The increasing prevalence of antimicrobial resistance is an important challenge that warrants new approaches to antibiotic development. Currently, all antibiotics inhibit biological processes. To explore whether activation of a biochemical pathway can elicit bactericidal effects we engineered variants of Mycobacterium tuberculosis ATP-phosphoribosyltransferase (ATP-PRT) that are resistant to allosteric inhibition by L-histidine, leading to supraphysiological activation of ATP-PRT and L-histidine overproduction. Upregulation of L-histidine biosynthesis significantly reduces the growth of M. tuberculosis in culture and causes a loss of fitness owing to nutrient and energy depletion. Moreover, the expression of allosteric variants in M. tuberculosis significantly reduced infections in human macrophages and in a mouse model of infection. Thus, metabolic activation represents a new mycobactericidal mechanism that could be applied to antimycobacterial drug discovery.

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

Combination therapy has, to some extent, been successful in limiting the emergence of drug-resistant tuberculosis. Drug combinations achieve this advantage by simultaneously acting on different targets and metabolic pathways. Additionally, drug combination therapies are shown to shorten the duration of therapy for tuberculosis. As new drugs are being developed, to overcome the challenge of finding new and effective drug combinations, systems biology commonly uses approaches that analyse mycobacterial cellular processes. These approaches identify the regulatory networks, metabolic pathways, and signaling programs associated with M. tuberculosis infection and survival. Different preclinical models that assess anti-tuberculosis drug activity are available, but the combination of models that is most predictive of clinical treatment efficacy remains unclear. In this structured literature review, we appraise the options to accelerate the TB drug development pipeline through the evaluation of preclinical testing assays of drug combinations.

There is a pressing need for new drugs against tuberculosis (TB) to combat the growing resistance to current antituberculars. Herein a novel strategy is described for hit generation against promising TB targets involving X-ray crystallographic screening in combination with phenotypic screening. This combined approach (XP Screen) affords both a validation of target engagement as well as determination of in cellulo activity. The utility of this method is illustrated by way of an XP Screen against CYP121A1, a cytochrome P450 enzyme from Mycobacterium tuberculosis (Mtb) championed as a validated drug discovery target. A focused screening set was synthesized and tested by such means, with several members of the set showing promising activity against Mtb strain H37Rv. One compound was observed as an X-ray hit against CYP121A1 and showed improved activity against Mtb strain H37Rv under multiple assay conditions (pan-assay activity). Data obtained during X-ray crystallographic screening were utilized in a structure-based campaign to design a limited number of analogues (less than twenty), many of which also showed pan-assay activity against Mtb strain H37Rv. These included the benzo[b][1,4]oxazine derivative (MIC90 6.25 μM), a novel hit compound suitable as a starting point for a more involved hit to lead candidate medicinal chemistry campaign.

Background: The turnaround times for phenotypic tests used to monitor the bacterial load of Mycobacterium tuberculosis, in both clinical and preclinical studies, are delayed by the organism’s slow growth in culture media. The existence of differentially culturable populations of M.tuberculosis may result in an underestimate of the true number. Moreover, culture methods are susceptible to contamination resulting in loss of critical data points. Objectives: We report the adaptation of our robust, culture-free assay utilising 16S ribosomal RNA, developed for sputum, to enumerate the number of bacteria present in animal tissues as a tool to improve the read-outs in preclinical drug efficacy studies. Methods: Initial assay adaptation was performed using naïve mouse lungs spiked with known quantities of M. tuberculosis and an internal RNA control. Tissues were homogenised, total RNA extracted, and enumeration performed using RT-qPCR. We then evaluated the utility of the assay, in comparison to bacterial counts estimated using growth assays on solid and liquid media, to accurately inform bacterial load in tissues from M. tuberculosis-infected mice before and during treatment with a panel of drug combinations. Results: When tested on lung tissues derived from infected mice, the MBL assay produced comparable results to the bacterial counts in solid culture (colony forming units: CFU). Notably, under specific drug treatments, the MBL assay was able to detect a significantly higher number of M. tuberculosis compared to CFU, likely indicating the presence of bacteria that were unable to produce colonies in solid-based culture. Additionally, growth recovery in liquid media using the most probable number (MPN) assay was able to account for the discrepancy between the MBL assay and CFU number, suggesting that the MBL assay detects differentially culturable sub-populations of M. tuberculosis. Conclusions: The MBL assay can enumerate the bacterial load in animal tissues in real time without the need to wait for extended periods for cultures to grow. The readout correlates well with CFUs. Importantly, we have shown that the MBL is able to measure specific populations of bacteria not cultured on solid agar. The adaptation of this assay for preclinical studies has the potential to decrease the readout time of data acquisition from animal experiments and could represent a valuable tool for tuberculosis drug discovery and development.

