Joanna C. Evans

Recent and Rapid Emergence of W-Beijing Strains of Mycobacterium tuberculosis in Cape Town, South Africa

BACKGROUND There is increasing evidence of a strain-related variation in the virulence in Mycobacterium tuberculosis that may afford a selective advantage to certain strains. The W-Beijing strain family is globally distributed, highly virulent in animal models, associated with human immunodeficiency virus infection and drug resistance, and may be an emerging strain family. Our goal was to determine whether W-Beijing strains are expanding in a region of South Africa where rates of tuberculosis are among the highest in the world. METHODS We used spoligotyping and single nucleotide polymorphism analysis to genotype all strains of tuberculosis from children presenting to the major pediatric referral hospital in Cape Town, South Africa over a period of 4 years and strains present in 352 archived histological samples from over a 76-year period. RESULTS The proportion of W-Beijing strains from children increased from 13% to 33% from 2000 to 2003 (P= .026). With regard to the histological samples, W-Beijing strains were absent in the samples from the period 1930-1965 and rare in the samples from the period 1966-1995 (2.8% of samples), but they were increasingly common in samples from the period 1996-2005 (20% of samples; P= .001). CONCLUSIONS The rapid expansion of W-Beijing strains in a region with a very high background incidence of tuberculosis suggests that these strains have a significant selective advantage. The biological reasons for this observation remain unclear but warrant further study. The rapid spread of this virulent strain lineage is likely to present additional challenges for tuberculosis control.

Rapid genotypic assays to identify drug-resistant Mycobacterium tuberculosis in South Africa

OBJECTIVES Molecular assays to detect drug resistance in Mycobacterium tuberculosis are more rapid than standard drug susceptibility testing. To evaluate the efficacy of such assays in this setting, the GenoType MTBDRplus assay (HAIN Lifescience) and multiplex allele-specific PCR assays were carried out. METHODS The GenoType MTBDRplus assay was evaluated for the detection of rifampicin and isoniazid resistance in 223 M. tuberculosis isolates of known phenotypic drug sensitivity. The presence of KatG S315T and inhA C-15T mutations that confer isoniazid resistance was determined using multiplex allele-specific PCR assays. The relationship between isolate lineage and resistance determinant was investigated by spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat analysis. RESULTS The GenoType MTBDRplus assay detected multidrug-resistant, isoniazid-monoresistant and rifampicin-monoresistant isolates with sensitivities of 91.5%, 56.1% and 70%, respectively. Multiplex allele-specific PCR detected isoniazid resistance in 91.5% of the MDR isolates and 53.7% of the isoniazid-monoresistant isolates. The W-Beijing lineage was overrepresented in the MDR subgroup of strains (odds ratio, 3.29; 95% confidence interval, 1.76-6.16). CONCLUSIONS A proportion of isoniazid resistance, particularly in isoniazid-monoresistant isolates of lineage X3, is due to resistance determinants other than KatG S315T and inhA C-15T. The fact that these isolates will be indicated as drug susceptible highlights the need for determining local patterns of resistance mutations to provide users with information regarding the capabilities of rapid genotypic assays.

Novel multiplex allele-specific PCR assays for the detection of resistance to second-line drugs in Mycobacterium tuberculosis

OBJECTIVES The use of rapid molecular assays for the detection of resistance to second-line drugs would significantly decrease the time delay in diagnosing drug-resistant tuberculosis (TB) that is associated with conventional phenotypic drug susceptibility testing. In this study, multiplex allele-specific (MAS)-PCR assays designed to detect the GyrA D94G and rrs A1401G mutations were evaluated for detection of ofloxacin and kanamycin resistance. METHODS GyrA D94G and rrs A1401G MAS-PCR assays were carried out on 288 Mycobacterium tuberculosis isolates. Phenotypic drug susceptibility testing of ofloxacin and kanamycin was performed on selected multidrug-resistant TB isolates using the indirect proportions method. RESULTS MAS-PCR assays detected GyrA D94G and rrs A1401G mutations in phenotypically resistant isolates with clinical sensitivities of 54.5% (6 of 11) and 90.0% (9 of 10), respectively, and specificities of 100% were obtained for both assays. A GyrA A90V mutation was identified in 4 of 11 (36.4%) ofloxacin-resistant isolates that did not carry a D94G substitution. CONCLUSIONS Rapid genotypic assays designed to detect GyrA D94G and A90V mutations and rrs A1401G mutations could detect up to 90.0% of extensively drug-resistant (XDR)-TB in the Western Cape region. The use of these assays in the clinical setting would significantly reduce the time to diagnosis of XDR-TB, enabling the administration of appropriate treatment regimens at the outset of therapy.

