Biology subjects

Voskuil, M. I.

Publications and source records attributed to Voskuil, M. I..

10 recordsLinked to original sources

bioRxiv2026

Adaptive immunity shapes baseline physiology of M. tuberculosis in high-dose versus low-dose infection BALB/c mouse drug treatment models

Preclinical tuberculosis (TB) drug evaluation relies heavily on mouse infection models. Drug efficacy varies depending on inoculum and the timing of treatment initiation. These differences reflect, in part, physiological adaptations of Mycobacterium tuberculosis (Mtb) to host immune pressure. We used novel molecular markers of pathogen health (RS ratio and SEARCH-TB) to contrast Mtb phenotypes in the BALB/c high dose aerosol (HDA) subacute TB and low dose aerosol (LDA) chronic TB infection models, focusing on the transition from innate to adaptive immunity. We found that the onset of adaptive immunity coincided with a rapid reprogramming of Mtb physiology, characterized by suppressed respiration, metabolism, and biosynthesis, together with induction of stress and nutrient acquisition pathways. Changes in key Mtb processes were concordant with changes in host expression of canonical features of adaptive immunity. Our results also explain key model-specific differences in drug efficacy: when used for drug evaluation, the HDA model begins treatment during the innate immune phase when Mtb are metabolically active, whereas the LDA model initiates therapy after host adaptive immunity has already activated and the bacterial population is immune-constrained. The HDA model is shown to be a mixed model, spanning immune phases with initial treatment acting on replicating bacteria and later treatment acting on immune-constrained populations. Together, the two models offer complementary perspectives on therapeutic activity across the spectrum of bacterial states and provide a framework for designing regimens that are effective against both active and immune-constrained Mtb.

genomics↗

bioRxiv2025

Same class, different activity: Delamanid and pretomanid have comparable bactericidal activity but pretomanid potently inhibits Mycobacterium tuberculosis ribosomal rRNA synthesis

BackgroundThe nitroimidazoles delamanid and pretomanid play an important role in contemporary tuberculosis treatment. It is unclear whether delamanid and pretomanid have meaningfully different activity since both reduce Mycobacterium tuberculosis colony forming units (CFU) similarly in animal models. The RS ratio is a pharmacodynamic marker of ongoing rRNA synthesis that has been associated with treatment-shortening (i.e., sterilizing) activity. MethodsUsing Mycobacterium tuberculosis Erdman, we conducted dose-ranging studies in aerobic axenic culture and in the conventional BALB/c mouse high-dose aerosol infection model to compare bactericidal and RS ratio activity of delamanid and pretomanid. ResultsIn vitro concentration-response curves showed that delamanid and pretomanid had similar RS ratio effect at maximal concentration but pretomanid was more potent, achieving 90% of the maximal effect (RS-EC90) at a lower concentration (390 ng/mL) than delamanid (810 ng/mL). In mice, delamanid and pretomanid had similar effects on CFU. Human-equivalent doses of delamanid (6 mg/kg) and pretomanid (50 mg/kg) resulted in plasma Cmax concentrations well below (210 ng/mL) and well above (7,825 ng/mL) the RS-EC90, respectively. Delamanid displayed no discernable RS ratio response, even at 16-times the human-equivalent dose. Higher pretomanid doses resulted in significantly greater RS ratio effects. ConclusionsWe found that delamanid and pretomanid have similar bactericidal activity but pretomanid has superior RS ratio activity. Meaningful differences between drugs within the same class were not captured by conventional CFU-based pharmacodynamics, supporting the value of measuring orthogonal drug effects such as the RS ratio. LAY SUMMARYAntibiotics in the nitroimidazole class are used in treatment of drug-resistant tuberculosis. There are two approved nitroimidazole antibiotics: delamanid and pretomanid. For decades, it has been unclear whether delamanid and pretomanid are interchangeable or whether they affect the bacterium M. tuberculosis differently. Most studies of the effect of antibiotics count the number of bacterial colonies that form on a culture plate. "Colony forming units" tell us about change in bacterial burden but does not give information about bacterial health. A new way of thinking about antibiotic effect is the RS ratio. The RS ratio is a test that measures how much ribosomal RNA synthesis is ongoing. Ribosomal RNA synthesis is a "vital sign" of bacterial health and activity. The key finding of this study is that although the two nitroimdazole antibiotics look the same in terms of their effect on bacterial burden, they have different effects on bacterial health. This information deepens understanding of differences between two clinically important antibiotics. It also shows that antibiotics testing should consider not only bacterial burden but also new tests of bacterial health.