In this cohort study, we aim to compare outcomes from coronavirus disease 2019 (COVID-19) in people with severe epilepsy and other co-morbidities living in long-term care facilities which all implemented early preventative measures, but different levels of surveillance. During 25-week observation period (16 March-6 September 2020), we included 404 residents (118 children), and 1643 caregivers. We compare strategies for infection prevention, control, and containment, and related outcomes, across four UK long-term care facilities. Strategies included early on-site enhancement of preventative and infection control measures, early identification and isolation of symptomatic cases, contact tracing, mass surveillance of asymptomatic cases and contacts. We measured infection rate among vulnerable people living in the facilities and their caregivers, with asymptomatic and symptomatic cases, including fatality rate. We report 38 individuals (17 residents) who tested severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-positive, with outbreaks amongst residents in two facilities. At Chalfont Centre for Epilepsy (CCE), 10/98 residents tested positive: two symptomatic (one died), eight asymptomatic on weekly enhanced surveillance; 2/275 caregivers tested positive: one symptomatic, one asymptomatic. At St Elizabeth's (STE), 7/146 residents tested positive: four symptomatic (one died), one positive during hospital admission for symptoms unrelated to COVID-19, two asymptomatic on one-off testing of all 146 residents; 106/601 symptomatic caregivers were tested, 13 positive. In addition, during two cycles of systematically testing all asymptomatic carers, four tested positive. At The Meath (TM), 8/80 residents were symptomatic but none tested; 26/250 caregivers were tested, two positive. At Young Epilepsy (YE), 8/80 children were tested, all negative; 22/517 caregivers were tested, one positive. Infection outbreaks in long-term care facilities for vulnerable people with epilepsy can be quickly contained, but only if asymptomatic individuals are identified through enhanced surveillance at resident and caregiver level. We observed a low rate of morbidity and mortality, which confirmed that preventative measures with isolation of suspected and confirmed COVID-19 residents can reduce resident-to-resident and resident-to-caregiver transmission. Children and young adults appear to have lower infection rates. Even in people with epilepsy and multiple co-morbidities, we observed a high percentage of asymptomatic people suggesting that epilepsy-related factors (anti-seizure medications and seizures) do not necessarily lead to poor outcomes. (C) 2020 Elsevier Inc. All rights reserved.

The emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) has reinforced the need for the development of new anti-TB drugs. The first line drug isoniazid inhibits InhA. This is a prodrug requiring activation by the enzyme KatG. Mutations in KatG have largely contributed to clinical isoniazid resistance. We aimed to design new ‘direct’ InhA inhibitors that obviate the need for activation by KatG, circumventing pre-existing resistance. In silico molecular modelling was used as part of a rational structure-based drug-design approach involving inspection of protein crystal structures of InhA:inhibitor complexes, including the broad spectrum antibiotic triclosan (TCS). One crystal structure exhibited the unusual presence of two triclosan molecules within the Mycobacterium tuberculosis InhA binding site. This became the basis of a strategy for the synthesis of novel inhibitors. A series of new, flexible ligands were designed and synthesised, expanding on the triclosan structure. Low Minimum Inhibitory Concentrations (MICs) were obtained for benzylphenyl compounds (12, 43 and 44) and di-triclosan derivative (39), against Mycobacterium bovis BCG although these may also be inhibiting other enzymes. The ether linked di-triclosan derivative (38) displayed excellent in vitro isolated enzyme inhibition results comparable with triclosan, but at a higher MIC (125 µg mL−1). These compounds offer good opportunities as leads for further optimisation.

