Currently there are a dozen or so of new vaccine candidates in clinical trials for prevention of tuberculosis (TB) and each formulation attempts to elicit protection by enhancement of cell-mediated immunity (CMI). In contrast, most approved vaccines against other bacterial pathogens are believed to mediate protection by eliciting antibody responses. However, it has been difficult to apply this formula to TB because of the difficulty in reliably eliciting protective antibodies. Here, we developed capsular polysaccharide conjugates by linking mycobacterial capsular arabinomannan (AM) to either Mtb Ag85b or B. anthracis protective antigen (PA). Further, we studied their immunogenicity by ELISA and AM glycan microarrays and protection efficacy in mice. Immunization with either Abg85b-AM or PA-AM conjugates elicited an AM-specific antibody response in mice. AM binding antibodies stimulated transcriptional changes in Mtb. Sera from AM conjugate immunized mice reacted against a broad spectrum of AM structural variants and specifically recognized arabinan fragments. Conjugate vaccine immunized mice infected with Mtb had lower bacterial numbers in lungs and spleen, and lived longer than control mice. These findings provide additional evidence that humoral immunity can contribute to protection against Mtb. Vaccine design in the TB field has been driven by the imperative of attempting to elicit strong cell-mediated responses. However, in recent decades evidence has accumulated that humoral immunity can protect against many intracellular pathogens through numerous mechanisms. In this work, we demonstrate that immunization with mycobacterial capsular arabinomannan (AM) conjugates elicited responses that contributed to protection against Mtb infection. We developed two different conjugates including capsular AM linked to the Mtb related protein Ag85b or the Mtb unrelated PA from B. anthracis and found that immunization with AM conjugates elicited antibody populations with different specificities. These surface-specific antibodies could directly modify the transcriptional profile and metabolism of mycobacteria. In addition, we observed a prolonged survival and a reduction in bacterial numbers in lungs and spleen in mice immunized with Ag85b-AM conjugates after infection with Mtb and that the presence of AM-binding antibodies was associated with modest prolongation in survival and a marked reduction in mycobacterial dissemination. Finally, we show that AM is antigenically variable and could potentially form the basis for a serological characterization of mycobacteria based on serotypes.

The heterogeneity of the clinical outcome of Mycobacterium tuberculosis (Mtb) infection may be due in part to different strategies used by circulating strains to cause disease. This heterogeneity is one of the main limitations to eradicate tuberculosis disease. In this study, we have compared the transcriptional response of two closely related Colombian clinical isolates (UT127 and UT205) of the LAM family under two axenic media conditions. These clinical isolates are phenotypically different at the level of cell death, cytokine production, growth kinetics upon in vitro infection of human tissue macrophages, and membrane vesicle secretion upon culture in synthetic medium. Using RNA-seq, we have identified different pathways that account for two different strategies to cope with the stressful condition of a carbon-poor media such as Sauton’s. We showed that the clinical isolate UT205 focus mainly in the activation of virulence systems such as the ESX-1, synthesis of diacyl-trehalose, polyacyl-trehalose, and sulfolipids, while UT127 concentrates its efforts mainly in the survival mode by the activation of the DNA replication, cell division, and lipid biosynthesis. This is an example of two Mtb isolates that belong to the same family and lineage, and even though they have a very similar genome, its transcriptional regulation showed important differences. This results in summary highlight the necessity to reach a better understanding of the heterogeneity in the behavior of these circulating Mtb strains which may help us to design better treatments and vaccines and to identify new targets for drugs.

