Characterizing the impact of Enterococcus cecorum infection during late embryogenesis on disease progression, cecal microbiome composition, and early performance in broiler chickens.

Enterococcus cecorum (EC) has been associated with septicemia and early mortality in broiler chickens. There is limited research investigating the pathogenicity of EC field strains obtained from affected birds. The purpose of this study was to evaluate the effect of in-ovo administration into the amnion with different EC field isolates at d 18 of embryogenesis (DOE18). In Exp 1, 7 EC field isolates alone or in combination (EC1-EC3, EC4-EC5, EC6, and EC7) were selected based on phenotypic characteristics and evaluated at different concentrations (1 × 102, 1 × 104, and 1 × 106 CFU/200 µL/embryo) to assess the impact on early performance and macroscopic lesions. Three isolates (n = 3; EC2, EC5, EC7) were selected for additional evaluation based on the significant (P < 0.05) BWG reduction (d 0-21) compared to the negative control (NC) and the presence of macroscopic lesions observed during posting sessions at d 14 and d 21. An additional isolate associated with enterococcal spondylitis was included in Exp 2 (EC11B). Treatment groups for Exp 2 include: 1) NC, 2) EC2, 3) EC5, 4) EC7, and 5) EC11B (n = 90-120/embryos/group). Groups 2 to 5 were challenged at 1 × 102 CFU/200 µL/embryo by in-ovo injection into the amnion at DOE18. Chicks were placed in battery cages for the duration of the study (21 d), and pen weights were recorded at d 0, d 7, d 14, and d 21 to calculate average BW and BWG. At d 14 and d 21 posthatch, liver, spleen, free thoracic vertebrae (FTV), and femoral head (FH) were aseptically collected to enumerate Enterococcus spp. using Chromagar Orientation as the selective media. Cecal contents were collected at d 21 to evaluate the effect of EC challenge on the cecal microbiome composition. There was a significant (P < 0.05) reduction in BW at d 21, and BWG from d 14 to 21 and d 0 to 21, for EC7 and EC11B. Enterococcus cecorum was recovered from the FTV of all challenged groups at d 14 and d 21. The most representative lesions were pericarditis, hydropericardium, focal heart necrosis, and FH osteomyelitis. However, lesions were not uniform across challenged groups or ages (d 14 and d 21). Alpha diversity of the cecal contents was markedly lower in EC5 and EC11B compared to all treatment groups suggesting that EC exposure during late embryogenesis affect the cecal microbiome up to 21 d posthatch. Additionally, these results highlight the differences in pathogenicity of EC strains isolated from field cases and suggest that hatchery exposure to EC during late embryogenesis is a potential route of introduction into a flock.

Reducing face touching through haptic feedback: A treatment evaluation against fomite-mediated self-infection.

Fomite-mediated self-infection via face touching is an understudied transmission pathway for infectious diseases. We evaluated the effect of computer-mediated vibrotactile cues (presented through experimental bracelets located on one or both hands of the participant) on the frequency of face touching among eight healthy adults in the community. We conducted a treatment evaluation totaling over 25,000 min of video observation. The treatment was evaluated through a multiple-treatment design and hierarchical linear modeling. The one-bracelet intervention did not produce significantly lower levels of face touching across both hands, whereas the two-bracelet intervention did result in significantly lower face touching. The effect increased over repeated presentations of the two-bracelet intervention, with the second implementation producing, on average, 31 fewer face-touching percentual points relative to baseline levels. Dependent on the dynamics of fomite-mediated self-infection via face touching, treatment effects could be of public health significance. The implications for research and practice are discussed.

Enabling community input to improve equity in and access to translational research: The Community Coalition for Equity in Research.

