Metabologenomics reveals strain-level genetic and chemical diversity of Microcystis secondary metabolism.

Microcystis spp. are renowned for producing the hepatotoxin microcystin in freshwater cyanobacterial harmful algal blooms around the world, threatening drinking water supplies and public and environmental health. However, Microcystis genomes also harbor numerous biosynthetic gene clusters (BGCs) encoding the biosynthesis of other secondary metabolites, including many with toxic properties. Most of these BGCs are uncharacterized and currently lack links to biosynthesis products. However, recent field studies show that many of these BGCs are abundant and transcriptionally active in natural communities, suggesting potentially important yet unknown roles in bloom ecology and water quality. Here, we analyzed 21 xenic Microcystis cultures isolated from western Lake Erie to investigate the diversity of the biosynthetic potential of this genus. Through metabologenomic and in silico approaches, we show that these Microcystis strains contain variable BGCs, previously observed in natural populations, and encode distinct metabolomes across cultures. Additionally, we find that the majority of metabolites and gene clusters are uncharacterized, highlighting our limited understanding of the chemical repertoire of Microcystis spp. Due to the complex metabolomes observed in culture, which contain a wealth of diverse congeners as well as unknown metabolites, these results underscore the need to deeply explore and identify secondary metabolites produced by Microcystis beyond microcystins to assess their impacts on human and environmental health.IMPORTANCEThe genus Microcystis forms dense cyanobacterial harmful algal blooms (cyanoHABs) and can produce the toxin microcystin, which has been responsible for drinking water crises around the world. While microcystins are of great concern, Microcystis also produces an abundance of other secondary metabolites that may be of interest due to their potential for toxicity, ecological importance, or pharmaceutical applications. In this study, we combine genomic and metabolomic approaches to study the genes responsible for the biosynthesis of secondary metabolites as well as the chemical diversity of produced metabolites in Microcystis strains from the Western Lake Erie Culture Collection. This unique collection comprises Microcystis strains that were directly isolated from western Lake Erie, which experiences substantial cyanoHAB events annually and has had negative impacts on drinking water, tourism, and industry.

Discovering type I cis-AT polyketides through computational mass spectrometry and genome mining with Seq2PKS.

Type 1 polyketides are a major class of natural products used as antiviral, antibiotic, antifungal, antiparasitic, immunosuppressive, and antitumor drugs. Analysis of public microbial genomes leads to the discovery of over sixty thousand type 1 polyketide gene clusters. However, the molecular products of only about a hundred of these clusters are characterized, leaving most metabolites unknown. Characterizing polyketides relies on bioactivity-guided purification, which is expensive and time-consuming. To address this, we present Seq2PKS, a machine learning algorithm that predicts chemical structures derived from Type 1 polyketide synthases. Seq2PKS predicts numerous putative structures for each gene cluster to enhance accuracy. The correct structure is identified using a variable mass spectral database search. Benchmarks show that Seq2PKS outperforms existing methods. Applying Seq2PKS to Actinobacteria datasets, we discover biosynthetic gene clusters for monazomycin, oasomycin A, and 2-aminobenzamide-actiphenol.

N-to-S Acyl Transfer as an Enabling Strategy in Asymmetric and Chemoenzymatic Synthesis.

The observation of thioester-mediated acyl transfer processes in nature has inspired the development of novel protein synthesis and functionalization methodologies. The chemoselective transfer of an acyl group from S-to-N is the basis of several powerful ligation strategies. In this work, we sought to apply the reverse process, the transfer of an acyl group from N-to-S, as a method to convert stable chiral amides into more reactive thioesters. To this end, we developed a novel cysteine-derived oxazolidinone that serves as both a chiral imide auxiliary and an acyl transfer agent. This auxiliary combines the desirable features of rigid chiral imides as templates for asymmetric transformations with the synthetic applicability of thioesters. We demonstrate that the auxiliary can be applied in a range of highly selective asymmetric transformations. Subsequent intramolecular N-to-S acyl transfer of the chiral product and in situ trapping of the resulting thioester provides access to diverse carboxylic acid derivatives under mild conditions. The oxazolidinone thioester products can also be isolated and used in Pd-mediated transformations to furnish highly valuable chiral scaffolds, such as noncanonical amino acids, cyclic ketones, tetrahydropyrones, and dihydroquinolinones. Finally, we demonstrate that the oxazolidinone thioesters can also serve as a surrogate for SNAC-thioesters, enabling their seamless use as non-native substrates in biocatalytic transformations.

