Nicholas P. West

A uridyl amino acid was incorporated into an mRNA display library to identify macrocyclic peptide ligands targeting Mycobacterium tuberculosis MurF. Several peptides exhibited nanomolar binding affinity, with one exhibiting an IC ₅₀ of 3 μM.

Mycobacterium tuberculosis (Mtb) is a highly adapted human pathogen capable of manipulating host immunity. This study demonstrates that PPE19, a member of the PE/PPE protein family, facilitates Mtb adhesion to, and invasion of murine macrophages. PPE19-coated microspheres showed enhanced uptake by macrophages compared to control beads, while Mtb overexpressing ppe19 (Rv1361c) was phagocytosed at a significantly greater rate than WT Mtb. ppe19 is identified as pH responsive and displays reduced expression following macrophage entry. CRISPR interference-mediated knockdown of two highly related PPE proteins, ppe18 (Rv1196) and ppe60 (Rv3478), revealed an additive reduction in Mtb’s ability to invade host macrophages, indicating a potential functional relationship. Furthermore, the absence of an in vivo phenotype following murine infection with a ppe19 knockout strain suggests functional redundancy within this PPE protein family. Finally, PE13 has been identified here as a binding partner for PPE19, characterizing another relationship presumed important for successful PPE secretion. These findings reveal PPE19 as a secreted effector protein used by Mtb to modulate important early interactions with the innate immune system, enhancing entry into host macrophages. IMPORTANCE: Tuberculosis remains a leading infectious disease killer worldwide, with approximately one-quarter of the global population infected with Mycobacterium tuberculosis (Mtb). Understanding how this pathogen initially establishes infection is crucial for developing more effective vaccines and treatments. This study identifies PPE19, a previously uncharacterized bacterial protein, as a key factor that helps Mtb invade and colonize human immune cells called macrophages during the earliest stages of infection. The research shows that PPE19 acts like a molecular “key” that facilitates bacterial entry into host cells but is then downregulated once the bacteria are safely inside. Importantly, PPE19 belongs to a family of similar proteins that can compensate for each other, explaining why targeting individual members may not be sufficient for treatment. These findings provide new insights into tuberculosis pathogenesis and suggest that early infection factors like PPE19 could serve as targets for next-generation vaccines designed to prevent initial infection rather than just disease progression.

Tuberculosis (TB) remains as a leading cause of morbidity and mortality, accounting for ∼1.3 million fatalities worldwide per year. There are two major concerns: (i) the rise in the number of multi- and extensively drug-resistant strains of TB and (ii) the significant side-effects related to the use of many of the current therapies to treat drug-resistant and drug-sensitive TB alike. Thus, there is an ongoing need to discover new drugs and drug targets to combat this disease. Here, acetohydroxyacid synthase (AHAS), the first enzyme in the branched-chain amino acids (BCAAs) biosynthesis pathway, is comprehensively investigated as such a drug target. All five chemical classes of plant AHAS inhibitors, established as commercial herbicides, were assessed as leads. Members of the triazolopyrimidine family (e.g., metosulam, penoxsulam, and florasulam) are the most potent inhibitors of Mycobacterium tuberculosis AHAS (MtbAHAS) with K_i values as low as 20 nM. These compounds also exhibit the property of accumulative time-dependent inhibition, a feature that appears to be crucial for herbicidal activity and more generally for biocidal activity. Of these, the anti-TB activity of florasulam was the most effective, with an MIC of 500 nM against virulent Mtb grown in culture. This compound is also effective in killing intramacrophage Mtb and reduces bacterial load, as compared to vehicle-only by 13-fold in the lungs of mice infected with Mtb. Thus, triazolopyrimidines as AHAS inhibitors, and in-particular florasulam, represents a promising new class of leads for anti-TB drug development.

