Novel extracellular products of Mycobacterium tuberculosis: Composition, synthesis, and relevance to disease

Mycobacterium tuberculosis (Mtb) is a bacterium causing great morbidity and mortality especially in developing countries. In order to identify possible areas of intervention to positively alter the history of the disease, a better identification and characterization of Mtb virulence determinants is required. Specifically, biosynthetic routes for these virulence determinants should be pursued. Furthermore, the interaction between the host and Mtb virulence determinants should be characterized at a molecular level. It is hoped that unraveling these pathogenesis mechanisms could lead to novel strategies to combat the infection. In Chapter II, the identification of secreted Mtb molecules that induce macrophage apoptosis was performed. Apoptosis is a mechanism of host cell death and in the life cycle of Mtb, different modalities of host cell death have been suggested to tip the balance between bacterial eradication and multiplication. However, a systematic approach to identify and characterize secreted Mtb molecules that modulate host cell death, has not been performed. Surprisingly, extracellular Mtb RNA fragments were identified as a potent inducer of host cell apoptosis. This extracellular RNA was identified as predominantly rRNA and tRNA fragments that accumulated early during in vitro culture of Mtb. Mechanistic studies determined that the Mtb RNA induced macrophage apoptosis through a caspase-8-dependent, TNF-α-independent mechanism. Importantly, Mtb RNA abrogated the macrophage's ability to control an Mtb infection. In Chapter II, the first description of an extracellular Mtb RNA with potent biological activity was performed. This opens an exciting field in research of host interactions with pathogen nucleic acids. Chapters III and IV were devoted to identifying the biochemical pathway involved in α-L-polyGlutamine (α-L-polyGln) biosynthesis and determining its role in pathogenesis in the murine model of TB. α-L-polyGln is an Mtband Mycobacterium bovis (M. bovis) specific product and its presence in virulent Mycobacterium spp., suggest that it could play an important role in pathogenesis. Bacillus anthracis (B. anthracis) synthesizes γ-D-polyGlutamate (γ-D-polyGlu), an amino acid polymer that is present in its capsule and is absolutely required for pathogenicity. As the pathway for B. anthracis γ-D-polyGlu biosynthesis has been well characterized, it was used as a model to start elucidating the Mtb α-L-polyGln biosynthetic pathway. Bioinformatics analysis suggested that Rv0574c and Rv2394 are the Mtb homologues for B. anthracis CapA and CapD, respectively. In Chapter III, a complete biochemical characterization of Rv2394 was performed. Similar to other γ-glutamyltranspeptidases (GGTs), Rv2394 had a conserved catalytic motif consisting of a Threonine (Thr) residue. Mutating this Thr residue to Alanine (Ala) abrogated the enzymatic activity of Rv2394, including its autocatalytic activation. In contrast to eukaryote GGT, Rv2394 was able to perform a GGT activity in the presence of physiological relevant acceptors such as di- or oligopeptides containing Glutamate (Glu) or Glutamine (Gln). In addition to its autocatalytic activation, Rv2394 was shown to be post-translationally modified with hexose residues. A putative phosphorylation and acylation modification also seemed to be present in Rv2394. In Chapter IV, Mtb mutants for rv0574c and rv2394 were engineered and characterized biochemically to determine if the concentration of α-L-polyGln had been altered. Furthermore, the mutant's virulence was evaluated in the murine model of TB. Consistent with a putative role in α-L-polyGln, both mutants had reduced concentrations of Glu and ammonia in the cell wall. Furthermore, preliminary analysis suggested that the apolar lipid profiles were also altered by these mutations. In the murine model, Mtb mutants had a tendency to grow faster in the initial stages of disease. However, the difference between wild type (WT) and mutant strains was not statistically significant and normalized during the later stages of disease. Furthermore, mutant Mtb also seemed to induce more lung damage. In contrast to bacterial burden, this difference persisted throughout the course of the study. Altogether, these results suggest that Rv0574c and Rv2394 participate in the biosynthesis of α-L-polyGln. Remarkably, similar biochemical and phenotypic results were obtained for both mutants despite being encoded in different loci. These initial results provide the foundation for future studies characterizing the biochemical pathway involved in α-L-polyGln biosynthesis