microbiology↗

bioRxiv2025

Mind the gap: Understanding discordance between culture- and a non-culture-based measure of bacterial burden in murine tuberculosis treatment models

The standard pharmacodynamic marker in murine tuberculosis drug studies is colony-forming units (CFU). A faster PCR-based marker of bacterial burden is 16S rRNA. For unclear reasons, treatment reduces CFU more than 16S rRNA. We evaluated this CFU-16S gap and estimated the fraction potentially attributable to slow decay of 16S rRNA from dead Mycobacterium tuberculosis (Mtb) versus transition to a viable but not culturable on solid agar (VBNCSA) population. We quantified the CFU-16S gap during and following treatment in six BALB/c mouse studies and one in vitro study. Applying a two-population ordinary differential equation-based model of Mtb death and 16S rRNA decay, we estimated the fraction of the gap potentially attributable to dead Mtb. Using meta-regression, we estimated the association between CFU or 16S rRNA with relapse. For all regimens, CFU fell more than 16S rRNA, ranging from isoniazid-rifampin-pyrazinamide-ethambutol (CFU decreased 39-times more than 16S rRNA at week 4) to bedaquiline-pretomanid-moxifloxacin-pyrazinamide (CFU decreased >500,000-times more). The two-population model suggested that the fraction of the CFU-16S gap attributable to residual 16S rRNA from dead Mtb is modest and decreases over time. After treatment, 16S rRNA often fell while CFU rose. Four-week CFU change explained most variation in relapse (R{superscript 2}=0.90) while four-week 16S rRNA change did not (R{superscript 2}=0.24). CFU-16S gap is only partially explained by slow decay of residual 16S, suggesting development of a VBNCSA population. However, continued decrease in 16S rRNA after treatment cessation and its limited association with relapse suggests VBNCSA may be a transient rather than persistent state.

microbiology↗

bioRxiv2025

Deconvoluting drug interactions based on M. tuberculosis physiologic processes:Transcriptional disaggregation of the BPaL regimen in vivo

Identification of optimal antibiotic combination treatments for tuberculosis (TB) in preclinical studies is impeded by the limited information conventional pharmacodynamic (PD) markers provide about drug interactions. Measurement of individual drug activity based on colony forming units (CFU) does not reliably predict the activity of drug combinations, potentially because one drug may affect the physiology of Mycobacterium tuberculosis (Mtb) in a way that either favors or disfavors the activity of a second drug. SEARCH-TB is a novel candidate PD approach which uses targeted in vivo transcriptional profiling to evaluate the effects of drugs on Mtb physiology. To test the capacity of SEARCH-TB to elucidate drug interactions, we deconstructed the BPaL (bedaquiline, pretomanid, linezolid) regimen in the BALB/c high-dose aerosol mouse infection model, measuring the effect of 2, 7, and 14-day treatment with drugs in monotherapy, pairwise combinations, and as a 3-drug combination. Monotherapy rapidly induced drug-specific Mtb transcriptional responses by day 2 with continued evolution over 14 days. Bedaquiline dominated pairwise combinations with both pretomanid and linezolid. The pretomanid-linezolid combination gave a blended response, inducing transcriptional profiles "intermediate" between either drug. In the 3-drug BPaL regimen, the addition of both pretomanid and linezolid to bedaquiline yielded a greater transcriptional response than expected based on pairwise results. This work demonstrates that physiologic perturbations induced by a single drug may be modified in complex ways when drugs are combined. This establishes proof of concept that SEARCH-TB is a highly granular readout of drug interactions in vivo, providing information distinct from CFU burden and suggesting a future in which regimen selection is informed by in vivo molecular measures of Mtb physiology.

genomics↗

bioRxiv2025

Physiologic recovery of Mycobacterium tuberculosis from drug injury: A molecular study of post antibiotic effect in mice

