Development of an In Vitro Assay for Detection of Drug-Induced Resuscitation-Promoting-Factor-Dependent Mycobacteria.

Tuberculosis is a major infectious disease that requires prolonged chemotherapy with a combination of four drugs. Here we present data suggesting that treatment of Mycobacterium tuberculosis, the causative agent of tuberculosis, and Mycobacterium smegmatis, a model organism widely used for the screening of antituberculosis agents, with first-line drugs resulted in the generation of substantial populations that could be recovered only by the addition of a culture supernatant from growing mycobacteria. These bacilli failed to grow in standard media, resulting in significant underestimation of the numbers of viable mycobacteria in treated samples. We generated M. smegmatis strains overexpressing M. tuberculosis resuscitation-promoting factors (Rpfs) and demonstrated their application for the detection of Rpf-dependent mycobacteria generated after drug exposure. Our data offer novel opportunities for validation of the sterilizing activity of antituberculosis agents

Differentially Culturable Tubercule Bacilli are Generated During Non-pulmonary Tuberculosis Infection.

Differentially Culturable Tubercule Bacilli are Generated During Non-pulmonary Tuberculosis Infection

A nested case-control study of predictors for tuberculosis recurrence in a large UK Centre

Background: Tuberculosis (TB) recurrence represents a challenge to control programs. In low incidence countries, the prevailing risk factors leading to recurrence are poorly characterised. Methods: We conducted a nested case-control study using the Leicester TB service TBIT database. Cases were identified from database notifications between 1994 and 2014. Controls had one episode and were matched to cases on a ratio of two to one by the date of notification. Multiple imputation was used to account for missing data. Multivariate conditional logistic regression analysis was employed to identify clinical, sociodemographic and TB specific risk factors for recurrence. Results: From a cohort of 4628 patients, 82 TB recurrences occurred (1.8%). Nineteen of 82 patients had paired isolates with MIRU-VNTR strain type profiles available, of which 84% were relapses and 16% reinfections. On multivariate analysis, smoking (OR 3.8; p = 0.04), grade 3/4 adverse drug reactions (OR 5.6; p = 0.02), ethnicity 'Indian subcontinent' (OR 8.5; p = <0.01), ethnicity 'other' (OR 31.2; p = 0.01) and receipt of immunosuppressants (OR 6.8; p = <0.01) were independent predictors of TB recurrence. CONCLUSIONS: Within this UK setting, the rate of TB recurrence was low, predominantly due to relapse. The identification of an elevated recurrence risk amongst the ethnic group contributing most cases to the national TB burden presents an opportunity to improve individual and population health

Phenotypically adapted Mycobacterium tuberculosis populations from sputum are tolerant to first line drugs

Tuberculous sputum contains multiple Mycobacterium tuberculosis (Mtb) populations with different requirements for isolation in vitro. These include cells forming colonies on solid media (platable Mtb), cells requiring standard liquid medium for growth (non-platable Mtb), and cells requiring supplementation of liquid medium with culture supernatant for growth (SN-dependent Mtb). Here we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of these Mtb populations within sputum. Our results show that first line drugs achieved only modest cidal effects on all three populations over 7 days (1-2.5xlog10 reductions) and SN-dependent Mtb were more tolerant to streptomycin and isoniazid compared to platable and non-platable Mtb. Susceptibility of platable Mtb to bactericidal drugs was significantly increased after passage in vitro, thus tolerance observed in the sputum populations was likely associated with mycobacterial adaptation to the host environment at some time prior to expectoration. Our findings support the use of a simple ex vivo system for testing drug efficacies against mycobacteria phenotypically adapted during tuberculosis infection

Oleoyl coenzyme A regulates interaction of transcriptional regulator RaaS (Rv1219c) with DNA in mycobacteria

We have recently shown that RaaS (regulator of antimicrobial-assisted survival), encoded by Rv1219c in Mycobacterium tuberculosis and by bcg_1279c in Mycobacterium bovis bacillus Calmette-Guérin, plays an important role in mycobacterial survival in prolonged stationary phase and during murine infection. Here, we demonstrate that long chain acyl-CoA derivatives (oleoyl-CoA and, to lesser extent, palmitoyl-CoA) modulate RaaS binding to DNA and expression of the downstream genes that encode ATP-dependent efflux pumps. Moreover, exogenously added oleic acid influences RaaS-mediated mycobacterial improvement of survival and expression of the RaaS regulon. Our data suggest that long chain acyl-CoA derivatives serve as biological indicators of the bacterial metabolic state. Dysregulation of efflux pumps can be used to eliminate non-growing mycobacteria

The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth

PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture

Two faces of CwlM, an essential PknB substrate, in Mycobacterium tuberculosis

Tuberculosis claims over one million lives annually and its causative agent Mycobacterium tuberculosis is a highly successful pathogen. Protein kinase B (PknB) is reported to be critical for mycobacterial growth. Here, we demonstrate that PknB-depleted M. tuberculosis can replicate normally and can synthesise peptidoglycan in osmoprotective medium. Comparative phosphoproteomics of PknB-producing and PknB-depleted mycobacteria identifies CwlM, an essential regulator of peptidoglycan synthesis, as a major PknB substrate. Our complementation studies of a cwlM mutant of M. tuberculosis support CwlM phosphorylation as a likely molecular basis for PknB being essential for mycobacterial growth. We demonstrate that growing mycobacteria produce two forms of CwlM : a nonphosphorylated membrane-associated form and a PknB-phosphorylated cytoplasmic form. Furthermore we show that the partner proteins for the phosphorylated and nonphosphorylated forms of CwlM are FhaA, a fork-head-associated domain protein, and MurJ, a proposed Lipid II flippase, respectively. From our results, we propose a model in which CwlM potentially regulates both the biosynthesis of peptidoglycan precursors and their transport across the cytoplasmic membrane