Mononuclear phagocytes such as monocytes, tissue-specific macrophages, and dendritic cells are primary actors in both innate and adaptive immunity. These professional phagocytes can be parasitized by intracellular bacteria, turning them from housekeepers to hiding places and favoring chronic and/or disseminated infection. One of the most infamous is the bacteria that cause tuberculosis (TB), which is the most pandemic and one of the deadliest diseases, with one-third of the world's population infected and an average of 1.8 million deaths/year worldwide. Here we demonstrate the effective targeting and intracellular delivery of antibiotics to infected macrophages both in vitro and in vivo, using pH-sensitive nanoscopic polymersomes made of PMPC-PDPA block copolymer. Polymersomes showed the ability to significantly enhance the efficacy of the antibiotics killing Mycobacterium bovis, Mycobacterium tuberculosis, and another established intracellular pathogen, Staphylococcus aureus. Moreover, they demonstrated to easily access TB-like granuloma tissues-one of the harshest environments to penetrate-in zebrafish models. We thus successfully exploited this targeting for the effective eradication of several intracellular bacteria, including M. tuberculosis, the etiological agent of human TB.

Background The rise of antimicrobial drug resistance in Mycobacterium tuberculosis coupled with the shortage of new antibiotics has elevated TB to a major global health priority. Repurposing drugs developed or used for other conditions has gained special attention in the current scenario of accelerated drug development for several global infectious diseases. In a similar effort, previous studies revealed that carprofen, a non-steroidal anti-inflammatory drug, selectively inhibited the growth of replicating, non-replicating and MDR clinical isolates of M. tuberculosis. Objectives We aimed to reveal the whole-cell phenotypic and transcriptomic effects of carprofen in mycobacteria. Methods Integrative molecular and microbiological approaches such as resazurin microtitre plate assay, high-throughput spot-culture growth inhibition assay, whole-cell efflux inhibition, biofilm inhibition and microarray analyses were performed. Analogues of carprofen were also synthesized and assessed for their antimycobacterial activity. Results Carprofen was found to be a bactericidal drug that inhibited mycobacterial drug efflux mechanisms. It also restricted mycobacterial biofilm growth. Transcriptome profiling revealed that carprofen likely acts by targeting respiration through the disruption of membrane potential. The pleiotropic nature of carprofen’s anti-TB action may explain why spontaneous drug-resistant mutants could not be isolated in practice. Conclusions This immunomodulatory drug and its chemical analogues have the potential to reverse TB antimicrobial drug resistance, offering a swift path to clinical trials of novel TB drug combinations.

In this comparative biomarker study, we analysed 1768 serial sputum samples from 178 patients at 4 sites in Southeast Africa. We show that tuberculosis Molecular Bacterial Load Assay (TB-MBLA) reduces time-to-TB-bacillary-load-result from days/weeks by culture to hours and detects early patient treatment response. By day 14 of treatment, 5% of patients had cleared bacillary load to zero, rising to 58% by 12th week of treatment. Fall in bacillary load correlated with mycobacterial growth indicator tube culture time-to-positivity (Spearmans r=−0.51, 95% CI (−0.56 to −0.46), p<0.0001). Patients with high pretreatment bacillary burdens (above the cohort bacillary load average of 5.5log10eCFU/ml) were less likely to convert-to-negative by 8th week of treatment than those with a low burden (below cohort bacillary load average), p=0.0005, HR 3.1, 95% CI (1.6 to 5.6) irrespective of treatment regimen. TB-MBLA distinguished the bactericidal effect of regimens revealing the moxifloxacin—20 mg rifampicin regimen produced a shorter time to bacillary clearance compared with standard-of-care regimen, p=0.008, HR 2.9, 95% CI (1.3 to 6.7). Our data show that the TB-MBLA could inform clinical decision making in real-time and expedite drug TB clinical trials.

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for six decades without significant appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We therefore investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question, we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro, in macrophages and in mice. Altogether, our results suggest that the ultra-low rate of emergence of D-cycloserine resistance mutations is the dominant biological factor delaying the appearance of clinical resistance to this antibiotic. Furthermore, we also identified potential compensatory mechanisms able to minimize the severe fitness costs of primary D-cycloserine resistance conferring mutations.

