Distinct Bacterial Pathways Influence the Efficacy of Antibiotics against Mycobacterium tuberculosis

Effective tuberculosis treatment requires at least 6 months of combination therapy. Alterations in the physiological state of the bacterium during infection are thought to reduce drug efficacy and prolong the necessary treatment period, but the nature of these adaptations remain incompletely defined. To identify specific bacterial functions that limit drug effects during infection, we employed a comprehensive genetic screening approach to identify mutants with altered susceptibility to the first-line antibiotics in the mouse model. We identified many mutations that increase the rate of bacterial clearance, suggesting new strategies for accelerating therapy. In addition, the drug-specific effects of these mutations suggested that different antibiotics are limited by distinct factors. Rifampin efficacy is inferred to be limited by cellular permeability, whereas isoniazid is preferentially affected by replication rate. Many mutations that altered bacterial clearance in the mouse model did not have an obvious effect on drug susceptibility using in vitro assays, indicating that these chemical-genetic interactions tend to be specific to the in vivo environment. This observation suggested that a wide variety of natural genetic variants could influence drug efficacy in vivo without altering behavior in standard drug-susceptibility tests. Indeed, mutations in a number of the genes identified in our study are enriched in drug-resistant clinical isolates, identifying genetic variants that may influence treatment outcome. Together, these observations suggest new avenues for improving therapy, as well as the mechanisms of genetic adaptations that limit it. IMPORTANCE Understanding how Mycobacterium tuberculosis survives during antibiotic treatment is necessary to rationally devise more effective tuberculosis (TB) chemotherapy regimens. Using genome-wide mutant fitness profiling and the mouse model of TB, we identified genes that alter antibiotic efficacy specifically in the infection environment and associated several of these genes with natural genetic variants found in drug-resistant clinical isolates. These data suggest strategies for synergistic therapies that accelerate bacterial clearance, and they identify mechanisms of adaptation to drug exposure that could influence treatment outcome

Therapeutic Monoclonal Antibodies to Prevent Tuberculosis Infection

Mycobacteria tuberculosis (Mtb) is a major cause of human morbidity and mortality. Transmission occurs through inhalation of aerosolized Mtb and the initial infection is believed to occur primarily in the alveolar macrophage, although Mtb can infect other cells residing in the lung including dendritic cells, pneumocytes and M cells. Several molecules derived from Mtb are involved in the attachment of the organism to host receptors (opsonic and non-opsonic), which have been reasonably well elucidated. However, a complete understanding of how Mtb attaches to the host and the relative importance of each mechanism on the outcome of infection remains elusive. We hypothesize that protection from infection is possible by blocking the critical initial surface interactions of the organism with the host cell using specific monoclonal antibodies (mAbs). To develop effective mAbs, the outermost layers of Mtb, the capsule and outer membrane, were isolated and characterized by protein gel and LC/MS/MS. Approximately 1000 different proteins were identified in the isolations, of which ~25% were unique to one of the two fractions. The capsule or outer membrane preparations were used as antigens to immunize CD1 mice for up to 12 weeks to generate antibodies via traditional hybridoma generation. Antibodies were screened, selected and characterized by their ability to bind whole cell Mtb by ELISA, demonstration of unique heavy chain variable region sequence and binding specificity by Western Blot. Of approximately 1500 screened hybridomas, 30 lead mAbs have been isolated with specificity to various targets. Preliminary results suggest several of the lead mAb candidates are able to prevent Mtb-induced macrophage cell death in vitro. Future studies will attempt to confirm efficacy in vivo after aerosolized infection in mice with mAb-coated Mtb or parenteral administration of mAb(s). Targets of functional mAbs will be determined and these antigens could serve as viable candidates for vaccine development

Functionally Overlapping Variants Control Tuberculosis Susceptibility in Collaborative Cross Mice

