Tools to develop antibiotic combinations that target drug tolerance in Mycobacterium tuberculosis

Combination therapy is necessary to treat tuberculosis to decrease the rate of disease relapse and prevent the acquisition of drug resistance, and shorter regimens are urgently needed. The adaptation of Mycobacterium tuberculosis to various lesion microenvironments in infection induces various states of slow replication and non-replication and subsequent antibiotic tolerance. This non-heritable tolerance to treatment necessitates lengthy combination therapy. Therefore, it is critical to develop combination therapies that specifically target the different types of drug-tolerant cells in infection. As new tools to study drug combinations earlier in the drug development pipeline are being actively developed, we must consider how to best model the drug-tolerant cells to use these tools to design the best antibiotic combinations that target those cells and shorten tuberculosis therapy. In this review, we discuss the factors underlying types of drug tolerance, how combination therapy targets these populations of bacteria, and how drug tolerance is currently modeled for the development of tuberculosis multidrug therapy. We highlight areas for future studies to develop new tools that better model drug tolerance in tuberculosis infection specifically for combination therapy testing to bring the best drug regimens forward to the clinic

Lyapunov exponents and phase diagrams reveal multi-factorial control over TRAIL-induced apoptosis

Kinetic modeling, phase diagrams analysis, and quantitative single-cell experiments are combined to investigate how multiple factors, including the XIAP:caspase-3 ratio and ligand concentration, regulate receptor-mediated apoptosis

Stress-Induced Reorganization of the Mycobacterial Membrane Domain

Cell elongation occurs primarily at the mycobacterial cell poles, but the molecular mechanisms governing this spatial regulation remain elusive. We recently reported the presence of an intracellular membrane domain (IMD) that was spatially segregated from the conventional plasma membrane in Mycobacterium smegmatis. The IMD is enriched in the polar region of actively elongating cells and houses many essential enzymes involved in envelope biosynthesis, suggesting its role in spatially restricted elongation at the cell poles. Here, we examined reorganization of the IMD when the cells are no longer elongating. To monitor the IMD, we used a previously established reporter strain expressing fluorescent IMD markers and grew it to the stationary growth phase or exposed the cells to nutrient starvation. In both cases, the IMD was delocalized from the cell pole and distributed along the sidewall. Importantly, the IMD could still be isolated biochemically by density gradient fractionation, indicating its maintenance as a membrane domain. Chemical and genetic inhibition of peptidoglycan biosynthesis led to the delocalization of the IMD, suggesting the suppression of peptidoglycan biosynthesis as a trigger of spatial IMD rearrangement. Starved cells with a delocalized IMD can resume growth upon nutrient repletion, and polar enrichment of the IMD coincides with the initiation of cell elongation. These data reveal that the IMD is a membrane domain with the unprecedented capability of subcellular repositioning in response to the physiological conditions of the mycobacterial cell. IMPORTANCE Mycobacteria include medically important species, such as the human tuberculosis pathogen Mycobacterium tuberculosis. The highly impermeable cell envelope is a hallmark of these microbes, and its biosynthesis is a proven chemotherapeutic target. Despite the accumulating knowledge regarding the biosynthesis of individual envelope components, the regulatory mechanisms behind the coordinated synthesis of the complex cell envelope remain elusive. We previously reported the presence of a metabolically active membrane domain enriched in the elongating poles of actively growing mycobacteria. However, the spatiotemporal dynamics of the membrane domain in response to stress have not been examined. Here, we show that the membrane domain is spatially reorganized when growth is inhibited in the stationary growth phase, under nutrient starvation, or in response to perturbation of peptidoglycan biosynthesis. Our results suggest that mycobacteria have a mechanism to spatiotemporally coordinate the membrane domain in response to metabolic needs under different growth conditions

Stable Regulation of Cell Cycle Events in Mycobacteria: Insights From Inherently Heterogeneous Bacterial Populations

Model bacteria, such as E. coli and B. subtilis, tightly regulate cell cycle progression to achieve consistent cell size distributions and replication dynamics. Many of the hallmark features of these model bacteria, including lateral cell wall elongation and symmetric growth and division, do not occur in mycobacteria. Instead, mycobacterial growth is characterized by asymmetric polar growth and division. This innate asymmetry creates unequal birth sizes and growth rates for daughter cells with each division, generating a phenotypically heterogeneous population. Although the asymmetric growth patterns of mycobacteria lead to a larger variation in birth size than typically seen in model bacterial populations, the cell size distribution is stable over time. Here, we review the cellular mechanisms of growth, division, and cell cycle progression in mycobacteria in the face of asymmetry and inherent heterogeneity. These processes coalesce to control cell size. Although Mycobacterium smegmatis and Mycobacterium bovis Bacillus Calmette-Guérin (BCG) utilize a novel model of cell size control, they are similar to previously studied bacteria in that initiation of DNA replication is a key checkpoint for cell division. We compare the regulation of DNA replication initiation and strategies used for cell size homeostasis in mycobacteria and model bacteria. Finally, we review the importance of cellular organization and chromosome segregation relating to the physiology of mycobacteria and consider how new frameworks could be applied across the wide spectrum of bacterial diversity

Prediction of ultra-high-order antibiotic combinations based on pairwise interactions.

