrophages. In contrast, blockade of Rv2346c induced oxidative stress by treatment with ROS inhibitor N-acetyl-L-cysteine prevented the host cell death, autophagy induction and genomic instability in infected macrophages. Conversely, MtbΔRv2346c mutant did not show any difference in intracellular survival and oxidative stress responses. We envision that Mtb ESAT-6 family protein Rv2346c dampens antibacterial effector functions namely by inducing oxidative stress mediated genomic instability in infected macrophages, while loss of Rv2346c gene function may be compensated by other redundant ESAT-6 family proteins. Thus EsxO plays an important role in mycobacterial pathogenesis in the context of innate immunity.

Deletion of zmp1 improves Mycobacterium bovis BCG-mediated protection in a guinea pig model of tuberculosis.

Having demonstrated previously that deletion of zinc metalloprotease zmp1 in Mycobacterium bovis BCG increased immunogenicity of BCG vaccines, we here investigated the protective efficacy of BCG zmp1 deletion mutants in a guinea pig model of tuberculosis infection. zmp1 deletion mutants of BCG provided enhanced protection by reducing the bacterial load of tubercle bacilli in the lungs of infected guinea pigs. The increased efficacy of BCG due to zmp1 deletion was demonstrated in both BCG Pasteur and BCG Denmark indicating that the improved protection by zmp1 deletion is independent from the BCG sub-strain. In addition, unmarked BCG Δzmp1 mutant strains showed a better safety profile in a CB-17 SCID mouse survival model than the parental BCG strains. Together, these results support the further development of BCG Δzmp1 for use in clinical trials.