Engineered Mycobacteriophage TM4::GeNL Rapidly Determines Bedaquiline, Pretomanid, Linezolid, Rifampicin, and Clofazimine Sensitivity in Mycobacterium tuberculosis Clinical Isolates.

Background: Drug-resistant tuberculosis is a growing public health threat, and early characterization of the resistance phenotype is essential for guiding treatment and mitigating the high mortality associated with the disease. However, the slow growth rate of Mycobacterium tuberculosis, the causative agent of tuberculosis, necessitates several weeks for conventional culture-dependent drug susceptibility testing (DST). In addition, there are no widely available molecular diagnostic assays for evaluating resistance to newer tuberculosis drugs or drugs with complex resistance mechanisms.

Intravenous Immunization with Triple Auxotrophs of : A novel vaccine strategy against tuberculosis.

Tuberculosis, caused by ( ), remains a leading infectious cause of mortality worldwide despite widespread use of the BCG vaccine and the availability of sterilizing pharmacopoeia. Recent research indicates that the intravenous administration of BCG confers sterilizing immunity against pulmonary challenge in non-human primates. However, while BCG is relatively safe, complications such as disseminated BCGosis have been observed in immunocompromised individuals.

A viral vaccine design harnessing prior BCG immunization confers protection against Ebola virus.

Previous studies have demonstrated the efficacy and feasibility of an anti-viral vaccine strategy that takes advantage of pre-existing CD4 helper T (Th) cells induced by bacille Calmette-Guérin (BCG) vaccination. This strategy uses immunization with recombinant fusion proteins comprised of a cell surface expressed viral antigen, such as a viral envelope glycoprotein, engineered to contain well-defined BCG Th cell epitopes, thus rapidly recruiting Th cells induced by prior BCG vaccination to provide intrastructural help to virus-specific B cells. In the current study, we show that Th cells induced by BCG were localized predominantly outside of germinal centers and promoted antibody class switching to isotypes characterized by strong Fc receptor interactions and effector functions.

Boosting Immunogenicity of a Recombinant Strain via Zinc-Dependent Ribosomal Proteins.

Tuberculosis (TB) continues to be a major global health burden and kills over a million people annually. New immunization strategies are required for the development of an efficacious TB vaccine that can potentially induce sterilizing immunity. In this study, we first confirmed that a live vaccine strain of , previously designated as IKEPLUS, conferred a higher survival benefit than the Bacillus Calmette-Guerin (BCG) in a murine model of intravenous (Mtb) infection.

The SapM phosphatase can arrest phagosome maturation in an ESX-1 independent manner in and BCG.

() is an intracellular pathogen that survives and grows in macrophages. A mechanism used by to achieve intracellular survival is to secrete effector molecules that arrest the normal process of phagosome maturation. Through phagosome maturation arrest (PMA), remains in an early phagosome and avoids delivery to degradative phagolysosomes.

A viral vaccine design harnessing prior BCG immunization confers protection against Ebola virus.

Previous studies have demonstrated the efficacy and feasibility of an anti-viral vaccine strategy that takes advantage of pre-existing CD4 helper T (Th) cells induced by bacille Calmette-Guérin (BCG) vaccination. This strategy uses immunization with recombinant fusion proteins comprised of a cell surface expressed viral antigen, such as a viral envelope glycoprotein, engineered to contain well-defined BCG Th cell epitopes, thus rapidly recruiting Th cells induced by prior BCG vaccination to provide intrastructural help to virus-specific B cells. In the current study, we show that Th cells induced by BCG were localized predominantly outside of germinal centers and promoted antibody class switching to isotypes characterized by strong Fc receptor interactions and effector functions.

Propionate prevents loss of the PDIM virulence lipid in Mycobacterium tuberculosis.

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of Mycobacterium tuberculosis. In vitro culturing rapidly selects for spontaneous PDIM-negative mutants that have attenuated virulence and increased cell wall permeability, thus impacting the relevance of experimental findings. PDIM loss can also reduce the efficacy of the BCG Pasteur vaccine.

Boosting bactericidal immunity of a recombinant strain via zinc-dependent ribosomal proteins.

Tuberculosis (TB) continues to be a major global health burden and kills over a million people annually. New immunization strategies are required for the development of an efficacious TB vaccine that can potentially induce sterilizing immunity. In this study, we first confirmed that various strains of the IKEPLUS vaccine confer a higher survival benefit than BCG in a murine model of intravenous (Mtb) infection.

Loss-of-function mutations in do not confer delamanid, ethionamide, isoniazid, or pretomanid resistance in .

Results from clinical strains and knockouts of the H37Rv and CDC1551 laboratory strains demonstrated that () is not a resistance-conferring gene for isoniazid, ethionamide, delamanid, or pretomanid in . This difference in the susceptibility to NAD-adduct-forming drugs compared with other mycobacteria may be driven by differences in the absolute intrabacterial NADH concentration.

The PDIM paradox of : new solutions to a persistent problem.

Phthiocerol dimycocerosate (PDIM) is an essential virulence lipid of . culturing rapidly selects for spontaneous mutations that cause PDIM loss leading to virulence attenuation and increased cell wall permeability. We discovered that PDIM loss is due to a metabolic deficiency of methylmalonyl-CoA that impedes the growth of PDIM-producing bacilli.

The Mycobacterium tuberculosis genome at 25 years: lessons and lingering questions.