Single Leg Hop Performance After Anterior Cruciate Ligament Reconstruction: Ready for Landing but Cleared for Take-Off?

CONTEXT: While the landing phases of the single-leg hop for distance (SLHD) are commonly assessed, limited work reflects how the take-off phase influences hop performance in patients with anterior cruciate ligament reconstruction (ACLR). OBJECTIVE: To compare trunk and lower extremity biomechanics between individuals with ACLR and matched uninjured controls during take-off of the SLHD. DESIGN: Cross-sectional study design. SETTING: Laboratory setting. PATIENTS OR OTHER PARTICIPANTS: 16 individuals with ACLR and 18 uninjured controls. MAIN OUTCOME MEASURES: Normalized quadriceps isokinetic torque, hop distance, and respective limb symmetry indices (LSI) were collected for each participant. Sagittal and frontal kinematics and kinetics of the trunk, hip, knee, and ankle, as well as vertical and horizontal ground reaction forces (GRF) were recorded for loading and propulsion of the take-off phase of the SLHD. RESULTS: Those with ACLR had weaker quadriceps peak torque in the involved limb (p=0.001) and greater strength asymmetry (p<0.001) compared to controls. Normalized hop distance was not statistically different between limbs or between groups (p>0.05) and hop distance symmetry was not different between groups (p>0.05). During loading, the involved limb demonstrated lesser knee flexion angles (p=0.030) and knee power (p=0.007) compared to the uninvolved limb, and lesser knee extension moments compared to the uninvolved limb (p=0.001) and controls (p=0.005). During propulsion, the involved limb demonstrated lesser knee extension moment (p=0.027), knee power (p=0.010), knee (p=0.032) and ankle work (p=0.032), anterior- posterior GRF (p=0.047), and greater knee (p=0.016) abduction excursions compared to the uninvolved limb. CONCLUSIONS: Between-limb differences in SLHD take-off suggest a knee underloading strategy in the involved limb. These results provide further evidence that distance covered during SLHD assessment can overestimate function and fail to identify compensatory biomechanical strategies.

Interpersonal Coordination between Female Soccer Players: Leader-Follower Roles within a Collision-Avoidance Task.

Background/Purpose: Return to sport decision-making may be improved by assessing an athlete's ability to coordinate movement with opponents in sport. The purpose was to investigate whether previous injuries associated with female soccer players' interpersonal coordination during a collision avoidance task. The authors hypothesized that external perturbations would disrupt the strength and stability of coordinated movement, and that individuals with a history of injury would be less likely to recover coordinated movement. Study Design: Cross-Sectional. Methods: Nine female athletes with a history of lower extremity injuries and nine without injuries were paired into dyads. Each dyad completed twenty trials of an externally paced collision-avoidance agility task with an unanticipated perturbation. Participant trajectories were digitized and analyzed using cross-recurrence quantification analysis (CRQA) to determine the strength and stability of interpersonal coordination dynamics. Trials in which participants with injury history assumed leader or follower roles within each dyad were then used to study how dyadic coordination varied across task stages (early, perturbation, and late) using linear mixed effect models. Cohen's d effect sizes were calculated to demonstrate magnitude of differences. In exploratory analysis, psychological readiness (i.e., self-reported knee functioning, fear of injury, and risk-taking propensity) was evaluated for their association with leader-follower status. Results: Perturbation disrupted the strength (R2=0.65, p<0.001, early=49.7±1.7, perturbation=41.1±1.7, d=0.39) and stability (R2=0.71, p < 0.001, early=65.0±1.6, perturbation=58.0±1.7, d=0.38) of interpersonal coordination regardless of leader-follower status. Individuals with injury history failed to restore coordination after the perturbation compared to control participants (injury=44.2.0±2.1, control=50.8±2.6, d=0.39). Neither demographic nor psychological measures were associated with leader-follower roles (B=0.039, p=0.224). Conclusion: Individuals with a history of lower extremity injury may have a diminished ability to adapt interpersonal coordination to perturbations, possibly contributing to a higher risk of re-injury. Level of Evidence: 3.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Nonalcoholic fatty liver disease, recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD. Despite its discovery 20 y ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.

Revealing the dynamics and mechanisms of bacterial interactions in cheese production with metabolic modelling.