Host genetics plays an important role in determining the outcome of Mycobacterium tuberculosis infection. We previously found that Collaborative Cross (CC) mouse strains differ in their susceptibility to M. tuberculosis and that the CC042/GeniUnc (CC042) strain suffered from a rapidly progressive disease and failed to produce the protective cytokine gamma interferon (IFN-gamma) in the lung. Here, we used parallel genetic and immunological approaches to investigate the basis of CC042 mouse susceptibility. Using a population derived from a CC001/Unc (CC001) x CC042 intercross, we mapped four quantitative trait loci (QTL) underlying tuberculosis immunophenotypes (Tip1 to Tip4). These included QTL that were associated with bacterial burden, IFN-gamma production following infection, and an IFN-gamma-independent mechanism of bacterial control. Further immunological characterization revealed that CC042 animals recruited relatively few antigen-specific T cells to the lung and that these T cells failed to express the integrin alpha L (alphaL; i.e., CD11a), which contributes to T cell activation and migration. These defects could be explained by a CC042 private variant in the Itgal gene, which encodes CD11a and is found within the Tip2 interval. This 15-bp deletion leads to aberrant mRNA splicing and is predicted to result in a truncated protein product. The Itgal(CC042) genotype was associated with all measured disease traits, indicating that this variant is a major determinant of susceptibility in CC042 mice. The combined effect of functionally distinct Tip variants likely explains the profound susceptibility of CC042 mice and highlights the multigenic nature of tuberculosis control in the Collaborative Cross. IMPORTANCE The variable outcome of Mycobacterium tuberculosis infection observed in natural populations is difficult to model in genetically homogeneous small-animal models. The newly developed Collaborative Cross (CC) represents a reproducible panel of genetically diverse mice that display a broad range of phenotypic responses to infection. We explored the genetic basis of this variation, focusing on a CC line that is highly susceptible to M. tuberculosis infection. This study identified multiple quantitative trait loci associated with bacterial control and cytokine production, including one that is caused by a novel loss-of-function mutation in the Itgal gene, which is necessary for T cell recruitment to the infected lung. These studies verify the multigenic control of mycobacterial disease in the CC panel, identify genetic loci controlling diverse aspects of pathogenesis, and highlight the utility of the CC resource

Host-pathogen genetic interactions underlie tuberculosis susceptibility in genetically diverse mice [preprint]

The outcome of an encounter with Mycobacterium tuberculosis (Mtb) depends on the pathogen’s ability to adapt to the heterogeneous immune response of the host. Understanding this interplay has proven difficult, largely because experimentally tractable small animal models do not recapitulate the heterogenous disease observed in natural infections. We leveraged the genetically diverse Collaborative Cross (CC) mouse panel in conjunction with a library of Mtb mutants to associate bacterial genetic requirements with host genetics and immunity. We report that CC strains vary dramatically in their susceptibility to infection and represent reproducible models of qualitatively distinct immune states. Global analysis of Mtb mutant fitness across the CC panel revealed that a large fraction of the pathogen’s genome is necessary for adaptation to specific host microenvironments. Both immunological and bacterial traits were associated with genetic variants distributed across the mouse genome, elucidating the complex genetic landscape that underlies host-pathogen interactions in a diverse population

Common Variants in the Glycerol Kinase Gene Reduce Tuberculosis Drug Efficacy

Despite the administration of multiple drugs that are highly effective in vitro, tuberculosis (TB) treatment requires prolonged drug administration and is confounded by the emergence of drug-resistant strains. To understand the mechanisms that limit antibiotic efficacy, we performed a comprehensive genetic study to identify Mycobacterium tuberculosis genes that alter the rate of bacterial clearance in drug-treated mice. Several functionally distinct bacterial genes were found to alter bacterial clearance, and prominent among these was the glpK gene that encodes the glycerol-3-kinase enzyme that is necessary for glycerol catabolism. Growth on glycerol generally increased the sensitivity of M. tuberculosis to antibiotics in vitro, and glpK-deficient bacteria persisted during antibiotic treatment in vivo, particularly during exposure to pyrazinamide-containing regimens. Frameshift mutations in a hypervariable homopolymeric region of the glpK gene were found to be a specific marker of multidrug resistance in clinical M. tuberculosis isolates, and these loss-of-function alleles were also enriched in extensively drug-resistant clones. These data indicate that frequently observed variation in the glpK coding sequence produces a drug-tolerant phenotype that can reduce antibiotic efficacy and may contribute to the evolution of resistance. IMPORTANCE: TB control is limited in part by the length of antibiotic treatment needed to prevent recurrent disease. To probe mechanisms underlying survival under antibiotic pressure, we performed a genetic screen for M. tuberculosis mutants with altered susceptibility to treatment using the mouse model of TB. We identified multiple genes involved in a range of functions which alter sensitivity to antibiotics. In particular, we found glycerol catabolism mutants were less susceptible to treatment and that common variation in a homopolymeric region in the glpK gene was associated with drug resistance in clinical isolates. These studies indicate that reversible high-frequency variation in carbon metabolic pathways can produce phenotypically drug-tolerant clones and have a role in the development of resistance