Post-antibiotic effect (PAE) describes the delayed recovery of bacteria following antibiotic exposure. PAE is thought to underlie tuberculosis (TB) treatment forgiveness, i.e. the capacity of regimens to tolerate non-adherence. The basis of PAE in Mycobacterium tuberculosis (Mtb) remains poorly understood, partly because PAE has conventionally been measured based on change in Mtb burden in vitro rather than change in Mtb physiology in vivo. We investigated Mtb physiologic recovery in the BALB/c mouse model following sub-curative 2- and 4-week durations of the standard isoniazid, rifampin, pyrazinamide, ethambutol (HRZE) treatment. Measurement of rRNA synthesis via the RS ratio(R) and the entire transcriptome via SEARCH-TB elucidated the dynamics of physiologic recovery. Mtb burden did not increase over 28 days of drug-free post-treatment recovery, indicating prolonged PAE in vivo. The RS ratio indicated that Mtb ribosomal RNA synthesis resumed within four days of treatment interruption. However, transcriptional changes indicative of metabolic reactivation were delayed for over two weeks. Processes critical for replication, including expression of genes involved in protein and cell wall synthesis, remained suppressed throughout 28 days post-treatment. Longer treatment induced more extensive physiologic perturbation and was associated with slower and less complete recovery. Expression of processes that are typically induced by environmental stress (e.g., DosR regulon, universal stress proteins, and heat shock proteins) exhibited the reverse, decreasing during drug treatment and rising during recovery. These findings provide a new basis for understanding PAE based on drug-induced injury and physiologic recovery. Following relatively short durations of HRZE, physiologic recovery of Mtb was a slow, sequential and incomplete process in vivo. Our observation that longer treatment resulted in even slower recovery suggests that Mtb may progressively lose capacity to recover. This work establishes a tractable experimental framework for quantifying the forgiveness of new TB treatment regimens in vivo.

microbiology↗

bioRxiv2024

Emergence of antibiotic-specific Mycobacterium tuberculosis phenotypes during prolonged treatment of mice

A major challenge in tuberculosis (TB) therapeutics is that antibiotic exposure leads to changes in the physiologic state of M. tuberculosis (Mtb) which may enable the pathogen to withstand treatment. While antibiotic-treated Mtb have been evaluated in short-term in vitro experiments, it is unclear if and how long-term in vivo treatment with diverse antibiotics with varying treatment-shortening activity (sterilizing activity) affect Mtb physiologic states differently. Here, we used SEARCH-TB, a pathogen-targeted RNA-sequencing platform, to characterize the Mtb transcriptome in the BALB/c high-dose aerosol infection mouse model following 4-week treatment with three sterilizing and three non-sterilizing antibiotics. Certain transcriptional changes were concordant among most antibiotics, including decreased expression of genes associated with protein synthesis and metabolism, and the induction of certain genes associated with stress responses. However, the magnitude of this concordant response differed between antibiotics. Sterilizing antibiotics rifampin, pyrazinamide, and bedaquiline generated a more quiescent Mtb state than did non-sterilizing antibiotics isoniazid, ethambutol, and streptomycin, as indicated by decreased expression of genes associated with translation, transcription, secretion of immunogenic proteins, metabolism, and cell wall synthesis. Additionally, we identified distinguishing transcriptional effects specific to each antibiotic, indicating that different mechanisms of action induce distinct patterns of cellular injury. In addition to elucidating Mtb physiologic changes associated with antibiotic stress, this study demonstrates the value of SEARCH-TB as a highly granular pharmacodynamic assay that reveals antibiotic effects that are not apparent based on culture alone.

genomics↗

bioRxiv2023

Transcriptional adaptation of drug-tolerant Mycobacterium tuberculosis in mice

Transcriptome evaluation of Mycobacterium tuberculosis in the lungs of laboratory animals during long-term treatment has been limited by extremely low abundance of bacterial mRNA relative to eukaryotic RNA. Here we report a targeted amplification RNA sequencing method called SEARCH-TB. After confirming that SEARCH-TB recapitulates conventional RNA-seq in vitro, we applied SEARCH-TB to Mycobacterium tuberculosis-infected BALB/c mice treated for up to 28 days with the global standard isoniazid, rifampin, pyrazinamide, and ethambutol regimen. We compared results in mice with 8-day exposure to the same regimen in vitro. After treatment of mice for 28 days, SEARCH-TB suggested broad suppression of genes associated with bacterial growth, transcription, translation, synthesis of rRNA proteins and immunogenic secretory peptides. Adaptation of drug-stressed Mycobacterium tuberculosis appeared to include a metabolic transition from ATP-maximizing respiration towards lower-efficiency pathways, modification and recycling of cell wall components, large-scale regulatory reprogramming, and reconfiguration of efflux pumps expression. Despite markedly different expression at pre-treatment baseline, murine and in vitro samples had broadly similar transcriptional change during treatment. The differences observed likely indicate the importance of immunity and pharmacokinetics in the mouse. By elucidating the long-term effect of tuberculosis treatment on bacterial cellular processes in vivo, SEARCH-TB represents a highly granular pharmacodynamic monitoring tool with potential to enhance evaluation of new regimens and thereby accelerate progress towards a new generation of more effective tuberculosis treatment.