Cheese taste and flavour properties result from complex metabolic processes occurring in microbial communities. A deeper understanding of such mechanisms makes it possible to improve both industrial production processes and end-product quality through the design of microbial consortia. In this work, we caracterise the metabolism of a three-species community consisting of Lactococcus lactis, Lactobacillus plantarum and Propionibacterium freudenreichii during a seven-week cheese production process. Using genome-scale metabolic models and omics data integration, we modeled and calibrated individual dynamics using monoculture experiments, and coupled these models to capture the metabolism of the community. This model accurately predicts the dynamics of the community, enlightening the contribution of each microbial species to organoleptic compound production. Further metabolic exploration revealed additional possible interactions between the bacterial species. This work provides a methodological framework for the prediction of community-wide metabolism and highlights the added value of dynamic metabolic modeling for the comprehension of fermented food processes.

Structure-Activity Relationships of Natural and Semisynthetic Plecomacrolides Suggest Distinct Pathways for HIV-1 Immune Evasion and Vacuolar ATPase-Dependent Lysosomal Acidification.

The human immunodeficiency virus (HIV)-encoded accessory protein Nef enhances pathogenicity by reducing major histocompatibility complex I (MHC-I) cell surface expression, protecting HIV-infected cells from immune recognition. Nef-dependent downmodulation of MHC-I can be reversed by subnanomolar concentrations of concanamycin A (1), a well-known inhibitor of vacuolar ATPase, at concentrations below those that interfere with lysosomal acidification or degradation. We conducted a structure-activity relationship study that assessed 76 compounds for Nef inhibition, 24 and 72 h viability, and lysosomal neutralization in Nef-expressing primary T cells. This analysis demonstrated that the most potent compounds were natural concanamycins and their derivatives. Comparison against a set of new, semisynthetic concanamycins revealed that substituents at C-8 and acylation of C-9 significantly affected Nef potency, target cell viability, and lysosomal neutralization. These findings provide important progress toward understanding the mechanism of action of these compounds and the identification of an advanced lead anti-HIV Nef inhibitory compound.

Engagement in Meaningful Activity Mediates the Relationship Between Stressful Life Events and Functional Resilience.

Resilience during stressful life events is a priority for administering the most client-centered care as possible. Occupational therapy practitioners have the unique opportunity to support resilience through promoting meaningful participation. The current study aims to understand the associations between meaningful activity engagement, resilience, and stressful life events. We specifically focused on answering if meaningful participation mediates the relationship between stressful life events and resilience. 492 participants from a non-clinical convenience sample of Amazon's MTurk completed the study. Participants completed an online survey and reported their experiences of stressful life events, resilience, well-being, and meaningful participation. We used SPSS and PROCESS to analyze our data. Stressful event severity and resilience were inversely related. When accounting for the effect of meaningful participation, the relationship became non-significant, indicating evidence of mediation. Implications: Focusing on measuring meaningful participation may be worth studying in further research.

Engagement in Meaningful Activity Mediates the Relationship between Stressful Life Events and Functional ResilienceSupporting recovery both psychologically and physically after a stressful life event is an important aspect of providing client-centered care. By supporting patients' and clients' engagement in meaningful participation, therapists can promote resilient outcomes for all patients and clients. The current study explored the relationship between resilience and meaningful activity participation during stressful life events. The sample was comprised of a convenience sample of 492 participants who were recruited from the online crowdsourcing platform MTurk. The results of the study include that when the stressful life experience was more severe for an individual, they reported lower resilience scores. When meaningful activity participation was accounted for, the relationship between stressful life event severity and resilience became non-existent. The results of the study suggest the importance of using and measuring meaningful activity when supporting recovery after an stressful and adverse life event.

A Portable, Neurostimulation-Integrated, Force Measurement Platform for the Clinical Assessment of Plantarflexor Central Drive.