A Novel Insertion Sequence, ISPa26, in oprD of Pseudomonas aeruginosa Is Associated with Carbapenem Resistance

Carbapenems are frequently used to treat Pseudomonas aeruginosa; however, resistance to the carbapenems is emerging rapidly (9). Mutations or deletions in oprD (3, 2, 8) commonly result in lack of OprD expression, blocking the entry of imipenem into the cell and resulting in resistance to this drug (6). More recently, insertional inactivation of oprD by the insertion sequence (IS) elements ISPa1328 and ISPa1635 was described (7). Previously, we showed that imipenem resistance in P. aeruginosa was due to point mutations and deletions in oprD; IS elements were not detected in the isolates (3). To determine whether IS elements play a role in imipenem resistance in more recent isolates, we screened for their presence in oprD in isolates from two local hospitals.

Validation of CoaBC as a Bactericidal Target in the Coenzyme A Pathway of Mycobacterium tuberculosis

Mycobacterium tuberculosis relies on its own ability to biosynthesize coenzyme A to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the essential pantothenate and coenzyme A biosynthesis pathways have attracted attention as targets for tuberculosis drug development. To identify the optimal step for coenzyme A pathway disruption in M. tuberculosis, we constructed and characterized a panel of conditional knockdown mutants in coenzyme A pathway genes. Here, we report that silencing of coaBC was bactericidal in vitro, whereas silencing of panB, panC, or coaE was bacteriostatic over the same time course. Silencing of coaBC was likewise bactericidal in vivo, whether initiated at infection or during either the acute or chronic stages of infection, confirming that CoaBC is required for M. tuberculosis to grow and persist in mice and arguing against significant CoaBC bypass via transport and assimilation of host-derived pantetheine in this animal model. These results provide convincing genetic validation of CoaBC as a new bactericidal drug target.

The application of tetracyclineregulated gene expression systems in the validation of novel drug targets in Mycobacterium tuberculosis

Although efforts to identify novel therapies for the treatment of tuberculosis have led to the identification of several promising drug candidates, the identification of high-quality hits from conventional whole-cell screens remains disappointingly low. The elucidation of the genome sequence of Mycobacterium tuberculosis (Mtb) facilitated a shift to target-based approaches to drug design but these efforts have proven largely unsuccessful. More recently, regulated gene expression systems that enable dose-dependent modulation of gene expression have been applied in target validation to evaluate the requirement of individual genes for the growth of Mtb both in vitro and in vivo. Notably, these systems can also provide a measure of the extent to which putative targets must be depleted in order to manifest a growth inhibitory phenotype. Additionally, the successful implementation of Mtb strains engineered to under-express specific molecular targets in whole-cell screens has enabled the simultaneous identification of cell-permeant inhibitors with defined mechanisms of action. Here, we review the application of tetracycline-regulated gene expression systems in the validation of novel drug targets in Mtb, highlighting both the strengths and limitations associated with this approach to target validation.

Reaction intermediate analogues as bisubstrate inhibitors of pantothenate synthetase

The biosynthesis of pantothenate, the core of coenzyme A (CoA), has been considered an attractive target for the development of antimicrobial agents since this pathway is essential in prokaryotes, but absent in mammals. Pantothenate synthetase, encoded by the gene panC, catalyzes the final condensation of pantoic acid with β-alanine to afford pantothenate via an intermediate pantoyl adenylate. We describe the synthesis and biochemical characterization of five PanC inhibitors that mimic the intermediate pantoyl adenylate. These inhibitors are competitive inhibitors with respect to pantoic acid and possess submicromolar to micromolar inhibition constants. The observed SAR is rationalized through molecular docking studies based on the reported co-crystal structure of 1a with PanC. Finally, whole cell activity is assessed against wild-type Mtb as well as a PanC knockdown strain where PanC is depleted to less than 5% of wild-type levels.