Background: The COVID vaccine trials illustrated the critical need for the development of mechanisms to serve as a bridge between least advantaged communities and researchers. Such mechanisms would increase the number of studies that are designed with community needs and interests in mind, in ways that will close gaps rather than widen them. This paper reports on the creation of the Community Coalition for Equity in Research, a community-driven resource designed to build community capacity to provide researchers with credible and actionable input on study design and implementation and increase researchers' understanding of factors that influence community support of research. Methods and Results: We provide a description of the Coalition's structure and process and an evaluation of its first year of operation. Researchers rated their experience very positively and reported that the Coalition's review will improve their research. Coalition members reported high levels of satisfaction with their participation and the processes set up for them to engage with researchers. Members also largely agreed that their participation has value for their community, and that it has increased their interest in research and the likelihood that they would recommend research participation to others. Conclusions: The Coalition represents a model for increasing two-way engagement between researchers and the larger community. We are optimistic that the Coalition will continue to develop and grow into a vibrant entity that will bring value to both investigators and our local communities and will increase the consideration of equity as a foundational principle in all translational research.

Chemical-genetic interaction mapping links carbon metabolism and cell wall structure to tuberculosis drug efficacy.

Current chemotherapy against Mycobacterium tuberculosis (Mtb), an important human pathogen, requires a multidrug regimen lasting several months. While efforts have been made to optimize therapy by exploiting drug­drug synergies, testing new drug combinations in relevant host environments remains arduous. In particular, host environments profoundly affect the bacterial metabolic state and drug efficacy, limiting the accuracy of predictions based on in vitro assays alone. In this study, we utilized conditional Mtb knockdown mutants of essential genes as an experimentally tractable surrogate for drug treatment and probe the relationship between Mtb carbon metabolism and chemical­genetic interactions (CGIs). We examined the antitubercular drugs isoniazid, rifampicin, and moxifloxacin and found that CGIs are differentially responsive to the metabolic state, defining both environment-independent and -dependent interactions. Specifically, growth on the in vivo­relevant carbon source, cholesterol, reduced rifampicin efficacy by altering mycobacterial cell surface lipid composition. We report that a variety of perturbations in cell wall synthesis pathways restore rifampicin efficacy during growth on cholesterol, and that both environment-independent and cholesterol-dependent in vitro CGIs could be leveraged to enhance bacterial clearance in the mouse infection model. Our findings present an atlas of chemical­genetic­environmental interactions that can be used to optimize drug­drug interactions, as well as provide a framework for understanding in vitro correlates of in vivo efficacy.

Genetic models of latent tuberculosis in mice reveal differential influence of adaptive immunity.

Studying latent Mycobacterium tuberculosis (Mtb) infection has been limited by the lack of a suitable mouse model. We discovered that transient depletion of biotin protein ligase (BPL) and thioredoxin reductase (TrxB2) results in latent infections during which Mtb cannot be detected but that relapse in a subset of mice. The immune requirements for Mtb control during latency, and the frequency of relapse, were strikingly different depending on how latency was established. TrxB2 depletion resulted in a latent infection that required adaptive immunity for control and reactivated with high frequency, whereas latent infection after BPL depletion was independent of adaptive immunity and rarely reactivated. We identified immune signatures of T cells indicative of relapse and demonstrated that BCG vaccination failed to protect mice from TB relapse. These reproducible genetic latency models allow investigation of the host immunological determinants that control the latent state and offer opportunities to evaluate therapeutic strategies in settings that mimic aspects of latency and TB relapse in humans.

Genome-wide gene expression tuning reveals diverse vulnerabilities of M. tuberculosis.

Antibacterial agents target the products of essential genes but rarely achieve complete target inhibition. Thus, the all-or-none definition of essentiality afforded by traditional genetic approaches fails to discern the most attractive bacterial targets: those whose incomplete inhibition results in major fitness costs. In contrast, gene "vulnerability" is a continuous, quantifiable trait that relates the magnitude of gene inhibition to the effect on bacterial fitness. We developed a CRISPR interference-based functional genomics method to systematically titrate gene expression in Mycobacterium tuberculosis (Mtb) and monitor fitness outcomes. We identified highly vulnerable genes in various processes, including novel targets unexplored for drug discovery. Equally important, we identified invulnerable essential genes, potentially explaining failed drug discovery efforts. Comparison of vulnerability between the reference and a hypervirulent Mtb isolate revealed incomplete conservation of vulnerability and that differential vulnerability can predict differential antibacterial susceptibility. Our results quantitatively redefine essential bacterial processes and identify high-value targets for drug development.