Plantarflexor central drive is a promising biomarker of neuromotor impairment; however, routine clinical assessment is hindered by the unavailability of force measurement systems with integrated neurostimulation capabilities. In this study, we evaluate the accuracy of a portable, neurostimulation-integrated, plantarflexor force measurement system we developed to facilitate the assessment of plantarflexor neuromotor function in clinical settings. Two experiments were conducted with the Central Drive System (CEDRS). To evaluate accuracy, experiment #1 included 16 neurotypical adults and used intra-class correlation (ICC2,1) to test agreement of plantarflexor strength capacity measured with CEDRS versus a stationary dynamometer. To evaluate validity, experiment #2 added 26 individuals with post-stroke hemiparesis and used one-way ANOVAs to test for between-limb differences in CEDRS' measurements of plantarflexor neuromotor function, comparing neurotypical, non-paretic, and paretic limb measurements. The association between paretic plantarflexor neuromotor function and walking function outcomes derived from the six-minute walk test (6MWT) were also evaluated. CEDRS' measurements of plantarflexor neuromotor function showed high agreement with measurements made by the stationary dynamometer (ICC = 0.83, p < 0.001). CEDRS' measurements also showed the expected between-limb differences (p's < 0.001) in maximum voluntary strength (Neurotypical: 76.21 ± 13.84 ft-lbs., Non-paretic: 56.93 ± 17.75 ft-lbs., and Paretic: 31.51 ± 14.08 ft-lbs.), strength capacity (Neurotypical: 76.47 ± 13.59 ft-lbs., Non-paretic: 64.08 ± 14.50 ft-lbs., and Paretic: 44.55 ± 14.23 ft-lbs.), and central drive (Neurotypical: 88.73 ± 1.71%, Non-paretic: 73.66% ± 17.74%, and Paretic: 52.04% ± 20.22%). CEDRS-measured plantarflexor central drive was moderately correlated with 6MWT total distance (r = 0.69, p < 0.001) and distance-induced changes in speed (r = 0.61, p = 0.002). CEDRS is a clinician-operated, portable, neurostimulation-integrated force measurement platform that produces accurate measurements of plantarflexor neuromotor function that are associated with post-stroke walking ability.

Community-scale models of microbiomes: Articulating metabolic modelling and metagenome sequencing.

Building models is essential for understanding the functions and dynamics of microbial communities. Metabolic models built on genome-scale metabolic network reconstructions (GENREs) are especially relevant as a means to decipher the complex interactions occurring among species. Model reconstruction increasingly relies on metagenomics, which permits direct characterisation of naturally occurring communities that may contain organisms that cannot be isolated or cultured. In this review, we provide an overview of the field of metabolic modelling and its increasing reliance on and synergy with metagenomics and bioinformatics. We survey the means of assigning functions and reconstructing metabolic networks from (meta-)genomes, and present the variety and mathematical fundamentals of metabolic models that foster the understanding of microbial dynamics. We emphasise the characterisation of interactions and the scaling of model construction to large communities, two important bottlenecks in the applicability of these models. We give an overview of the current state of the art in metagenome sequencing and bioinformatics analysis, focusing on the reconstruction of genomes in microbial communities. Metagenomics benefits tremendously from third-generation sequencing, and we discuss the opportunities of long-read sequencing, strain-level characterisation and eukaryotic metagenomics. We aim at providing algorithmic and mathematical support, together with tool and application resources, that permit bridging the gap between metagenomics and metabolic modelling.

Corticomuscular cross-recurrence analysis reveals between-limb differences in motor control among individuals with ACL reconstruction.

Surgical reconstruction of the anterior cruciate ligament (ACL) and subsequent physical therapy can help athletes return to competition; however, re-injury rates remain disproportionately high due, in part, to lingering biomechanical and neurological factors that are not fully addressed during rehabilitation. Prior reports indicate that individuals exhibit altered electrical activity in both brain and muscle after ACL reconstruction (ACLR). In this investigation, we aimed to extend existing approaches by introducing a novel non-linear analysis of corticomuscular dynamics, which does not assume oscillatory coupling between brain and muscle: Corticomuscular cross-recurrence analysis (CM-cRQA). Our findings indicate that corticomuscular dynamics vary significantly between involved (injured) and uninvolved legs of participants with ACLR during voluntary isometric contractions between the brain and both the vastus medialis and lateralis. This finding points to a potential lingering neural deficit underlying re-injury for athletes after surgical reconstruction, namely the dynamical structure of neuromuscular (brain to quad muscle) coordination, which is significantly asymmetric, between limbs, in those who have ACLR.

Molecular detection of pre-ribosomal RNAs of Mycobacterium bovis bacille Calmette-Guérin and Mycobacterium tuberculosis to enhance pre-clinical tuberculosis drug and vaccine development.