Tuberculosis susceptibility and vaccine protection are independently controlled by host genotype

The outcome of Mycobacterium tuberculosis infection and the immunological response to the bacillus Calmette-Guerin (BCG) vaccine are highly variable in humans. Deciphering the relative importance of host genetics, environment, and vaccine preparation for the efficacy of BCG has proven difficult in natural populations. We developed a model system that captures the breadth of immunological responses observed in outbred individual mice, which can be used to understand the contribution of host genetics to vaccine efficacy. This system employs a panel of highly diverse inbred mouse strains, consisting of the founders and recombinant progeny of the "Collaborative Cross" project. Unlike natural populations, the structure of this panel allows the serial evaluation of genetically identical individuals and the quantification of genotype-specific effects of interventions such as vaccination. When analyzed in the aggregate, our panel resembled natural populations in several important respects: the animals displayed a broad range of susceptibility to M. tuberculosis, differed in their immunological responses to infection, and were not durably protected by BCG vaccination. However, when analyzed at the genotype level, we found that these phenotypic differences were heritable. M. tuberculosis susceptibility varied between lines, from extreme sensitivity to progressive M. tuberculosis clearance. Similarly, only a minority of the genotypes was protected by vaccination. The efficacy of BCG was genetically separable from susceptibility to M. tuberculosis, and the lack of efficacy in the aggregate analysis was driven by nonresponsive lines that mounted a qualitatively distinct response to infection. These observations support an important role for host genetic diversity in determining BCG efficacy and provide a new resource to rationally develop more broadly efficacious vaccines. IMPORTANCE Tuberculosis (TB) remains an urgent global health crisis, and the efficacy of the currently used TB vaccine, M. bovis BCG, is highly variable. The design of more broadly efficacious vaccines depends on understanding the factors that limit the protection imparted by BCG. While these complex factors are difficult to disentangle in natural populations, we used a model population of mice to understand the role of host genetic composition in BCG efficacy. We found that the ability of BCG to protect mice with different genotypes was remarkably variable. The efficacy of BCG did not depend on the intrinsic susceptibility of the animal but, instead, correlated with qualitative differences in the immune responses to the pathogen. These studies suggest that host genetic polymorphism is a critical determinant of vaccine efficacy and provide a model system to develop interventions that will be useful in genetically diverse populations.This work, including the efforts of Hardy Kornfeld, was funded by HHS | National Institutes of Health (NIH) (HL081149). This work, including the efforts of Sam Behar, was funded by HHS | National Institutes of Health (NIH) (AI123286-01). This work, including the efforts of Clare Margaret Smith and Christopher Sassetti, was funded by Howard Hughes Medical Institute (HHMI)

Why humans kill animals and why we cannot avoid it

Killing animals has been a ubiquitous human behaviour throughout history, yet it is becoming increasingly controversial and criticised in some parts of contemporary human society. Here we review 10 primary reasons why humans kill animals, discuss the necessity (or not) of these forms of killing, and describe the global ecological context for human killing of animals. Humans historically and currently kill animals either directly or indirectly for the following reasons: (1) wild harvest or food acquisition, (2) human health and safety, (3) agriculture and aquaculture, (4) urbanisation and industrialisation, (5) invasive, overabundant or nuisance wildlife control, (6) threatened species conservation, (7) recreation, sport or entertainment, (8) mercy or compassion, (9) cultural and religious practice, and (10) research,education and testing. While the necessity of some forms of animal killing is debatable and further depends on individual values, we emphasise that several of these forms of animal killing are a necessary component of our inescapable involvement in a single, functioning, finite, global food web. We conclude that humans (and all other animals) cannot live in a way that does not require animal killing either directly or indirectly, but humans can modify some of these killing behaviours in ways that improve the welfare of animals while they are alive, or to reduce animal suffering whenever they must be killed. We encourage a constructive dialogue that (1) accepts and permits human participation in one enormous global food web dependent on animal killing and (2) focuses on animal welfare and environmental sustainability. Doing so will improve the lives of both wild and domestic animals to a greater extent than efforts to avoid, prohibit or vilify human animal-killing behaviour. Animal ethics Conservation biology Culling Factory farmingpublishedVersio