Antigen Presentation of mRNA-Based and Virus-Vectored SARS-CoV-2 Vaccines

Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which has reached pandemic proportions. A number of effective vaccines have been produced, including mRNA vaccines and viral vector vaccines, which are now being implemented on a large scale in order to control the pandemic. The mRNA vaccines are composed of viral Spike S1 protein encoding mRNA incorporated in a lipid nanoparticle and stabilized by polyethylene glycol (PEG). The mRNA vaccines are novel in many respects, including cellular uptake and the intracellular routing, processing, and secretion of the viral protein. Viral vector vaccines have incorporated DNA sequences, encoding the SARS-CoV-2 Spike protein into (attenuated) adenoviruses. The antigen presentation routes in MHC class I and class II, in relation to the induction of virus-neutralizing antibodies and cytotoxic T-lymphocytes, will be reviewed. In rare cases, mRNA vaccines induce unwanted immune mediated side effects. The mRNA-based vaccines may lead to an anaphylactic reaction. This reaction may be triggered by PEG. The intracellular routing of PEG and potential presentation in the context of CD1 will be discussed. Adenovirus vector-based vaccines have been associated with thrombocytopenic thrombosis events. The anti-platelet factor 4 antibodies found in these patients could be generated due to conformational changes of relevant epitopes presented to the immune system

Mycobacterium bovis BCG-Mediated Protection against W-Beijing Strains of Mycobacterium tuberculosis Is Diminished Concomitant with the Emergence of Regulatory T Cells▿†

Despite issues relating to variable efficacy in the past, the Mycobacterium bovis BCG vaccine remains the basis for new-generation recombinant vaccines currently in clinical trials. To date, vaccines have been tested mostly against laboratory strains and not against the newly emerging clinical strains. In this study, we evaluated the ability of BCG Pasteur to protect mice from aerosol infections with two highly virulent W-Beijing clinical strains, HN878 and SA161. In a conventional 30-day protection assay, BCG was highly protective against both strains, but by day 60 of the assay, this protection was diminished. Histological examination of the lungs of vaccinated animals showed reduced lung consolidation and smaller and more-organized granulomas in the vaccinated mice after 30 days, but in both cases, these tissues demonstrated worsening pathology over time. Effector T cell responses were increased in the vaccinated mice infected with HN878, but these diminished in number after day 30 of the infections concomitant with increased CD4+ Foxp3+ T cells in the lungs, draining lymph nodes, and the spleen. Given the concomitant decrease in effector immunity and continued expansion of regulatory Foxp3+ cells observed here, it is reasonable to hypothesize that downregulation of effector immunity by these cells may be a serious impediment to the efficacy of BCG-based vaccines

A new antibiotic with potent activity targets MscL

The growing problem of antibiotic-resistant bacteria is a major threat to human health. Paradoxically, new antibiotic discovery is declining, with most of the recently approved antibiotics corresponding to new uses for old antibiotics or structurally similar derivatives of known antibiotics. We used an in silico approach to design a new class of nontoxic antimicrobials for the bacteria-specific mechanosensitive ion channel of large conductance, MscL. One antimicrobial of this class, compound 10, is effective against methicillin-resistant Staphylococcus aureus with no cytotoxicity in human cell lines at the therapeutic concentrations. As predicted from in silico modeling, we show that the mechanism of action of compound 10 is at least partly dependent on interactions with MscL. Moreover we show that compound 10 cured a methicillin-resistant S. aureus infection in the model nematode Caenorhabditis elegans. Our work shows that compound 10, and other drugs that target MscL, are potentially important therapeutics against antibiotic-resistant bacterial infections

Beijing sublineages of Mycobacterium tuberculosis differ in pathogenicity in the Guinea pig

The Beijing family of Mycobacterium tuberculosis strains is part of lineage 2 (also known as the East Asian lineage). In clinical studies, we have observed that isolates from the sublineage RD207 of lineage 2 were more readily transmitted among humans. To investigate the basis for this difference, we tested representative strains with the characteristic Beijing spoligotype from four of the five sublineages of lineage 2 in the guinea pig model and subjected these strains to comparative whole-genome sequencing. The results of these studies showed that all of the clinical strains were capable of growing and causing lung pathology in guinea pigs after low-dose aerosol exposure. Differences between the abilities of the four sublineages to grow in the lungs of these animals were not overt, but members of RD207 were significantly more pathogenic, resulting in severe lung damage. The RD207 strains also induced much higher levels of markers associated with regulatory T cells and showed a significant loss of activated T cells in the lungs over the course of the infections. Whole-genome sequencing of the strains revealed mutations specific for RD207 which may explain this difference. Based on these data, we hypothesize that the sublineages of M. tuberculosis are associated with distinct pathological and clinical phenotypes and that these differences influence the transmissibility of particular M. tuberculosis strains in human population