SummaryBiosynthesis of the nucleotide sugar precursor dTDP-L-rhamnose is critical for the viability and virulence of many human pathogenic bacteria, includingStreptococcus pyogenes(Group AStreptococcus; GAS) andStreptococcus mutans. Those pathogens require dTDP-L-rhamnose for the production of structurally similar rhamnose polysaccharides in their cell wall. Via heterologous expression inS. mutans, we confirm that GAS RmlB and RmlC are critical for dTDP-L-rhamnose biosynthesis through their action as dTDP-glucose-4,6-dehydratase and dTDP-4-keto-6-deoxyglucose-3,5-epimerase enzymes, respectively. Complementation with GAS RmlB and RmlC containing specific point mutations corroborated the conservation of previous identified catalytic residues in these enzymes. Bio-layer interferometry was used to identify and confirm inhibitory lead compounds that bind to GAS dTDP-rhamnose biosynthesis enzymes RmlB, RmlC and GacA. One of the identified compounds, Ri03, inhibited growth of GAS as well as several other rhamnose-dependent streptococcal pathogens with an MIC50of 120-410 μM. We therefore conclude that inhibition of dTDP-L-rhamnose biosynthesis such as Ri03 affect streptococcal viability and can serve as a lead compound for the development of a new class of antibiotics that targets dTDP-rhamnose biosynthesis in pathogenic bacteria.

Objectives Better outcomes in tuberculosis require new diagnostic and treatment monitoring tools. In this paper we evaluated the utility of a marker of M. tuberculosis viable count, the Molecular Bacterial Load assay (MBLA) for diagnosis and treatment monitoring of tuberculosis in a high burden setting.Methods Patients with smear positive pulmonary tuberculosis from two sites in Tanzania and one each in Malawi and Mozambique. Sputum samples were taken weekly for the first 12 weeks of treatment and evaluated by MBLA and mycobacterial growth indicator tube method (MGIT).Results The results of high and low positive control samples confirmed inter site reproducibility. Over the 12 weeks of treatment there was a steady decline in the viable bacterial load as measured by the MBLA that corresponds to rise in time to a positive result (TTP) in the Mycobacterial Growth Indicator Tube. Both MBLA and MGIT provided similar time to test negativity. Importantly, as treatment progressed samples in MGIT were increasingly likely to be contaminated, which compromised the acquisition of results but did not affect MBLA samples.Conclusions MBLA produces a reproducible measure of Mtb viable count comparable to that of MGIT that is not compromised by contamination in a real-world setting. As a molecular test, the results can be available in as little as four hours and could allow health care professionals to identify rapidly patients who are failing therapy.

The initial adaptive transcriptional response to nitrogen (N) starvation in Escherichia coli involves large-scale alterations to the transcriptome mediated by the transcriptional activator, NtrC. One of these NtrC-activated genes is yeaG , which encodes a conserved bacterial kinase. Although it is known that YeaG is required for optimal survival under sustained N starvation, the molecular basis by which YeaG benefits N starved E. coli remains elusive. By combining transcriptomics with targeted metabolomics analyses, we demonstrate that the methionine biosynthesis pathway becomes transcriptionally dysregulated in Δ yeaG bacteria experiencing sustained N starvation. It appears the ability of MetJ, the master transcriptional repressor of methionine biosynthesis genes, to effectively repress transcription of genes under its control is compromised in Δ yeaG bacteria under sustained N starvation, resulting in transcriptional derepression of MetJ-regulated genes. Although the aberrant biosynthesis does not appear to be a contributing factor for the compromised viability of Δ yeaG bacteria experiencing sustained N starvation, this study identifies YeaG as a novel regulatory factor in E. coli affecting the transcription of methionine biosynthesis genes under sustained N starvation.

Drug resistant infections represent one of the most challenging medical problems of our time. D-cycloserine is an antibiotic used for decades without appearance and dissemination of antibiotic resistant strains, making it an ideal model compound to understand what drives resistance evasion. We investigated why Mycobacterium tuberculosis fails to become resistant to D-cycloserine. To address this question we employed a combination of bacterial genetics, genomics, biochemistry and fitness analysis in vitro , in macrophages and in mice. Altogether, our results suggest that the ultra-low mutation frequency associated with D-cycloserine resistance is the dominant factor delaying the appearance of clinical resistance to this antibiotic in bacteria infecting humans, and not lack of potential compensatory mechanisms. One Sentence Summary We show that the lack of D-cycloserine resistance in Mycobacterium tuberculosis is due its ultra-low mutation frequency rather than lack of compensatory mechanisms.