TSH Level and Risk of Malignancy in Patients with Bethesda Category IV Thyroid Nodules.

Fine needle aspiration biopsy does not permit to distinguish between benign and malignant follicular thyroid lesions (category IV in the Bethesda Cytopathology System). Some reports have suggested an association between increased serum TSH levels and thyroid cancer, so the aim of this study was to investigate the association between TSH levels and malignancy in patients with follicular thyroid nodules. Therefore, we conducted a retrospective study of all subjects who underwent surgical treatment for Bethesda IV thyroid nodules in a single center (years 2012-2017). A total of 127 patients were analyzed, and malignancy was present in 38.6% of the patients. Using ROC analysis, the best TSH cut-off point to differentiate benign from malignant disease was 2.1 mU/l and the age cut-off with better sensitivity and specificity was 47 years. The proportion of subjects with TSH ≥ 2.1 mU/l was greater among subjects with cancer than in those with benign diseases (65.3 vs 44.9%, P = 0.029). The concurrence of both cut-off points (TSH ≥ 2.1 mU/l and age ≥ 47 years) showed a higher diagnostic accuracy than either of the two variables separately. Therefore, the present study supports an association between serum concentrations of TSH and risk of malignancy among subjects with Bethesda IV thyroid nodules. TSH levels could modify the diagnostic and therapeutic approach of patients with Bethesda IV nodules.

Tissue Distribution of Doxycycline in Animal Models of Tuberculosis.

Doxycycline, an FDA-approved tetracycline, is used in tuberculosis in vivo models for the temporal control of mycobacterial gene expression. In these models, animals are infected with recombinant Mycobacterium tuberculosis carrying genes of interest under transcriptional control of the doxycycline-responsive TetR-tetO unit. To minimize fluctuations of plasma levels, doxycycline is usually administered in the diet. However, tissue penetration studies to identify the minimum doxycycline content in food achieving complete repression of TetR-controlled genes in tuberculosis (TB)-infected organs and lesions have not been conducted. Here, we first determined the tetracycline concentrations required to achieve silencing of M. tuberculosis target genes in vitro Next, we measured doxycycline concentrations in plasma, major organs, and lung lesions in TB-infected mice and rabbits and compared these values to silencing concentrations measured in vitro We found that 2,000 ppm doxycycline supplemented in mouse and rabbit feed is sufficient to reach target concentrations in TB lesions. In rabbit chow, the calcium content had to be reduced 5-fold to minimize chelation of doxycycline and deliver adequate oral bioavailability. Clearance kinetics from major organs and lung lesions revealed that doxycycline levels fall below concentrations that repress tet promoters within 7 to 14 days after doxycycline is removed from the diet. In summary, we have shown that 2,000 ppm doxycycline supplemented in standard mouse diet and in low-calcium rabbit diet delivers concentrations adequate to achieve full repression of tet promoters in infected tissues of mice and rabbits.

Large-scale chemical-genetics yields new M. tuberculosis inhibitor classes.