Tuberculosis (TB) caused by Mycobacterium tuberculosis has been a threat to human health for thousands of years and still leads to millions of deaths each year. TB is a disease that is refractory to treatment, partially due to its capacity for in-host persistence. The cell wall of mycobacteria, rich in mycolic acid, is broadly associated with bacterial persistence together with antimicrobial and immunological resistance. Enzymes for the biosynthesis of bacterial peptidoglycan, an essential component of the cell wall, have been addressed and considered as appealing drug targets in pathogens. Significant effort has been dedicated to finding inhibitors that hinder peptidoglycan biosynthesis, many with demonstrated enzymatic inhibition in vitro being published. One family of critical biosynthetic enzymes are the Mur enzymes, with many enzyme specific inhibitors having been reported. However, a lesser developed strategy which may have positive clinical implications is to take advantage of the common structural and catalytic characteristics among Mur enzymes and to allow simultaneous, multiple Mur inhibition, and avert the development of drug resistance. M. tuberculosis relies on these essential Mur enzymes, with the best-known subset being Mur ligases, but also utilizes unique functions of atypical transpeptidases resulting in peptidoglycan peptide cross-linking beneficial to the bacteria’s capacity for chronic persistence in humans. A systematic review is now needed, with an emphasis on M. tuberculosis. The urgent development of novel anti-TB agents to counter rapidly developing drug resistance requires a revisit of the literature, past successes and failures, in an attempt to reveal liabilities in critical cellular functions and drive innovation.

Drug-resistant strains of Mycobacterium tuberculosis are a major global health problem. Resistance to the front-line antibiotic isoniazid is often associated with mutations in the katG-encoded bifunctional catalase-peroxidase. We hypothesise that perturbed KatG activity would generate collateral vulnerabilities in isoniazid-resistant katG mutants, providing potential pathway targets to combat isoniazid resistance. Whole genome CRISPRi screens, transcriptomics, and metabolomics were used to generate a genome-wide map of cellular vulnerabilities in an isoniazid-resistant katG mutant strain of M. tuberculosis. Here, we show that metabolic and transcriptional remodelling compensates for the loss of KatG but in doing so generates vulnerabilities in respiration, ribosome biogenesis, and nucleotide and amino acid metabolism. Importantly, these vulnerabilities are more sensitive to inhibition in an isoniazid-resistant katG mutant and translated to clinical isolates. This work highlights how changes in the physiology of drug-resistant strains generates druggable vulnerabilities that can be exploited to improve clinical outcomes.

Tuberculosis remains a leading cause of death by infectious disease. The long treatment regimen and the spread of drug‐resistant strains of the causative agent Mycobacterium tuberculosis (Mtb) necessitates the development of new treatment options. In a phenotypic screen, nitrofuran‐resorufin conjugate 1 was identified as a potent sub‐micromolar inhibitor of whole cell Mtb. Complete loss of activity was observed for this compound in Mtb mutants affected in enzyme cofactor F₄₂₀ biosynthesis (fbiC), suggesting that 1 undergoes prodrug activation in a manner similar to anti‐tuberculosis prodrug pretomanid. Exploration of the structure‐activity relationship led to the discovery of novel resorufin analogues that do not rely on the deazaflavin‐dependent nitroreductase (Ddn) bioactivation pathway for their antimycobacterial activity. These analogues are of interest as they work through an alternative, currently unknown mechanism that may expand our chemical arsenal towards the treatment of this devastating disease.