First achieved in 1998 by Cole et al., the complete genome sequence of Mycobacterium tuberculosis continues to provide an invaluable resource to understand tuberculosis (TB), the leading cause of global infectious disease mortality. At the 25-year anniversary of this accomplishment, we describe how insights gleaned from the M.

Central Metabolism Is Key Regulator of Macrophage Pyroptosis and Host Immunity.

Metabolic dysregulation in results in increased macrophage apoptosis or pyroptosis. However, mechanistic links between virulence and bacterial metabolic plasticity remain ill defined. In this study, we screened random transposon insertions of BCG to identify mutants that induce pyroptotic death of the infected macrophage.

MAT Gain of Activity Mutation in Is Associated with Resistance to MTAN Transition State Analogues.

is found in the gut lining of more than half of the world's population, causes gastric ulcers, and contributes to stomach cancers. Menaquinone synthesis in relies on the rare futalosine pathway, where 5'-methylthioadenosine nucleosidase (MTAN) is proposed to play an essential role. Transition state analogues of MTAN, including BuT-DADMe-ImmA (BTDIA) and MeT-DADMe-ImmA (MTDIA), exhibit bacteriostatic action against numerous diverse clinical isolates of with minimum inhibitory concentrations (MIC's) of <2 ng/mL.

Mycobacterium smegmatis: The Vanguard of Mycobacterial Research.

The genus Mycobacterium contains several slow-growing human pathogens, including Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium. Mycobacterium smegmatis is a nonpathogenic and fast growing species within this genus. In 1990, a mutant of M.

Strain Ravenel Is Attenuated in Cattle.

variant (MBO) has one of the widest known mammalian host ranges, including humans. Despite the characterization of this pathogen in the 1800s and whole genome sequencing of a UK strain (AF2122) nearly two decades ago, the basis of its host specificity and pathogenicity remains poorly understood. Recent experimental calf infection studies show that MBO strain Ravenel (MBO Ravenel) is attenuated in the cattle host compared to other pathogenic strains of MBO.

A world without tuberculosis: moving from imagination to reality.

Tuberculosis (TB), caused by Mycobacterium tuberculosis infection, remains a leading cause of death from an infectious agent, resulting in more than a million deaths per year. Despite vaccines and chemotherapies, patients often harbor persister M. tuberculosis cells that resist immune assault and chemotherapeutic treatments, resulting in a latent TB infection (LTBI).

Capturing Structural Variants of Herpes Simplex Virus Genome in Full Length by Oxford Nanopore Sequencing.

Genome sequencing and assembly of viral genomes within the family, particularly herpes simplex virus (HSV), have been challenging due to the large size (~154 Kb), high GC content (68%), and nucleotide variations arising during replication. Oxford Nanopore Technology (ONT) has been successful in obtaining read lengths ranging from 100 Kb up to 2.3 Mb.

Commonalities of Transcriptomes in Response to Defined Persisting Macrophage Stresses.

As the goal of a bacterium is to become bacteria, evolution has imposed continued selections for gene expression. The intracellular pathogen , the causative agent of tuberculosis, has adopted a fine-tuned response to survive its host's methods to aggressively eradicate invaders. The development of microarrays and later RNA sequencing has led to a better understanding of biological processes controlling the relationship between host and pathogens.

Multiple genetic paths including massive gene amplification allow to overcome loss of ESX-3 secretion system substrates.

() possesses five type VII secretion systems (T7SS), virulence determinants that include the secretion apparatus and associated secretion substrates. strains deleted for the genes encoding substrates of the ESX-3 T7SS, or , require iron supplementation for in vitro growth and are highly attenuated in vivo. In a subset of infected mice, suppressor mutants of or deletions were isolated, which enabled growth to high titers or restored virulence.

Elimination of PknL and MSMEG_4242 in alters the character of the outer cell envelope and selects for mutations in Lsr2.

Four serine/threonine kinases are present in all mycobacteria: PknA, PknB, PknG and PknL. PknA and PknB are essential for growth and replication, PknG regulates metabolism, but little is known about PknL. Inactivation of and adjacent regulator 2 in rough colony mc155 produced both smooth and rough colonies.

The promises and limitations of acetylcysteine as a potentiator of first-line and second-line tuberculosis drugs.

acetylcysteine (NAC) is most commonly used for the treatment of acetaminophen overdose and acetaminophen-induced liver injury. In patients infected with , the causative agent of tuberculosis (TB), NAC is given to treat hepatotoxicity induced by TB drugs. We had previously shown that cysteine, a derivative of NAC, potentiated the activity of isoniazid, a first-line TB drug, by preventing the emergence of INH resistance and persistence in in vitro.

Sterilization by Adaptive Immunity of a Conditionally Persistent Mutant of Mycobacterium tuberculosis.

(), the causative agent of tuberculosis, can enter into a persistent state that confers resistance to antibacterial agents. Many observations suggest that persistent cells also evade the antimycobacterial immune mechanisms, thereby reducing the effectiveness of the current tuberculosis vaccine. Understanding the factors that contribute to persistence may enable the rational design of vaccines that stimulate effective immune killing mechanisms against persister cells.

Drivers and sites of diversity in the DNA adenine methylomes of 93 complex clinical isolates.

This study assembles DNA adenine methylomes for 93 complex (MTBC) isolates from seven lineages paired with fully-annotated, finished, de novo assembled genomes. Integrative analysis yielded four key results. First, methyltransferase allele-methylome mapping corrected methyltransferase variant effects previously obscured by reference-based variant calling.