New antibiotics are needed to combat rising levels of resistance, with new Mycobacterium tuberculosis (Mtb) drugs having the highest priority. However, conventional whole-cell and biochemical antibiotic screens have failed. Here we develop a strategy termed PROSPECT (primary screening of strains to prioritize expanded chemistry and targets), in which we screen compounds against pools of strains depleted of essential bacterial targets. We engineered strains that target 474 essential Mtb genes and screened pools of 100-150 strains against activity-enriched and unbiased compound libraries, probing more than 8.5 million chemical-genetic interactions. Primary screens identified over tenfold more hits than screening wild-type Mtb alone, with chemical-genetic interactions providing immediate, direct target insights. We identified over 40 compounds that target DNA gyrase, the cell wall, tryptophan, folate biosynthesis and RNA polymerase, as well as inhibitors that target EfpA. Chemical optimization yielded EfpA inhibitors with potent wild-type activity, thus demonstrating the ability of PROSPECT to yield inhibitors against targets that would have eluded conventional drug discovery.

Identification of Enolase as the Target of 2-Aminothiazoles in Mycobacterium tuberculosis.

Tuberculosis is a massive global burden and Mycobacterium tuberculosis is increasingly resistant to first- and second-line drugs. There is an acute need for new anti-mycobacterial drugs with novel targets. We previously evaluated a series of 2-aminothiazoles with activity against Mycobacterium tuberculosis. In this study, we identify the glycolytic enzyme enolase as the target of these molecules using pull down studies. We demonstrate that modulation of the level of enolase expression affects sensitivity to 2-aminothiazoles; increased expression leads to resistance while decreased protein levels increase sensitivity. Exposure to 2-aminothiazoles results in increased levels of metabolites preceding the action of enolase in the glycolytic pathway and decreased ATP levels. We demonstrate that 2-aminothiazoles inhibit the activity of the human α-enolase, which could also account for the cytotoxicity of some of those molecules. If selectivity for the bacterial enzyme over the human enzyme could be achieved, enolase would represent an attractive target for M. tuberculosis drug discovery and development efforts.

Glyoxylate detoxification is an essential function of malate synthase required for carbon assimilation in Mycobacterium tuberculosis.

The glyoxylate shunt is a metabolic pathway of bacteria, fungi, and plants used to assimilate even-chain fatty acids (FAs) and has been implicated in persistence of Mycobacterium tuberculosis (Mtb). Recent work, however, showed that the first enzyme of the glyoxylate shunt, isocitrate lyase (ICL), may mediate survival of Mtb during the acute and chronic phases of infection in mice through physiologic functions apart from fatty acid metabolism. Here, we report that malate synthase (MS), the second enzyme of the glyoxylate shunt, is essential for in vitro growth and survival of Mtb on even-chain fatty acids, in part, for a previously unrecognized activity: mitigating the toxicity of glyoxylate excess arising from metabolism of even-chain fatty acids. Metabolomic profiling revealed that MS-deficient Mtb cultured on fatty acids accumulated high levels of the ICL aldehyde endproduct, glyoxylate, and increased levels of acetyl phosphate, acetoacetyl coenzyme A (acetoacetyl-CoA), butyryl CoA, acetoacetate, and ß-hydroxybutyrate. These changes were indicative of a glyoxylate-induced state of oxaloacetate deficiency, acetate overload, and ketoacidosis. Reduction of intrabacterial glyoxylate levels using a chemical inhibitor of ICL restored growth of MS-deficient Mtb, despite inhibiting entry of carbon into the glyoxylate shunt. In vivo depletion of MS resulted in sterilization of Mtb in both the acute and chronic phases of mouse infection. This work thus identifies glyoxylate detoxification as an essential physiologic function of Mtb malate synthase and advances its validation as a target for drug development.

Fumarase Deficiency Causes Protein and Metabolite Succination and Intoxicates Mycobacterium tuberculosis.

Enzymes of central carbon metabolism are essential mediators of Mycobacterium tuberculosis (Mtb) physiology and pathogenicity, but are often perceived to lack sufficient species selectivity to be pursued as potential drug targets. Fumarase (Fum) is an enzyme of the canonical tricarboxylic acid cycle and is dispensable in many organisms. Transposon mutagenesis studies in Mtb, however, indicate that Fum is required for optimal growth. Here, we report the generation and characterization of a genetically engineered Mtb strain in which Fum expression is conditionally regulated. This revealed that Fum deficiency is bactericidal in vitro and during both the acute and chronic phases of mouse infection. This essentiality is linked to marked accumulations of fumarate resulting in protein and metabolite succination, a covalent modification of cysteine thiol residues. These results identify Mtb Fum as a potentially species-specific drug target whose inactivation may kill Mtb through a covalently irreversible form of metabolic toxicity.

