Martin Gengenbacher

Mycobacterium tuberculosis: Success through dormancy

Tuberculosis (TB) remains a major health threat, killing nearly 2 million individuals around this globe, annually. The only vaccine, developed almost a century ago, provides limited protection only during childhood. After decades without the introduction of new antibiotics, several candidates are currently undergoing clinical investigation. Curing TB requires prolonged combination of chemotherapy with several drugs. Moreover, monitoring the success of therapy is questionable owing to the lack of reliable biomarkers. To substantially improve the situation, a detailed understanding of the cross-talk between human host and the pathogen Mycobacterium tuberculosis (Mtb) is vital. Principally, the enormous success of Mtb is based on three capacities: first, reprogramming of macrophages after primary infection/phagocytosis to prevent its own destruction; second, initiating the formation of well-organized granulomas, comprising different immune cells to create a confined environment for the host-pathogen standoff; third, the capability to shut down its own central metabolism, terminate replication, and thereby transit into a stage of dormancy rendering itself extremely resistant to host defense and drug treatment. Here, we review the molecular mechanisms underlying these processes, draw conclusions in a working model of mycobacterial dormancy, and highlight gaps in our understanding to be addressed in future research.

The Mtb Proteome Library: A Resource of Assays to Quantify the Complete Proteome of Mycobacterium tuberculosis

Research advancing our understanding of Mycobacterium tuberculosis (Mtb) biology and complex host-Mtb interactions requires consistent and precise quantitative measurements of Mtb proteins. We describe the generation and validation of a compendium of assays to quantify 97% of the 4,012 annotated Mtb proteins by the targeted mass spectrometric method selected reaction monitoring (SRM). Furthermore, we estimate the absolute abundance for 55% of all Mtb proteins, revealing a dynamic range within the Mtb proteome of over four orders of magnitude, and identify previously unannotated proteins. As an example of the assay library utility, we monitored the entire Mtb dormancy survival regulon (DosR), which is linked to anaerobic survival and Mtb persistence, and show its dynamic protein-level regulation during hypoxia. In conclusion, we present a publicly available research resource that supports the sensitive, precise, and reproducible quantification of virtually any Mtb protein by a robust and widely accessible mass spectrometric method.

Absolute Proteome Composition and Dynamics during Dormancy and Resuscitation of Mycobacterium tuberculosis

Mycobacterium tuberculosis remains a health concern due to its ability to enter a non-replicative dormant state linked to drug resistance. Understanding transitions into and out of dormancy will inform therapeutic strategies. We implemented a universally applicable, label-free approach to estimate absolute cellular protein concentrations on a proteome-wide scale based on SWATH mass spectrometry. We applied this approach to examine proteomic reorganization of M. tuberculosis during exponential growth, hypoxia-induced dormancy, and resuscitation. The resulting data set covering >2,000 proteins reveals how protein biomass is distributed among cellular functions during these states. The stress-induced DosR regulon contributes 20% to cellular protein content during dormancy, whereas ribosomal proteins remain largely unchanged at 5%-7%. Absolute protein concentrations furthermore allow protein alterations to be translated into changes in maximal enzymatic reaction velocities, enhancing understanding of metabolic adaptations. Thus, global absolute protein measurements provide a quantitative description of microbial states, which can support the development of therapeutic interventions.

Reduced Drug Uptake in Phenotypically Resistant Nutrient-Starved Nonreplicating Mycobacterium tuberculosis

ABSTRACT During active tuberculosis a spectrum of physiologically different Mycobacterium tuberculosis bacilli reside in human tissues. Subpopulations of the pathogen survive antibiotic treatment for a prolonged time in a dormant state of phenotypic drug resistance, a phenomenon independent of genetic mutations. Here, we used an established culture model of nutrient deprivation to shift down M. tuberculosis from growth to nonreplicating survival, which is characterized by a drastic loss of drug susceptibility. Liquid chromatography coupled with mass spectrometry techniques were employed to quantify drug penetration in replicating and nutrient-starved nonreplicating bacilli. We found that intracellular concentrations of fluoroquinolones, rifamycins, and linezolid were lower in nonreplicating M. tuberculosis. Studies with pump inhibitors suggest that the observed differences were independent of efflux processes. We conclude that decreased drug permeability contributes to phenotypic drug resistance of dormant M. tuberculosis.

Recombinant live vaccine candidates against tuberculosis

Tuberculosis (TB) remains among the most deadly health threats to humankind despite availability of several potent antibiotics and a vaccine, bacille Calmette-Guérin (BCG). BCG partially protects children but not adults from the disease. Growing knowledge of the molecular basis of infection, immunity, and pathology in TB has driven various approaches, which strive to complement or replace BCG with more effective vaccines. Three recombinant live TB vaccine candidates have entered clinical trials. These candidates have been genetically engineered to be attenuated, to overexpress TB antigens and/or to secrete bacterial perforins, ultimately seeking to trigger a robust immune response thereby providing long-lasting protection against TB.

Central Memory CD4+ T Cells Are Responsible for the Recombinant Bacillus Calmette-Guérin ΔureC::hly Vaccine's Superior Protection Against Tuberculosis

Bacillus Calmette-Guérin (BCG) has been used for vaccination against tuberculosis for nearly a century. Here, we analyze immunity induced by a live tuberculosis vaccine candidate, recombinant BCG ΔureC::hly vaccine (rBCG), with proven preclinical and clinical safety and immunogenicity. We pursue in-depth analysis of the endogenous mycobacteria-specific CD4+ T-cell population, comparing the more efficacious rBCG with canonical BCG to determine which T-cell memory responses are prerequisites for superior protection against tuberculosis. rBCG induced higher numbers and proportions of antigen-specific memory CD4+ T cells than BCG, with a CXCR5+CCR7+ phenotype and low expression of the effector transcription factors T-bet and Bcl-6. We found that the superior protection of rBCG, compared with BCG, correlated with higher proportions and numbers of these central memory T cells and of T follicular helper cells associated with specific antibody responses. Adoptive transfer of mycobacteria-specific central memory T cells validated their critical role in protection against pulmonary tuberculosis.