Efforts are underway globally to develop effective vaccines and drugs against M. tuberculosis (Mtb) to reduce the morbidity and mortality of tuberculosis. Improving detection of slow-growing mycobacteria could simplify and accelerate efficacy studies of vaccines and drugs in animal models and human clinical trials. Here, a real-time reverse transcription PCR (RT-PCR) assay was developed to detect pre-ribosomal RNA (pre-rRNA) of Mycobacterium bovis bacille Calmette-Guérin (BCG) and Mtb. This pre-rRNA biomarker is indicative of bacterial viability. In two different mouse models, the presence of pre-rRNA from BCG and Mtb in ex vivo tissues showed excellent agreement with slower culture-based colony-forming unit assays. The addition of a brief nutritional stimulation prior to molecular viability testing further differentiated viable but dormant mycobacteria from dead mycobacteria. This research has set the stage to evaluate pre-rRNA as a BCG and/or Mtb infection biomarker in future drug and vaccine clinical studies.

The fatty liver disease-causing protein PNPLA3-I148M alters lipid droplet-Golgi dynamics.

Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic dysfunction-associated steatotic liver disease (MASLD), is a progressive metabolic disorder that begins with aberrant triglyceride accumulation in the liver and can lead to cirrhosis and cancer. A common variant in the gene PNPLA3, encoding the protein PNPLA3-I148M, is the strongest known genetic risk factor for MASLD to date. Despite its discovery twenty years ago, the function of PNPLA3, and now the role of PNPLA3-I148M, remain unclear. In this study, we sought to dissect the biogenesis of PNPLA3 and PNPLA3-I148M and characterize changes induced by endogenous expression of the disease-causing variant. Contrary to bioinformatic predictions and prior studies with overexpressed proteins, we demonstrate here that PNPLA3 and PNPLA3-I148M are not endoplasmic reticulum-resident transmembrane proteins. To identify their intracellular associations, we generated a paired set of isogenic human hepatoma cells expressing PNPLA3 and PNPLA3-I148M at endogenous levels. Both proteins were enriched in lipid droplet, Golgi, and endosomal fractions. Purified PNPLA3 and PNPLA3-I148M proteins associated with phosphoinositides commonly found in these compartments. Despite a similar fractionation pattern as the wild-type variant, PNPLA3-I148M induced morphological changes in the Golgi apparatus, including increased lipid droplet-Golgi contact sites, which were also observed in I148M-expressing primary human patient hepatocytes. In addition to lipid droplet accumulation, PNPLA3-I148M expression caused significant proteomic and transcriptomic changes that resembled all stages of liver disease. Cumulatively, we validate an endogenous human cellular system for investigating PNPLA3-I148M biology and identify the Golgi apparatus as a central hub of PNPLA3-I148M-driven cellular change.

Neural drive and motor unit characteristics after anterior cruciate ligament reconstruction: implications for quadriceps weakness.

Purpose: The purpose of this investigation was to compare the quality of neural drive and recruited quadriceps motor units' (MU) action potential amplitude (MUAPAMP) and discharge rate (mean firing rate (MFR)) relative to recruitment threshold (RT) between individuals with anterior cruciate ligament reconstruction (ACLR) and controls. Methods: Fourteen individuals with ACLR and 13 matched controls performed trapezoidal knee extensor contractions at 30%, 50%, 70%, and 100% of their maximal voluntary isometric contraction (MVIC). Decomposition electromyography (dEMG) and torque were recorded concurrently. The Hoffmann reflex (H-reflex) and central activation ratio (CAR) were acquired bilaterally to detail the proportion of MU pool available and volitionally activated. We examined MUAPAMP-RT and MFR-RT relationships with linear regression and extracted the regression line slope, y-intercept, and RT range for each contraction. Linear mixed effect modelling used to analyze the effect of group and limb on regression line slope and RT range. Results: Individuals with ACLR demonstrated lower MVIC torque in the involved limb compared to uninvolved limb. There were no differences in H-reflex or CAR between groups or limbs. The ACLR involved limb demonstrated smaller mass-normalized RT range and slower MU firing rates at high contraction intensities (70% and 100% MVIC) compared to uninvolved and control limbs. The ACLR involved limb also demonstrated larger MU action potentials in the VM compared to the contralateral limb. These differences were largely attenuated with relative RT normalization. Conclusions: These results suggest that persistent strength deficits following ACLR may be attributable to a diminished quadriceps motor neuron pool and inability to upregulate the firing rate of recruited MUs.