Metastatic Malignant Pilomatrixoma in an 8-Year-Old Girl Misdiagnosed as a Recurrent Pilomatrixoma.

Malignant pilomatrixoma or pilomatrix carcinoma is a rare, locally aggressive malignant neoplasm, derived from the hair follicle with a high propensity for local recurrence. Only a few cases of metastatic pilomatrixoma have been described in the literature. Till date, only 17 cases have been reported in the English-language medical literature, most commonly occurring associated with local recurrence in adults. We report the first case in children of a metastatic malignant pilomatrixoma in an 8-year-old girl presenting with a recurrent pilomatrix carcinoma of the forehead with metastases in cervical and parotid lymph nodes.

Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice.

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors.

Validation of CoaBC as a Bactericidal Target in the Coenzyme A Pathway of Mycobacterium tuberculosis.

Mycobacterium tuberculosis relies on its own ability to biosynthesize coenzyme A to meet the needs of the myriad enzymatic reactions that depend on this cofactor for activity. As such, the essential pantothenate and coenzyme A biosynthesis pathways have attracted attention as targets for tuberculosis drug development. To identify the optimal step for coenzyme A pathway disruption in M. tuberculosis, we constructed and characterized a panel of conditional knockdown mutants in coenzyme A pathway genes. Here, we report that silencing of coaBC was bactericidal in vitro, whereas silencing of panB, panC, or coaE was bacteriostatic over the same time course. Silencing of coaBC was likewise bactericidal in vivo, whether initiated at infection or during either the acute or chronic stages of infection, confirming that CoaBC is required for M. tuberculosis to grow and persist in mice and arguing against significant CoaBC bypass via transport and assimilation of host-derived pantetheine in this animal model. These results provide convincing genetic validation of CoaBC as a new bactericidal drug target.

Mycobacterium tuberculosis Thioredoxin Reductase Is Essential for Thiol Redox Homeostasis but Plays a Minor Role in Antioxidant Defense.

Mycobacterium tuberculosis (Mtb) must cope with exogenous oxidative stress imposed by the host. Unlike other antioxidant enzymes, Mtb's thioredoxin reductase TrxB2 has been predicted to be essential not only to fight host defenses but also for in vitro growth. However, the specific physiological role of TrxB2 and its importance for Mtb pathogenesis remain undefined. Here we show that genetic inactivation of thioredoxin reductase perturbed several growth-essential processes, including sulfur and DNA metabolism and rapidly killed and lysed Mtb. Death was due to cidal thiol-specific oxidizing stress and prevented by a disulfide reductant. In contrast, thioredoxin reductase deficiency did not significantly increase susceptibility to oxidative and nitrosative stress. In vivo targeting TrxB2 eradicated Mtb during both acute and chronic phases of mouse infection. Deliberately leaky knockdown mutants identified the specificity of TrxB2 inhibitors and showed that partial inactivation of TrxB2 increased Mtb's susceptibility to rifampicin. These studies reveal TrxB2 as essential thiol-reducing enzyme in Mtb in vitro and during infection, establish the value of targeting TrxB2, and provide tools to accelerate the development of TrxB2 inhibitors.

Inactivation of fructose-1,6-bisphosphate aldolase prevents optimal co-catabolism of glycolytic and gluconeogenic carbon substrates in Mycobacterium tuberculosis.