microbiology↗

bioRxiv2023

Lung microenvironments harbor Mycobacterium tuberculosis phenotypes with distinct treatment responses

Tuberculosis lung lesions are complex and harbor heterogeneous microenvironments that influence antibiotic effectiveness. Major strides have been made recently in understanding drug pharmacokinetics in pulmonary lesions, but the bacterial phenotypes that arise under these conditions and their contribution to drug tolerance is poorly understood. A pharmacodynamic marker called the RS ratio quantifies ongoing rRNA synthesis based on the abundance of newly-synthesized precursor rRNA relative to mature structural rRNA. Application of the RS ratio in the C3HeB/FeJ mouse model demonstrated that Mycobacterium tuberculosis populations residing in different tissue microenvironments are phenotypically distinct and respond differently to drug treatment with rifampin, isoniazid or bedaquiline. This work provides a foundational basis required to address how anatomic and pathologic microenvironmental niches may contribute to the long treatment duration and drug tolerance during treatment of human tuberculosis.

microbiology↗

bioRxiv2021

Combination of Mycobacterium tuberculosis RS ratio and CFU improves the ability of murine efficacy experiments to distinguish between drug treatments

Murine tuberculosis drug efficacy studies have historically monitored bacterial burden based on colony forming units of M. tuberculosis in lung homogenate. In an alternative approach, a recently described molecular pharmacodynamic marker called the RS ratio quantifies drug effect on a fundamental cellular process: ongoing ribosomal RNA synthesis. Here we evaluated the ability of different pharmacodynamic markers to distinguish between treatments in three BALB/c mouse experiments at two institutions. We confirmed that different pharmacodynamic markers measure distinct biological responses. We found that a combination of pharmacodynamic markers distinguishes between treatments better than any single marker. The combination of the RS ratio with colony forming units showed the greatest ability to recapitulate the rank order of regimen treatment-shortening activity, providing proof of concept that simultaneous assessment of pharmacodynamic markers measuring different properties will enhance insight gained from animal models and accelerate development of new combination regimens. These results suggest potential for a new era in which antimicrobial therapies are evaluated not only on culture-based measures of bacterial burden but also on molecular assays that indicate how drugs impact the physiological state of the pathogen.

microbiology↗

bioRxiv2019

Protein kinase B controls Mycobacterium tuberculosis growth via phosphorylation of the global transcriptional regulator Lsr2

Mycobacterium tuberculosis is able to persist in the body through months of multi-drug therapy. Mycobacteria possess a wide range of regulatory proteins, including the essential protein kinase B (PknB), that control transitions between growth states. Here, we establish that depletion of PknB in replicating M. tuberculosis results in transcriptional adaptations that implicate the DNA-binding protein Lsr2 in coordinating these changes. We show that Lsr2 is phosphorylated by PknB, and that phosphorylation of Lsr2 at threonine 112 is important for M. tuberculosis growth and survival under hypoxic conditions. Fluorescence anisotropy and electrophoretic mobility shift assays demonstrate that phosphorylation reduces Lsr2 binding to DNA, and ChIP-sequencing confirms increased DNA binding of a phosphoablative (T112A) Lsr2 mutant in M. tuberculosis. Altered expression of target genes in T112A Lsr2 compared to wild type Lsr2 M. tuberculosis offers further evidence that phosphorylation mediates expression of the Lsr2 regulon. Structural studies reveal increased dynamics of the Lsr2 DNA binding domain from a T112D phosphomimetic Lsr2 mutant, providing a molecular basis for decreased DNA binding by phosphorylated Lsr2. Our findings suggest that, the essential protein kinase, PknB controls M. tuberculosis growth and adaptations to the changing host environment by phosphorylating the global transcriptional regulator Lsr2.

microbiology↗