The plethora of genome sequences produced in the postgenomic age has not resolved many of our most pressing biological questions. Correlating gene expression with an interrogatable and easily observable characteristic such as the surrogate phenotype conferred by a reporter gene is a valuable approach to gaining insight into gene function. Many reporters including lacZ, amdS, and the fluorescent proteins mRuby3 and mNeonGreen have been used across all manners of organisms. Described here is an investigation into the creation of a robust, synthetic, fusion reporter system for Cryptococcus neoformans that combines some of the most useful fluorophores available in this system with the versatility of the counter-selectable nature of amdS. The reporters generated include multiple composition and orientation variants, all of which were investigated for differences in expression. Evaluation of known promoters from the TEF1 and GAL7 genes was undertaken, elucidating novel expression tendencies of these biologically relevant C. neoformans regulators of transcription. Smaller than lacZ but providing multiple useful surrogate phenotypes for interrogation, the fusion ORF serves as a superior whole-cell assay compared to traditional systems. Ultimately, the work described here bolsters the array of relevant genetic tools that may be employed in furthering manipulation and understanding of the WHO fungal priority group pathogen C. neoformans.

Tuberculosis remains a significant global health pandemic. There is an urgent need for new anti-tubercular agents to combat the rising incidence of drug resistance and to offer effective and additive therapeutic options. High-throughput screening of a subset of the NatureBank marine fraction library (n = 2000) identified a sample derived from an Australian marine sponge belonging to the order Haplosclerida that displayed promising anti-mycobacterial activity. Bioassay-guided fractionation of the organic extract from this Haplosclerida sponge led to the purification of previously identified antimicrobial pyrrole alkaloids, axinellamines A (1) and B (2). The axinellamine compounds were found to have a 90% minimum inhibitory concentration (MIC₉₀) of 18 µM and 15 µM, respectively. The removal of protein and complex carbon sources reduced the MIC₉₀ of 1 and 2 to 0.6 and 0.8 µM, respectively. The axinellamines were not toxic to mammalian cells at 25 µM and significantly reduced the intracellular bacterial load by >5-fold. These data demonstrate that axinellamines A and B are effective anti-tubercular agents and promising targets for future medicinal chemistry efforts.

The ability ofMycobacterium tuberculosis(Mtb) to persist in the host complicates and prolongs tuberculosis (TB) patient chemotherapy. Here we demonstrate that a neglected two-component system (TCS) of Mtb, TcrXY, is an autoregulated acid-sensing TCS that controls a functionally diverse 70-gene regulon required for bacterial persistence. Characterisation of two representatives of this regulon,Rv3706candRv3705A, implicate these genes as key determinants for the survival of Mtb in vivo by serving as important effectors to mitigate redox stress at acidic pH. We show that genetic silencing of the response regulatortcrXusing CRISPR interference attenuates the persistence of Mtb during chronic mouse infection and improves treatment with the two front-line anti-TB drugs, rifampicin and isoniazid. We propose that targeting TcrXY signal transduction blocks the ability of Mtb to sense and respond to acid stress, resulting in a disordered program of persistence to render the organism vulnerable to existing TB chemotherapy.

New control measures are urgently required to control tuberculosis (TB), as the current vaccine, Bacille Calmette–Guérin (BCG), has had a limited impact on disease spread. The identification of virulence mechanisms of Mycobacterium tuberculosis is an important strategy in vaccine design, as it permits the development of strains attenuated for growth that may have vaccine potential. In this report, we determined the role of the PdtaS response regulator in M. tuberculosis virulence and defined the vaccine potential of a pdtaS-deficient strain. Deletion of pdtaS (Mtb^ΔpdtaS) resulted in reduced persistence of M. tuberculosis within mouse organs, which was equivalent to the persistence of the BCG vaccine in the lung and liver of infected mice. However, the generation of effector CD4⁺ and CD8⁺ T cells (CD44⁺CD62L^loKLRG1⁺) was similar between wild-type M. tuberculosis and Mtb^ΔpdtaS and greater than that elicited by BCG. Heightened immunity induced by Mtb^ΔpdtaS compared to BCG was also observed by analysis of antigen-specific IFN-γ-secreting T cell responses induced by vaccination. Mtb^ΔpdtaS displayed improved protection against aerosol M. tuberculosis compared to BCG, which was most apparent in the lung at 20 weeks post-infection. These results suggest that the deletion of the PdtaS response regulator warrants further appraisal as a tool to combat TB in humans.