Developing novel antimicrobials by combining cancer chemotherapeutics with bacterial DNA repair inhibitors.

Cancer chemotherapeutics kill rapidly dividing cells, which includes cells of the immune system. The resulting neutropenia predisposes patients to infection, which delays treatment and is a major cause of morbidity and mortality. To tackle this problem, we have isolated several compounds that inhibit bacterial DNA repair, alone they are non-toxic, however in combination with DNA damaging anti-cancer drugs, they prevent bacterial growth. These compounds were identified through screening of an FDA-approved drug library in the presence of the anti-cancer compound cisplatin. Using a series of triage tests, the screen was reduced to a handful of drugs that were tested for specific activity against bacterial nucleotide excision DNA repair (NER). Five compounds emerged, of which three possess promising antimicrobial properties including cell penetrance, and the ability to block replication in a multi-drug resistant clinically relevant E. coli strain. This study suggests that targeting NER could offer a new therapeutic approach tailor-made for infections in cancer patients, by combining cancer chemotherapy with an adjuvant that targets DNA repair.

Optimal Intensity and Duration of Walking Rehabilitation in Patients With Chronic Stroke: A Randomized Clinical Trial.

Importance: For walking rehabilitation after stroke, training intensity and duration are critical dosing parameters that lack optimization. Objective: To assess the optimal training intensity (vigorous vs moderate) and minimum training duration (4, 8, or 12 weeks) needed to maximize immediate improvement in walking capacity in patients with chronic stroke. Design, Setting, and Participants: This multicenter randomized clinical trial using an intent-to-treat analysis was conducted from January 2019 to April 2022 at rehabilitation and exercise research laboratories. Survivors of a single stroke who were aged 40 to 80 years and had persistent walking limitations 6 months or more after the stroke were enrolled. Interventions: Participants were randomized 1:1 to high-intensity interval training (HIIT) or moderate-intensity aerobic training (MAT), each involving 45 minutes of walking practice 3 times per week for 12 weeks. The HIIT protocol used repeated 30-second bursts of walking at maximum safe speed, alternated with 30- to 60-second rest periods, targeting a mean aerobic intensity above 60% of the heart rate reserve (HRR). The MAT protocol used continuous walking with speed adjusted to maintain an initial target of 40% of the HRR, progressing up to 60% of the HRR as tolerated. Main Outcomes and Measures: The main outcome was 6-minute walk test distance. Outcomes were assessed by blinded raters after 4, 8, and 12 weeks of training. Results: Of 55 participants (mean [SD] age, 63 [10] years; 36 male [65.5%]), 27 were randomized to HIIT and 28 to MAT. The mean (SD) time since stroke was 2.5 (1.3) years, and mean (SD) 6-minute walk test distance at baseline was 239 (132) m. Participants attended 1675 of 1980 planned treatment visits (84.6%) and 197 of 220 planned testing visits (89.5%). No serious adverse events related to study procedures occurred. Groups had similar 6-minute walk test distance changes after 4 weeks (HIIT, 27 m [95% CI, 6-48 m]; MAT, 12 m [95% CI, -9 to 33 m]; mean difference, 15 m [95% CI, -13 to 42 m]; P = .28), but HIIT elicited greater gains after 8 weeks (58 m [95% CI, 39-76 m] vs 29 m [95% CI, 9-48 m]; mean difference, 29 m [95% CI, 5-54 m]; P = .02) and 12 weeks (71 m [95% CI, 49-94 m] vs 27 m [95% CI, 3-50 m]; mean difference, 44 m [95% CI, 14-74 m]; P = .005) of training; HIIT also showed greater improvements than MAT on some secondary measures of gait speed and fatigue. Conclusions and Relevance: These findings show proof of concept that vigorous training intensity is a critical dosing parameter for walking rehabilitation. In patients with chronic stroke, vigorous walking exercise produced significant and meaningful gains in walking capacity with only 4 weeks of training, but at least 12 weeks were needed to maximize immediate gains. Trial Registration: ClinicalTrials.gov Identifier: NCT03760016.

Identification of the target and mode of action for the prokaryotic nucleotide excision repair inhibitor ATBC.

In bacteria, nucleotide excision repair (NER) plays a major role in repairing DNA damage from a wide variety of sources. Therefore, its inhibition offers potential to develop a new antibacterial in combination with adjuvants, such as UV light. To date, only one known chemical inhibitor of NER is 2-(5-amino-1,3,4-thiadiazol-2-yl)benzo(f)chromen-3-one (ATBC) exists and targets Mycobacterium tuberculosis NER. To enable the design of future drugs, we need to understand its mechanism of action. To determine the mechanism of action, we used in silico structure-based prediction, which identified the ATP-binding pocket of Escherichia coli UvrA as a probable target. Growth studies in E. coli showed it was nontoxic alone, but able to impair growth when combined with DNA-damaging agents, and as we predicted, it reduced by an approximately 70% UvrA's ATPase rate. Since UvrA's ATPase activity is necessary for effective DNA binding, we used single-molecule microscopy to directly observe DNA association. We measured an approximately sevenfold reduction in UvrA molecules binding to a single molecule of dsDNA suspended between optically trapped beads. These data provide a clear mechanism of action for ATBC, and show that targeting UvrA's ATPase pocket is effective and ATBC provides an excellent framework for the derivation of more soluble inhibitors that can be tested for activity.