Why humans kill animals and why we cannot avoid it

Killing animals has been a ubiquitous human behaviour throughout history, yet it is becoming increasingly controversial and criticised in some parts of contemporary human society. Here we review 10 primary reasons why humans kill animals, discuss the necessity (or not) of these forms of killing, and describe the global ecological context for human killing of animals. Humans historically and currently kill animals either directly or indirectly for the following reasons: (1) wild harvest or food acquisition, (2) human health and safety, (3) agriculture and aquaculture, (4) urbanisation and industrialisation, (5) invasive, overabundant or nuisance wildlife control, (6) threatened species conservation, (7) recreation, sport or entertainment, (8) mercy or compassion, (9) cultural and religious practice, and (10) research,education and testing. While the necessity of some forms of animal killing is debatable and further depends on individual values, we emphasise that several of these forms of animal killing are a necessary component of our inescapable involvement in a single, functioning, finite, global food web. We conclude that humans (and all other animals) cannot live in a way that does not require animal killing either directly or indirectly, but humans can modify some of these killing behaviours in ways that improve the welfare of animals while they are alive, or to reduce animal suffering whenever they must be killed. We encourage a constructive dialogue that (1) accepts and permits human participation in one enormous global food web dependent on animal killing and (2) focuses on animal welfare and environmental sustainability. Doing so will improve the lives of both wild and domestic animals to a greater extent than efforts to avoid, prohibit or vilify human animal-killing behaviour. Animal ethics Conservation biology Culling Factory farmingpublishedVersio

Cause of Death and Predictors of All-Cause Mortality in Anticoagulated Patients With Nonvalvular Atrial Fibrillation : Data From ROCKET AF

M. Kaste on työryhmän ROCKET AF Steering Comm jäsen.Background-Atrial fibrillation is associated with higher mortality. Identification of causes of death and contemporary risk factors for all-cause mortality may guide interventions. Methods and Results-In the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF) study, patients with nonvalvular atrial fibrillation were randomized to rivaroxaban or dose-adjusted warfarin. Cox proportional hazards regression with backward elimination identified factors at randomization that were independently associated with all-cause mortality in the 14 171 participants in the intention-to-treat population. The median age was 73 years, and the mean CHADS(2) score was 3.5. Over 1.9 years of median follow-up, 1214 (8.6%) patients died. Kaplan-Meier mortality rates were 4.2% at 1 year and 8.9% at 2 years. The majority of classified deaths (1081) were cardiovascular (72%), whereas only 6% were nonhemorrhagic stroke or systemic embolism. No significant difference in all-cause mortality was observed between the rivaroxaban and warfarin arms (P=0.15). Heart failure (hazard ratio 1.51, 95% CI 1.33-1.70, P= 75 years (hazard ratio 1.69, 95% CI 1.51-1.90, P Conclusions-In a large population of patients anticoagulated for nonvalvular atrial fibrillation, approximate to 7 in 10 deaths were cardiovascular, whereasPeer reviewe

Omecamtiv mecarbil in chronic heart failure with reduced ejection fraction, GALACTIC‐HF: baseline characteristics and comparison with contemporary clinical trials

Aims: The safety and efficacy of the novel selective cardiac myosin activator, omecamtiv mecarbil, in patients with heart failure with reduced ejection fraction (HFrEF) is tested in the Global Approach to Lowering Adverse Cardiac outcomes Through Improving Contractility in Heart Failure (GALACTIC‐HF) trial. Here we describe the baseline characteristics of participants in GALACTIC‐HF and how these compare with other contemporary trials. Methods and Results: Adults with established HFrEF, New York Heart Association functional class (NYHA) ≥ II, EF ≤35%, elevated natriuretic peptides and either current hospitalization for HF or history of hospitalization/ emergency department visit for HF within a year were randomized to either placebo or omecamtiv mecarbil (pharmacokinetic‐guided dosing: 25, 37.5 or 50 mg bid). 8256 patients [male (79%), non‐white (22%), mean age 65 years] were enrolled with a mean EF 27%, ischemic etiology in 54%, NYHA II 53% and III/IV 47%, and median NT‐proBNP 1971 pg/mL. HF therapies at baseline were among the most effectively employed in contemporary HF trials. GALACTIC‐HF randomized patients representative of recent HF registries and trials with substantial numbers of patients also having characteristics understudied in previous trials including more from North America (n = 1386), enrolled as inpatients (n = 2084), systolic blood pressure < 100 mmHg (n = 1127), estimated glomerular filtration rate < 30 mL/min/1.73 m2 (n = 528), and treated with sacubitril‐valsartan at baseline (n = 1594). Conclusions: GALACTIC‐HF enrolled a well‐treated, high‐risk population from both inpatient and outpatient settings, which will provide a definitive evaluation of the efficacy and safety of this novel therapy, as well as informing its potential future implementation