Vitamin B6 biosynthesis is essential for survival and virulence of Mycobacterium tuberculosis

With 500 000 cases of multidrug‐resistant tuberculosis there is an urgent need for attractive targets to enable the discovery of novel antimycobacterials. The biosynthesis of essential cofactors is of particular interest as these pathways are absent in man and their inhibition is expected to affect the metabolism of Mycobacterium tuberculosis at multiple sites. Our data demonstrate that the pathogen synthesizes pyridoxal 5‐phosphate (PLP), the bioactive form of vitamin B6, by a heteromeric PLP synthase composed of Pdx1 (Rv2606c) and Pdx2 (Rv2604c). Disruption of the pdx1 gene generated a strictly B6 auxotrophic M. tuberculosis mutant, Δpdx1. Removal of the cofactor during exponential growth or stationary phase demonstrated the essentiality of vitamin B6 biosynthesis for growth and survival of the pathogen in culture. In a tuberculosis dormancy model based on gradual oxygen depletion, de novo biosynthesis of PLP was required for regrowth of the bacillus after direct oxygen exposure. The Δpdx1 mutant showed a severe growth defect in immunocompetent mice: bacilli applied intranasally failed to persist in host tissues and were quickly cleared. We conclude that vitamin B6 biosynthesis is required for survival of M. tuberculosis in vivo and thus might represent a candidate pathway for the development of new antitubercular agents.

The Recombinant BCG ΔureC::hly Vaccine Targets the AIM2 Inflammasome to Induce Autophagy and Inflammation

BACKGROUND The recombinant BCG ΔureC::hly (rBCG) vaccine candidate induces improved protection against tuberculosis over parental BCG (pBCG) in preclinical studies and has successfully completed a phase 2a clinical trial. However, the mechanisms responsible for the superior vaccine efficacy of rBCG are still incompletely understood. Here, we investigated the underlying biological mechanisms elicited by the rBCG vaccine candidate relevant to its protective efficacy. METHODS THP-1 macrophages were infected with pBCG or rBCG, and inflammasome activation and autophagy were evaluated. In addition, mice were vaccinated with pBCG or rBCG, and gene expression in the draining lymph nodes was analyzed by microarray at day 1 after vaccination. RESULTS BCG-derived DNA was detected in the cytosol of rBCG-infected macrophages. rBCG infection was associated with enhanced absent in melanoma 2 (AIM2) inflammasome activation, increased activation of caspases and production of interleukin (IL)-1β and IL-18, as well as induction of AIM2-dependent and stimulator of interferon genes (STING)-dependent autophagy. Similarly, mice vaccinated with rBCG showed early increased expression of Il-1β, Il-18, and Tmem173 (transmembrane protein 173; also known as STING). CONCLUSIONS rBCG stimulates AIM2 inflammasome activation and autophagy, suggesting that these cell-autonomous functions should be exploited for improved vaccine design.

The BCG replacement vaccine VPM1002: from drawing board to clinical trial

Tuberculosis remains a major health threat and vaccines better than bacillus Calmette-Guérin (BCG) are urgently required. Here we describe our experience with a recombinant BCG expressing listeriolysin and deficient in urease. This potential replacement vaccine has demonstrated superior efficacy and safety over BCG in Mycobacterium tuberculosis aerosol-challenged mice and was safe in numerous animal models including immune-deficient mice, guinea pigs, rabbits and nonhuman primates. Phase I clinical trials in adults in Germany and South Africa have proven safety and a current Phase IIa trial is under way to assess immunogenicity and safety in its target population, newborns in a high tuberculosis incidence setting, with promising early results. Second-generation candidates are being developed to improve safety and efficacy.

Antigen 85C inhibition restricts Mycobacterium tuberculosis growth through disruption of cord factor biosynthesis

ABSTRACT The antigen 85 (Ag85) protein family, consisting of Ag85A, -B, and -C, is vital for Mycobacterium tuberculosis due to its role in cell envelope biogenesis. The mycoloyl transferase activity of these proteins generates trehalose dimycolate (TDM), an envelope lipid essential for M. tuberculosis virulence, and cell wall arabinogalactan-linked mycolic acids. Inhibition of these enzymes through substrate analogs hinders growth of mycobacteria, but a link to mycolic acid synthesis has not been established. In this study, we characterized a novel inhibitor of Ag85C, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile (I3-AG85). I3-AG85 was isolated from a panel of four inhibitors that exhibited structure- and dose-dependent inhibition of M. tuberculosis division in broth culture. I3-AG85 also inhibited M. tuberculosis survival in infected primary macrophages. Importantly, it displayed an identical MIC against the drug-susceptible H37Rv reference strain and a panel of extensively drug-resistant/multidrug-resistant M. tuberculosis strains. Nuclear magnetic resonance analysis indicated binding of I3-AG85 to Ag85C, similar to its binding to the artificial substrate octylthioglucoside. Quantification of mycolic acid-linked lipids of the M. tuberculosis envelope showed a specific blockade of TDM synthesis. This was accompanied by accumulation of trehalose monomycolate, while the overall mycolic acid abundance remained unchanged. Inhibition of Ag85C activity also disrupted the integrity of the M. tuberculosis envelope. I3-AG85 inhibited the division of and reduced TDM synthesis in an M. tuberculosis strain deficient in Ag85C. Our results indicate that Ag85 proteins are promising targets for novel antimycobacterial drug design.