Table_2_Persistence of Mycobacterium tuberculosis in response to infection burden and host-induced stressors.docx

IntroductionAs infection with Mycobacterium tuberculosis progresses, the bacilli experience various degrees of host stressors in the macrophage phagosome such as low pH, nutrient deprivation, or exposure to toxic agents, which promotes cell-to-cell phenotypic variation. This includes a physiologically viable but non- or slowly replicating persister subpopulation, which is characterised by a loss of growth on solid media, while remaining metabolically active. Persisters additionally evade the host immune response and macrophage antimicrobial processes by adapting their metabolic pathways to maintain survival and persistence in the host.MethodsA flow cytometry-based dual-fluorescent replication reporter assay, termed fluorescence dilution, provided a culture-independent method to characterize the single-cell replication dynamics of M. tuberculosis persisters following macrophage infection. Fluorescence dilution in combination with reference counting beads and a metabolic esterase reactive probe, calcein violet AM, provided an effective approach to enumerate and characterize the phenotypic heterogeneity within M. tuberculosis following macrophage infection.ResultsPersister formation appeared dependent on the initial infection burden and intracellular bacterial burden. However, inhibition of phagocytosis by cytochalasin D treatment resulted in a significantly higher median percentage of persisters compared to inhibition of phagosome acidification by bafilomycin A1 treatment.DiscussionOur results suggest that different host factors differentially impact the intracellular bacterial burden, adaptive mechanisms and entry into persistence in macrophages.</p

Table_1_Persistence of Mycobacterium tuberculosis in response to infection burden and host-induced stressors.docx

IntroductionAs infection with Mycobacterium tuberculosis progresses, the bacilli experience various degrees of host stressors in the macrophage phagosome such as low pH, nutrient deprivation, or exposure to toxic agents, which promotes cell-to-cell phenotypic variation. This includes a physiologically viable but non- or slowly replicating persister subpopulation, which is characterised by a loss of growth on solid media, while remaining metabolically active. Persisters additionally evade the host immune response and macrophage antimicrobial processes by adapting their metabolic pathways to maintain survival and persistence in the host.MethodsA flow cytometry-based dual-fluorescent replication reporter assay, termed fluorescence dilution, provided a culture-independent method to characterize the single-cell replication dynamics of M. tuberculosis persisters following macrophage infection. Fluorescence dilution in combination with reference counting beads and a metabolic esterase reactive probe, calcein violet AM, provided an effective approach to enumerate and characterize the phenotypic heterogeneity within M. tuberculosis following macrophage infection.ResultsPersister formation appeared dependent on the initial infection burden and intracellular bacterial burden. However, inhibition of phagocytosis by cytochalasin D treatment resulted in a significantly higher median percentage of persisters compared to inhibition of phagosome acidification by bafilomycin A1 treatment.DiscussionOur results suggest that different host factors differentially impact the intracellular bacterial burden, adaptive mechanisms and entry into persistence in macrophages.</p

Image_1_Persistence of Mycobacterium tuberculosis in response to infection burden and host-induced stressors.jpeg

IntroductionAs infection with Mycobacterium tuberculosis progresses, the bacilli experience various degrees of host stressors in the macrophage phagosome such as low pH, nutrient deprivation, or exposure to toxic agents, which promotes cell-to-cell phenotypic variation. This includes a physiologically viable but non- or slowly replicating persister subpopulation, which is characterised by a loss of growth on solid media, while remaining metabolically active. Persisters additionally evade the host immune response and macrophage antimicrobial processes by adapting their metabolic pathways to maintain survival and persistence in the host.MethodsA flow cytometry-based dual-fluorescent replication reporter assay, termed fluorescence dilution, provided a culture-independent method to characterize the single-cell replication dynamics of M. tuberculosis persisters following macrophage infection. Fluorescence dilution in combination with reference counting beads and a metabolic esterase reactive probe, calcein violet AM, provided an effective approach to enumerate and characterize the phenotypic heterogeneity within M. tuberculosis following macrophage infection.ResultsPersister formation appeared dependent on the initial infection burden and intracellular bacterial burden. However, inhibition of phagocytosis by cytochalasin D treatment resulted in a significantly higher median percentage of persisters compared to inhibition of phagosome acidification by bafilomycin A1 treatment.DiscussionOur results suggest that different host factors differentially impact the intracellular bacterial burden, adaptive mechanisms and entry into persistence in macrophages.</p

Image_2_Persistence of Mycobacterium tuberculosis in response to infection burden and host-induced stressors.jpeg

IntroductionAs infection with Mycobacterium tuberculosis progresses, the bacilli experience various degrees of host stressors in the macrophage phagosome such as low pH, nutrient deprivation, or exposure to toxic agents, which promotes cell-to-cell phenotypic variation. This includes a physiologically viable but non- or slowly replicating persister subpopulation, which is characterised by a loss of growth on solid media, while remaining metabolically active. Persisters additionally evade the host immune response and macrophage antimicrobial processes by adapting their metabolic pathways to maintain survival and persistence in the host.MethodsA flow cytometry-based dual-fluorescent replication reporter assay, termed fluorescence dilution, provided a culture-independent method to characterize the single-cell replication dynamics of M. tuberculosis persisters following macrophage infection. Fluorescence dilution in combination with reference counting beads and a metabolic esterase reactive probe, calcein violet AM, provided an effective approach to enumerate and characterize the phenotypic heterogeneity within M. tuberculosis following macrophage infection.ResultsPersister formation appeared dependent on the initial infection burden and intracellular bacterial burden. However, inhibition of phagocytosis by cytochalasin D treatment resulted in a significantly higher median percentage of persisters compared to inhibition of phagosome acidification by bafilomycin A1 treatment.DiscussionOur results suggest that different host factors differentially impact the intracellular bacterial burden, adaptive mechanisms and entry into persistence in macrophages.</p

Forest plot of the association between <i>TLR2</i> rs5743708 and TB risk for all five models in the Asian subgroup.

<p>A) Allelic model, B) Homozygote comparison, C) Heterozygote comparison, D) Dominant model, E) Recessive model. OR: odds ratio; 95%CI: 95% confidence interval.</p

Forest plot of the association between <i>TLR9</i> rs352139 and TB risk for all five models.

<p>A) Allelic model, B) Homozygote comparison, C) Heterozygote comparison, D) Dominant model, E) Recessive model. OR: odds ratio; 95%CI: 95% confidence interval.</p

Forest plot of the association between <i>TLR4</i> rs4986791 and TB risk for all five models in the Asian subgroup.

<p>A) Allelic model, B) Homozygote comparison, C) Heterozygote comparison, D) Dominant model, E) Recessive model. OR: odds ratio; 95%CI: 95% confidence interval.</p

Forest plot of the association between <i>TLR2</i> rs3804100 and TB risk for all five models.

<p>A) Allelic model, B) Homozygote comparison, C) Heterozygote comparison, D) Dominant model, E) Recessive model. OR: odds ratio; 95%CI: 95% confidence interval.</p

Forest plot of the association between <i>TLR1</i> rs4833095 and TB risk for all five models.

<p>A) Allelic model, B) Homozygote comparison, C) Heterozygote comparison, D) Dominant model, E) Recessive model. OR: odds ratio; 95%CI: 95% confidence interval.</p

Forest plot of the association between <i>TLR2</i> rs5743708 and TB risk for all five models in the Hispanic subgroup.

<p>A) Allelic model, B) Homozygote comparison, C) Heterozygote comparison, D) Dominant model, E) Recessive model. OR: odds ratio; 95%CI: 95% confidence interval.</p