Topological analysis of a bacterial DedA protein associated with alkaline tolerance and antimicrobial resistance.

Maintaining membrane integrity is of paramount importance to the survival of bacteria as the membrane is the site of multiple crucial cellular processes including energy generation, nutrient uptake and antimicrobial efflux. The DedA family of integral membrane proteins are widespread in bacteria and are associated with maintaining the integrity of the membrane. In addition, DedA proteins have been linked to resistance to multiple classes of antimicrobials in various microorganisms. Therefore, the DedA family are attractive targets for the development of new antibiotics. Despite DedA family members playing a key physiological role in many bacteria, their structure, function and physiological role remain unclear. To help illuminate the structure of the bacterial DedA proteins, we performed substituted cysteine accessibility method (SCAM) analysis on the most comprehensively characterized bacterial DedA protein, YqjA from Escherichia coli. By probing the accessibility of 15 cysteine residues across the length of YqjA using thiol reactive reagents, we mapped the topology of the protein. Using these data, we experimentally validated a structural model of YqjA generated using evolutionary covariance, which consists of an α-helical bundle with two re-entrant hairpin loops reminiscent of several secondary active transporters. In addition, our cysteine accessibility data suggest that YqjA forms an oligomer wherein the protomers are arranged in a parallel fashion. This experimentally verified model of YqjA lays the foundation for future work in understanding the function and mechanism of this interesting and important family.

Cell-wall synthesis and ribosome maturation are co-regulated by an RNA switch in Mycobacterium tuberculosis.

The success of Mycobacterium tuberculosis relies on the ability to switch between active growth and non-replicating persistence, associated with latent TB infection. Resuscitation promoting factors (Rpfs) are essential for the transition between these states. Rpf expression is tightly regulated as these enzymes are able to degrade the cell wall, and hence potentially lethal to the bacterium itself. We have identified a regulatory element in the 5' untranslated region (UTR) of rpfB. We demonstrate that this element is a transcriptionally regulated RNA switch/riboswitch candidate, which appears to be restricted to pathogenic mycobacteria, suggesting a role in virulence. We have used translation start site mapping to re-annotate the RpfB start codon and identified and validated a ribosome binding site that is likely to be targeted by an rpfB antisense RNA. Finally, we show that rpfB is co-transcribed with ksgA and ispE downstream. ksgA encodes a universally conserved methyltransferase involved in ribosome maturation and ispE encodes an essential kinase involved in cell wall synthesis. This arrangement implies co-regulation of resuscitation, cell wall synthesis and ribosome maturation via the RNA switch.

Expression, maturation and turnover of DrrS, an unusually stable, DosR regulated small RNA in Mycobacterium tuberculosis.

Mycobacterium tuberculosis depends on the ability to adjust to stresses encountered in a range of host environments, adjustments that require significant changes in gene expression. Small RNAs (sRNAs) play an important role as post-transcriptional regulators of prokaryotic gene expression, where they are associated with stress responses and, in the case of pathogens, adaptation to the host environment. In spite of this, the understanding of M. tuberculosis RNA biology remains limited. Here we have used a DosR-associated sRNA as an example to investigate multiple aspects of mycobacterial RNA biology that are likely to apply to other M. tuberculosis sRNAs and mRNAs. We have found that accumulation of this particular sRNA is slow but robust as cells enter stationary phase. Using reporter gene assays, we find that the sRNA core promoter is activated by DosR, and we have renamed the sRNA DrrS for DosR Regulated sRNA. Moreover, we show that DrrS is transcribed as a longer precursor, DrrS+, which is rapidly processed to the mature and highly stable DrrS. We characterise, for the first time in mycobacteria, an RNA structural determinant involved in this extraordinary stability and we show how the addition of a few nucleotides can lead to acute destabilisation. Finally, we show how this RNA element can enhance expression of a heterologous gene. Thus, the element, as well as its destabilising derivatives may be employed to post-transcriptionally regulate gene expression in mycobacteria in combination with different promoter variants. Moreover, our findings will facilitate further investigations into the severely understudied topic of mycobacterial RNA biology and into the role that regulatory RNA plays in M. tuberculosis pathogenesis.

RfaH suppresses small RNA MicA inhibition of fimB expression in Escherichia coli K-12.

The phase variation (reversible on-off switching) of the type 1 fimbrial adhesin of Escherichia coli involves a DNA inversion catalyzed by FimB (switching in either direction) or FimE (on-to-off switching). Here, we demonstrate that RfaH activates expression of a FimB-LacZ protein fusion while having a modest inhibitory effect on a comparable fimB-lacZ operon construct and on a FimE-LacZ protein fusion, indicating that RfaH selectively controls fimB expression at the posttranscriptional level. Further work demonstrates that loss of RfaH enables small RNA (sRNA) MicA inhibition of fimB expression even in the absence of exogenous inducing stress. This effect is explained by induction of σ(E), and hence MicA, in the absence of RfaH. Additional work confirms that the procaine-dependent induction of micA requires OmpR, as reported previously (A. Coornaert et al., Mol. Microbiol. 76:467-479, 2010, doi:10.1111/j.1365-2958.2010.07115.x), but also demonstrates that RfaH inhibition of fimB transcription is enhanced by procaine independently of OmpR. While the effect of procaine on fimB transcription is shown to be independent of RcsB, it was found to require SlyA, another known regulator of fimB transcription. These results demonstrate a complex role for RfaH as a regulator of fimB expression.