Mycobacterium tuberculosis (Mtb) is still one of the primary pathogens of humans causing tuberculosis (TB) disease. Mtb embraces nine well-defined phylogenetic lineages with biological and geographical disparities. The lineage L4 is the most globally widespread of all lineages and was introduced to America with European colonization. Taking advantage of many genome projects available in public repositories, we undertake an evolutionary and comparative genomic analysis of 522 L4 Latin American Mtb genomes. Initially, we performed careful quality control of public read datasets and applied several thresholds to filter out low-quality data. Using a genome de novo assembly strategy and phylogenomic methods, we spotted novel south American clades that have not been revealed yet. Additionally, we describe genomic deletion profiles of these strains from an evolutionary perspective and report Mycobacterium tuberculosis L4 sublineages signature-like gene deletions, some of the novel. One is a specific deletion of 6.5 kbp that is only present in sublineage 4.1.2.1. This deletion affects a complex group of 10 genes with putative products annotated, among others, as a lipoprotein, transmembrane protein, and toxin/antitoxin system proteins. The second novel deletion spans for 4.9 kbp and specific of a particular clade of the 4.8 sublineage and affects 7 genes. The last novel deletion affects 4 genes, extends for 4.8 kbp., and is specific to some strains within the 4.1.2.1 sublineage that are present in Colombia, Peru and Brasil.

Up to date, Mycobacterium tuberculosis (Mtb) remains as the worst intracellular killer pathogen. To establish infection, inside the granuloma, Mtb reprograms its metabolism to support both growth and survival, keeping a balance between catabolism, anabolism and energy supply. Mtb knockouts with the faculty of being essential on a wide range of nutritional conditions are deemed as target candidates for tuberculosis (TB) treatment. Constraint-based genome-scale modeling is considered as a promising tool for evaluating genetic and nutritional perturbations on Mtb metabolic reprogramming. Nonetheless, few in silico assessments of the effect of nutritional conditions on Mtb’s vulnerability and metabolic adaptation have been carried out. A genome-scale model (GEM) of Mtb, modified from the H37Rv iOSDD890, was used to explore the metabolic reprogramming of two Mtb knockout mutants (pfkA- and icl-mutants), lacking key enzymes of central carbon metabolism, while exposed to changing nutritional conditions (oxygen, and carbon and nitrogen sources). A combination of shadow pricing, sensitivity analysis, and flux distributions patterns allowed us to identify metabolic behaviors that are in agreement with phenotypes reported in the literature. During hypoxia, at high glucose consumption, the Mtb pfkA-mutant showed a detrimental growth effect derived from the accumulation of toxic sugar phosphate intermediates (glucose-6-phosphate and fructose-6-phosphate) along with an increment of carbon fluxes towards the reductive direction of the tricarboxylic acid cycle (TCA). Furthermore, metabolic reprogramming of the icl-mutant (icl1&icl2) showed the importance of the methylmalonyl pathway for the detoxification of propionyl-CoA, during growth at high fatty acid consumption rates and aerobic conditions. At elevated levels of fatty acid uptake and hypoxia, we found a drop in TCA cycle intermediate accumulation that might create redox imbalance. Finally, findings regarding Mtb-mutant metabolic adaptation associated with asparagine consumption and acetate, succinate and alanine production, were in agreement with literature reports. This study demonstrates the potential application of genome-scale modeling, flux balance analysis (FBA), phenotypic phase plane (PhPP) analysis and shadow pricing to generate valuable insights about Mtb metabolic reprogramming in the context of human granulomas.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis (Mtb), leading to pulmonary and extrapulmonary TB, whereby Mtb is disseminated to many other organs and tissues. Dissemination occurs early during the disease, and bacteria can be found first in the lymph nodes adjacent to the lungs and then later in the extrapulmonary organs, including the spleen. The early global gene expression response of human tissue macrophages and intracellular clinical isolates of Mtb has been poorly studied. Using dual RNA-seq, we have explored the mRNA profiles of two closely related clinical strains of the Latin American and Mediterranean (LAM) family of Mtb in infected human splenic macrophages (hSMs). This work shows that these pathogens mediate a distinct host response despite their genetic similarity. Using a genome-scale host–pathogen metabolic reconstruction to analyze the data further, we highlight that the infecting Mtb strain also determines the metabolic response of both the host and pathogen. Thus, macrophage ontogeny and the genetic-derived program of Mtb direct the host–pathogen interaction.