Structure of a modular polyketide synthase reducing region.

The chemical scaffolds of numerous therapeutics are polyketide natural products, many formed by bacterial modular polyketide synthases (PKS). The large and flexible dimeric PKS modules have distinct extension and reducing regions. Structures are known for all individual enzyme domains and several extension regions. Here, we report the structure of the full reducing region from a modular PKS, the ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains of module 5 of the juvenimicin PKS. The modular PKS-reducing region has a different architecture than the homologous fatty acid synthase (FAS) and iterative PKS systems in its arrangement of domains and dimer interface. The structure reveals a critical role for linker peptides in the domain interfaces, leading to discovery of key differences in KR domains dependent on module composition. Finally, our studies provide insight into the mechanism underlying modular PKS intermediate shuttling by carrier protein (ACP) domains.

Quantitative measurement of the requirement of diverse protein degradation pathways in MHC class I peptide presentation.

Peptides from degradation of intracellular proteins are continuously displayed by major histocompatibility complex (MHC) class I. To better understand origins of these peptides, we performed a comprehensive census of the class I peptide repertoire in the presence and absence of ubiquitin-proteasome system (UPS) activity upon developing optimized methodology to enrich for and quantify these peptides. Whereas most class I peptides are dependent on the UPS for their generation, a surprising 30%, enriched in peptides of mitochondrial origin, appears independent of the UPS. A further ~10% of peptides were found to be dependent on the proteasome but independent of ubiquitination for their generation. Notably, clinically achievable partial inhibition of the proteasome resulted in display of atypical peptides. Our results suggest that generation of MHC class I•peptide complexes is more complex than previously recognized, with UPS-dependent and UPS-independent components; paradoxically, alternative protein degradation pathways also generate class I peptides when canonical pathways are impaired.

A Novel Tool to Identify Bactericidal Compounds against Vulnerable Targets in Drug-Tolerant M. tuberculosis found in Caseum.

Caseous necrosis is a hallmark of tuberculosis (TB) pathology and creates a niche for drug-tolerant persisters within the host. Cavitary TB and high bacterial burden in caseum require longer treatment duration. An in vitro model that recapitulates the major features of Mycobacterium tuberculosis (Mtb) in caseum would accelerate the identification of compounds with treatment-shortening potential. We have developed a caseum surrogate model consisting of lysed and denatured foamy macrophages. Upon inoculation of Mtb from replicating cultures, the pathogen adapts to the lipid-rich matrix and gradually adopts a nonreplicating state. We determined that the lipid composition of ex vivo caseum and the surrogate matrix are similar. We also observed that Mtb in caseum surrogate accumulates intracellular lipophilic inclusions (ILI), a distinctive characteristic of quiescent and drug-tolerant Mtb. Expression profiling of a representative gene subset revealed common signatures between the models. Comparison of Mtb drug susceptibility in caseum and caseum surrogate revealed that both populations are similarly tolerant to a panel of TB drugs. By screening drug candidates in the surrogate model, we determined that the bedaquiline analogs TBAJ876 and TBAJ587, currently in clinical development, exhibit superior bactericidal against caseum-resident Mtb, both alone and as substitutions for bedaquiline in the bedaquiline-pretomanid-linezolid regimen approved for the treatment of multidrug-resistant TB. In summary, we have developed a physiologically relevant nonreplicating persistence model that reflects the distinct metabolic and drug-tolerant state of Mtb in caseum. IMPORTANCE M. tuberculosis (Mtb) within the caseous core of necrotic granulomas and cavities is extremely drug tolerant and presents a significant hurdle to treatment success and relapse prevention. Many in vitro models of nonreplicating persistence have been developed to characterize the physiologic and metabolic adaptations of Mtb and identify compounds active against this treatment-recalcitrant population. However, there is little consensus on their relevance to in vivo infection. Using lipid-laden macrophage lysates, we have designed and validated a surrogate matrix that closely mimics caseum and in which Mtb develops a phenotype similar to that of nonreplicating bacilli in vivo. The assay is well suited to screen for bactericidal compounds against caseum-resident Mtb in a medium-throughput format, allowing for reduced reliance on resource intensive animal models that present large necrotic lesions and cavities. Importantly, this approach will aid the identification of vulnerable targets in caseum Mtb and can accelerate the development of novel TB drugs with treatment-shortening potential.