The initial adaptive transcriptional response to nitrogen (N) starvation in Escherichia coli involves large-scale alterations to the transcriptome mediated by the transcription activator, NtrC. One of the NtrC-activated genes is yeaG , which encodes a conserved bacterial kinase. Although it is known that YeaG is required for optimal survival under sustained N starvation, the molecular basis by which YeaG benefits N starved E. coli remains elusive. By combining transcriptomics with targeted metabolomics analyses, we demonstrate that the methionine biosynthesis pathway becomes transcriptionally dysregulated in ΔyeaG bacteria experiencing sustained N starvation. This results in the aberrant and energetically costly biosynthesis of methionine and associated metabolites under sustained N starvation with detrimental consequences to cell viability. It appears the activity of the master transcriptional repressor of methionine biosynthesis genes, MetJ, is compromised in ΔyeaG bacteria under sustained N starvation, resulting in transcriptional derepression of MetJ-regulated genes. The results suggest that YeaG is a novel regulatory factor and functions as a molecular brake in the transcriptional control of both the NtrC-regulon and methionine biosynthesis genes in E. coli experiencing sustained N starvation.

Disulfides from Allium stipitatum , commonly known as Persian shallot, were previously reported to possess antibacterial properties. Analogues of these compounds, produced by S -methylthiolation of appropriate thiols using S -methyl methanethiosulfonate, exhibited antimicrobial activity, with one compound inhibiting the growth of Mycobacterium tuberculosis at 17 µM (4 mg L −1 ) and other compounds inhibiting Escherichia coli and multi-drug-resistant (MDR) Staphylococcus aureus at concentrations ranging between 32–138 µM (8–32 mg L −1 ). These compounds also displayed moderate inhibitory effects on Klebsiella and Proteus species. Whole-cell phenotypic bioassays such as the spot-culture growth inhibition assay (SPOTi), drug efflux inhibition, biofilm inhibition and cytotoxicity assays were used to evaluate these compounds. Of particular note was their ability to inhibit mycobacterial drug efflux and biofilm formation, while maintaining a high selectivity towards M . tuberculosis H37Rv. These results suggest that methyl disulfides are novel scaffolds which could lead to the development of new drugs against tuberculosis (TB).

Background and PurposeWith the emergence of extensively drug‐resistant tuberculosis, there is a need for new anti‐tubercular drugs that work through novel mechanisms of action. The meta cleavage product hydrolase, HsaD, has been demonstrated to be critical for the survival of Mycobacterium tuberculosis in macrophages and is encoded in an operon involved in cholesterol catabolism, which is identical in M. tuberculosis and M. bovis BCG.Experimental ApproachWe generated a mutant strain of M. bovis BCG with a deletion of hsaD and tested its growth on cholesterol. Using a fragment based approach, over 1000 compounds were screened by a combination of differential scanning fluorimetry, NMR spectroscopy and enzymatic assay with pure recombinant HsaD to identify potential inhibitors. We used enzymological and structural studies to investigate derivatives of the inhibitors identified and to test their effects on growth of M. bovis BCG and M. tuberculosis.Key ResultsThe hsaD deleted strain was unable to grow on cholesterol as sole carbon source but did grow on glucose. Of seven chemically distinct ‘hits’ from the library, two chemical classes of fragments were found to bind in the vicinity of the active site of HsaD by X‐ray crystallography. The compounds also inhibited growth of M. tuberculosis on cholesterol. The most potent inhibitor of HsaD was also found to be the best inhibitor of mycobacterial growth on cholesterol‐supplemented minimal medium.Conclusions and ImplicationsWe propose that HsaD is a novel therapeutic target, which should be fully exploited in order to design and discover new anti‐tubercular drugs.Linked ArticlesThis article is part of a themed section on Drug Metabolism and Antibiotic Resistance in Micro‐organisms. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.14/issuetoc