Drug combinations are a promising approach to achieve high efficacy at low doses and to overcome resistance. Drug combinations are especially useful when drugs cannot achieve effectiveness at tolerable doses, as occurs in cancer and tuberculosis (TB). However, discovery of effective drug combinations faces the challenge of combinatorial explosion, in which the number of possible combinations increases exponentially with the number of drugs and doses. A recent advance, called the dose model, uses a mathematical formula to overcome combinatorial explosion by reducing the problem to a feasible quadratic one: using data on drug pairs at a few doses, the dose model accurately predicts the effect of combinations of three and four drugs at all doses. The dose model has not yet been tested on higher-order combinations beyond four drugs. To address this, we measured the effect of combinations of up to ten antibiotics on E. coli growth, and of up to five tuberculosis (TB) drugs on the growth of M. tuberculosis. We find that the dose model accurately predicts the effect of these higher-order combinations, including cases of strong synergy and antagonism. This study supports the view that the interactions between drug pairs carries key information that largely determines higher-order interactions. Therefore, systematic study of pairwise drug interactions is a compelling strategy to prioritize drug regimens in high-dimensional spaces

Data Availability: The a...

Mycobacterium tuberculosis (Mtb) remains a major public health problem, with an effective vaccine continuing to prove elusive. Progress in vaccination strategies has been hampered by a lack of appreciation of the bacterium’s response to dynamic changes in the host immune environment. Here, we utilize reporter Mtb strains that respond to specific host immune stresses such as hypoxia and nitric oxide (hspX9::GFP), and phagosomal maturation (rv2390c9::GFP), to investigate vaccine-induced alterations in the environmental niche during experimental murine infections. While vaccination undoubtedly decreased bacterial burden, we found that it also appeared to accelerate Mtb’s adoption of a phenotype better equipped to survive in its host. We subsequently utilized a novel replication reporter strain of Mtb to demonstrate that, in addition to these alterations in host stress response, there is a decreased percentage of actively replicating Mtb in vaccinated hosts. This observation was supported by the differential sensitivity of recovered bacteria to the front-line drug isoniazid. Our study documents the natural history of the impact that vaccination has on Mtb’s physiology and replication and highlights the value of reporter Mtb strains for probing heterogeneous Mtb populations in the context of a complex, whole animal model

Exploitation of <i>Mycobacterium tuberculosis</i> Reporter Strains to Probe the Impact of Vaccination at Sites of Infection

<div><p><i>Mycobacterium tuberculosis</i> (Mtb) remains a major public health problem, with an effective vaccine continuing to prove elusive. Progress in vaccination strategies has been hampered by a lack of appreciation of the bacterium's response to dynamic changes in the host immune environment. Here, we utilize reporter Mtb strains that respond to specific host immune stresses such as hypoxia and nitric oxide (<i>hspX</i>′::GFP), and phagosomal maturation (<i>rv2390c</i>′::GFP), to investigate vaccine-induced alterations in the environmental niche during experimental murine infections. While vaccination undoubtedly decreased bacterial burden, we found that it also appeared to accelerate Mtb's adoption of a phenotype better equipped to survive in its host. We subsequently utilized a novel replication reporter strain of Mtb to demonstrate that, in addition to these alterations in host stress response, there is a decreased percentage of actively replicating Mtb in vaccinated hosts. This observation was supported by the differential sensitivity of recovered bacteria to the front-line drug isoniazid. Our study documents the natural history of the impact that vaccination has on Mtb's physiology and replication and highlights the value of reporter Mtb strains for probing heterogeneous Mtb populations in the context of a complex, whole animal model.</p></div

Itay Katzir - Poster of BC2 .pdf

<p>Finding potent multi-drug combinations against cancer and bacterial infections is a pressing therapeutic challenge; however, screening all combinations is difficult because the number of experiments grows exponentially with the number of drugs and doses. To address this, we recently developed a mathematical model which predicts the effects of three or more antibiotics or anti-cancer drugs at all doses based only on measurements of drug pairs at a few doses, without need for mechanistic information. The model provides accurate predictions on previous data for up to four antibiotic combinations, and on experiments on the response matrix of three cancer drugs at eight doses per drug. </p><p>To further test the model beyond four drugs and for clinically relevant pathogens, we performed experiments on drug combinations at multiple doses in two organisms: <i>E. coli</i> and <i>M. tuberculosis</i>. We measured all 45 pair combinations of ten drugs in <i>E. coli</i> and <i>M. tuberculosis</i>, and tested predictions for combinations of three to five drugs. We find that the dose model works well in both <i>E. coli</i> and <i>M. tuberculosis</i>. We also use the model to find new synergistic combinations of three to five drugs for <i>M. tuberculosis</i>.</p><p> </p><p> </p

Characterization of a heat-killed Mtb vaccination model.

<p>(A) Kinetics of Mtb colonization in vaccinated or mock-treated mice lungs. C57BL/6J mice were intraperitoneally injected with 5×10⁵ CFUs of heat-killed Erdman or with sterile PBS (mock-treated). Four weeks post-vaccination, mice were challenged with 10³ CFUs of various reporter Mtb intranasally. At indicated time points post-challenge, mice were sacrificed and the bacterial burden in the lungs determined. The pooled data for each time point are shown as means ± SEM, from at least 3 animals per data point. * indicates p<0.05 (unpaired t-test). (B) Lung pathology of vaccinated and mock-treated mice. Lungs collected at indicated time points post-challenge were fixed in 4% paraformaldehyde and subjected to routine H&E staining. Scale bar 200 µm.</p