Metabolic pathways used by Mycobacterium tuberculosis (Mtb) to establish and maintain infections are important for our understanding of pathogenesis and the development of new chemotherapies. To investigate the role of fructose-1,6-bisphosphate aldolase (FBA), we engineered an Mtb strain in which FBA levels were regulated by anhydrotetracycline. Depletion of FBA resulted in clearance of Mtb in both the acute and chronic phases of infection in vivo, and loss of viability in vitro when cultured on single carbon sources. Consistent with prior reports of Mtb's ability to co-catabolize multiple carbon sources, this in vitro essentiality could be overcome when cultured on mixtures of glycolytic and gluconeogenic carbon sources, enabling generation of an fba knockout (Δfba). In vitro studies of Δfba however revealed that lack of FBA could only be compensated for by a specific balance of glucose and butyrate in which growth and metabolism of butyrate were determined by Mtb's ability to co-catabolize glucose. These data thus not only evaluate FBA as a potential drug target in both replicating and persistent Mtb, but also expand our understanding of the multiplicity of in vitro conditions that define the essentiality of Mtb's FBA in vivo.

An outer membrane channel protein of Mycobacterium tuberculosis with exotoxin activity.

The ability to control the timing and mode of host cell death plays a pivotal role in microbial infections. Many bacteria use toxins to kill host cells and evade immune responses. Such toxins are unknown in Mycobacterium tuberculosis. Virulent M. tuberculosis strains induce necrotic cell death in macrophages by an obscure molecular mechanism. Here we show that the M. tuberculosis protein Rv3903c (channel protein with necrosis-inducing toxin, CpnT) consists of an N-terminal channel domain that is used for uptake of nutrients across the outer membrane and a secreted toxic C-terminal domain. Infection experiments revealed that CpnT is required for survival and cytotoxicity of M. tuberculosis in macrophages. Furthermore, we demonstrate that the C-terminal domain of CpnT causes necrotic cell death in eukaryotic cells. Thus, CpnT has a dual function in uptake of nutrients and induction of host cell death by M. tuberculosis.

Triosephosphate isomerase is dispensable in vitro yet essential for Mycobacterium tuberculosis to establish infection.

ABSTRACT Triosephosphate isomerase (TPI) catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). This reaction is required for glycolysis and gluconeogenesis, and tpi has been predicted to be essential for growth of Mycobacterium tuberculosis. However, when studying a conditionally regulated tpi knockdown mutant, we noticed that depletion of TPI reduced growth of M. tuberculosis in media containing a single carbon source but not in media that contained both a glycolytic and a gluconeogenic carbon source. We used such two-carbon-source media to isolate a tpi deletion (Δtpi) mutant. The Δtpi mutant did not survive with single carbon substrates but grew like wild-type (WT) M. tuberculosis in the presence of both a glycolytic and a gluconeogenic carbon source. (13)C metabolite tracing revealed the accumulation of TPI substrates in Δtpi and the absence of alternative triosephosphate isomerases and metabolic bypass reactions, which confirmed the requirement of TPI for glycolysis and gluconeogenesis in M. tuberculosis. The Δtpi strain was furthermore severely attenuated in the mouse model of tuberculosis, suggesting that M. tuberculosis cannot simultaneously access sufficient quantities of glycolytic and gluconeogenic carbon substrates to establish infection in mice. IMPORTANCE The importance of central carbon metabolism for the pathogenesis of M. tuberculosis has recently been recognized, but the consequences of depleting specific metabolic enzymes remain to be identified for many enzymes. We investigated triosephosphate isomerase (TPI) because it is central to both glycolysis and gluconeogenesis and had been predicted to be essential for growth of M. tuberculosis. This work identified metabolic conditions that make TPI dispensable for M. tuberculosis growth in culture and proved that M. tuberculosis relies on a single TPI enzyme and has no metabolic bypass for the TPI-dependent interconversion of dihydroxyacetone phosphate and glyceraldehyde-3-phosphate in glycolysis and gluconeogenesis. Finally, we demonstrate that TPI is essential for growth of the pathogen in mouse lungs.