Identification of Aminopyrimidine-Sulfonamides as Potent Modulators of Wag31-mediated Cell Elongation in Mycobacteria

There is an urgent need to discover new anti‐tubercular agents with novel mechanisms of action in order to tackle the scourge of drug‐resistant tuberculosis. Here, we report the identification of such a molecule – an AminoPYrimidine‐Sulfonamide (APYS1) that has potent, bactericidal activity against M. tuberculosis. Mutations in APYS1‐resistant M. tuberculosis mapped exclusively to wag31, a gene that encodes a scaffolding protein thought to orchestrate cell elongation. Recombineering confirmed that a Gln201Arg mutation in Wag31 was sufficient to cause resistance to APYS1, however, neither overexpression nor conditional depletion of wag31 impacted M. tuberculosis susceptibility to this compound. In contrast, expression of the wildtype allele of wag31 in APYS1‐resistant M. tuberculosis was dominant and restored susceptibility to APYS1 to wildtype levels. Time‐lapse imaging and scanning electron microscopy revealed that APYS1 caused gross malformation of the old pole of M. tuberculosis, with eventual lysis. These effects resembled the morphological changes observed following transcriptional silencing of wag31 in M. tuberculosis. These data show that Wag31 is likely not the direct target of APYS1, but the striking phenotypic similarity between APYS1 exposure and genetic depletion of Wag31 in M. tuberculosis suggests that APYS1 might indirectly affect Wag31 through an as yet unknown mechanism.

The influence of HIV on the evolution of Mycobacterium tuberculosis

Abstract HIV significantly affects the immunological environment during tuberculosis coinfection, and therefore may influence the selective landscape upon which M. tuberculosis evolves. To test this hypothesis whole genome sequences were determined for 169 South African M. tuberculosis strains from HIV-1 coinfected and uninfected individuals and analyzed using two Bayesian codon-model based selection analysis approaches: FUBAR which was used to detect persistent positive and negative selection (selection respectively favoring and disfavoring nonsynonymous substitutions); and MEDS which was used to detect episodic directional selection specifically favoring nonsynonymous substitutions within HIV-1 infected individuals. Among the 25,251 polymorphic codon sites analyzed, FUBAR revealed that 189-fold more were detectably evolving under persistent negative selection than were evolving under persistent positive selection. Three specific codon sites within the genes celA2b, katG, and cyp138 were identified by MEDS as displaying significant evidence of evolving under directional selection influenced by HIV-1 coinfection. All three genes encode proteins that may indirectly interact with human proteins that, in turn, interact functionally with HIV proteins. Unexpectedly, epitope encoding regions were enriched for sites displaying weak evidence of directional selection influenced by HIV-1. Although the low degree of genetic diversity observed in our M. tuberculosis data set means that these results should be interpreted carefully, the effects of HIV-1 on epitope evolution in M. tuberculosis may have implications for the design of M. tuberculosis vaccines that are intended for use in populations with high HIV-1 infection rates.

Priming the tuberculosis drug pipeline: new antimycobacterial targets and agents

Claiming close to two million lives each year, tuberculosis is now the leading cause of death from an infectious disease. The rise in number of Mycobacterium tuberculosis (Mtb) strains resistant to existing TB drugs has underscored the urgent need to develop new antimycobacterials with novel mechanisms of action. To meet this need, a drug pipeline has been established that is populated with new and repurposed drugs. Recent advances in identifying molecules with inhibitory activity against Mtb under conditions modelled on those encountered during infection, and in elucidating their mechanisms of action, have primed the pipeline with promising drug/target couples, hit compounds and new targets. In this review, we highlight recent advances and emerging areas of opportunity in this field.