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

2013 Spring.Includes bibliographical references.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

Stable Extracellular RNA Fragments of Mycobacterium tuberculosis Induce Early Apoptosis in Human Monocytes via a Caspase-8 Dependent Mechanism

The molecular basis of pathogen-induced host cell apoptosis is well characterized for a number of microorganisms. Mycobacterium tuberculosis is known to induce apoptosis and it was shown that live but not heat killed M. tuberculosis stimulates this biological pathway in monocytes. The dependence of this activity on live bacilli led us to hypothesize that products released or secreted by M. tuberculosis are the primary apoptotic factors for human monocytes. Thus, the culture filtrate of in vitro grown M. tuberculosis strain H37Rv was fractioned by conventional chromatography and the apoptosis-inducing activity of individual fractions was measured on human monocytes. The tests employed included measurement of cell membrane damage, caspase activation, and cytokine release. Small molecular weight RNAs of M. tuberculosis were recognized as the predominant apoptosis inducing factors. The RNA was comprised primarily of tRNA and rRNA fragments that stably accumulate in the culture filtrate during early log-phase growth. The RNA fragments signaled through a caspase-8 dependent, caspase-1 and TNF-α independent pathway that ultimately compromised the human monocytes' ability to control M. tuberculosis infection. These studies provide the first report of bacterial RNA inducing apoptosis. They also provide a foundation to pursue pathways for secretion or release of nucleic acids from M. tuberculosis and the impact of secreted RNA fragments on pathogenesis

Uptake and Accumulation of Oxidized Low-Density Lipoprotein during Mycobacterium tuberculosis Infection in Guinea Pigs

The typical host response to infection of humans and some animals by M. tuberculosis is the accumulation of reactive oxygen species generating inflammatory cells into discrete granulomas, which frequently develop central caseous necrosis. In previous studies we showed that infection of immunologically naïve guinea pigs with M. tuberculosis leads to localized and systemic oxidative stress that results in a significant depletion of serum total antioxidant capacity and the accumulation of malondialdehyde, a bi-product of lipid peroxidation. Here we show that in addition, the generation of excessive reactive oxygen species in vivo resulted in the accumulation of oxidized low density lipoproteins (OxLDL) in pulmonary and extrapulmonary granulomas, serum and lung macrophages collected by bronchoalveolar lavage. Macrophages from immunologically naïve guinea pigs infected with M. tuberculosis also had increased surface expression of the type 1 scavenger receptors CD36 and LOX1, which facilitate the uptake of oxidized host macromolecules including OxLDL. Vaccination of guinea pigs with Bacillus Calmette Guerin (BCG) prior to aerosol challenge reduced the bacterial burden as well as the intracellular accumulation of OxLDL and the expression of macrophage CD36 and LOX1. In vitro loading of guinea pig lung macrophages with OxLDL resulted in enhanced replication of bacilli compared to macrophages loaded with non-oxidized LDL. Overall, this study provides additional evidence of oxidative stress in M. tuberculosis infected guinea pigs and the potential role OxLDL laden macrophages have in supporting intracellular bacilli survival and persistence

Gr1(int)CD11b+ myeloid-derived suppressor cells in Mycobacterium tuberculosis infection.

Tuberculosis is one of the world's leading killers, stealing 1.4 million lives and causing 8.7 million new and relapsed infections in 2011. The only vaccine against tuberculosis is BCG which demonstrates variable efficacy in adults worldwide. Human infection with Mycobacterium tuberculosis results in the influx of inflammatory cells to the lung in an attempt to wall off bacilli by forming a granuloma. Gr1(int)CD11b(+) cells are called myeloid-derived suppressor cells (MDSC) and play a major role in regulation of inflammation in many pathological conditions. Although MDSC have been described primarily in cancer their function in tuberculosis remains unknown. During M. tuberculosis infection it is crucial to understand the function of cells involved in the regulation of inflammation during granuloma formation. Understanding their relative impact on the bacilli and other cellular phenotypes is necessary for future vaccine and drug design.We compared the bacterial burden, lung pathology and Gr1(int)CD11b(+) myeloid-derived suppressor cell immune responses in M. tuberculosis infected NOS2-/-, RAG-/-, C3HeB/FeJ and C57/BL6 mice. Gr-1(+) cells could be found on the edges of necrotic lung lesions in NOS2-/-, RAG-/-, and C3HeB/FeJ, but were absent in wild-type mice. Both populations of Gr1(+)CD11b(+) cells expressed high levels of arginase-1, and IL-17, additional markers of myeloid derived suppressor cells. We then sorted the Gr1(hi) and Gr1(int) populations from M. tuberculosis infected NOS-/- mice and placed the sorted both Gr1(int) populations at different ratios with naïve or M. tuberculosis infected splenocytes and evaluated their ability to induce activation and proliferation of CD4+T cells. Our results showed that both Gr1(hi) and Gr1(int) cells were able to induce activation and proliferation of CD4+ T cells. However this response was reduced as the ratio of CD4(+) T to Gr1(+) cells increased. Our results illustrate a yet unrecognized interplay between Gr1(+) cells and CD4(+) T cells in tuberculosis

Gr1^int and Gr1^hi cells sorted from <i>M. tuberculosis</i> infected NOS-/- mice activate Th17 cells and induce proliferation of CD4⁺ T cells.

<p>Specific populations of Gr1^int or Gr1^hi cells were sorted from infected NOS-/- mice and placed into <i>in </i><i>vitro</i> cultures with increasing numbers of either stimulated naïve or infected splenocytes. After 2 days of incubation T cells were evaluated for activation and memory profiles (A-D). Panel A -D shows lower ratios of Gr1^high (A and C) or Gr1^int (B and D) induce increased the percentage of activation of CD4⁺CD69⁺IL-17⁺ and memory CD4⁺CD44⁺IL-17⁺, when co-incubated with naïve (A and B) or <i>M. tuberculosis</i> infected splenocytes (C and D), respectively. Panel E and F shows Gr1 ^high and Gr1^int induced of proliferating CD4 T cells. However, as the splenocyte to Gr1^high or Gr1^int ratio increased the percentage of CD4+ proliferation was reduced. Results are expressed as the mean values of mean percentage of CD4⁺CD69⁺IL-17⁺, CD4⁺CD44⁺IL-17⁺ and proliferation. ANOVA and the Tukey post-test. *p<0.05, **p<0.01, ***p<0.001.</p

Gr1^int CD11b⁺ FSC^low cells predominate in the lungs of NOS2-/- mice.

<p>Further analysis of CD11b⁺FSC^low and CD11b⁺FSC^high cells was performed by staining with anti-Gr1 antibody. As determined by mean fluorescence intensity, CD11b⁺FSC^high had significantly higher Gr1 expression than CD11b⁺FSC^low (A). Starting at early time points NOS2-/- mice but not WT C57BL/6 mice have a significant number of cells giving an intermediate staining pattern for Gr-1 (B). At later time points, the influx of CD11b⁺FSC^high cells into the lungs of NOS2 -/- mice is also enhanced (C). Results are expressed as the Log mean cell number (± SEM, n=5) in the lung. Gr1^high CD11b⁺ FSC^high cells express markers compatible with a monocytic lineage. Both populations of Gr1 expressing cells were further analyzed by flow cytometry using the monocytic markers Ly6C, CD14 and F4/80 (D). As determined by the mean fluorescence intensity for these markers, a more precise phenotype for these two cellular populations would be Gr1^high CD11b⁺ FSC^high Ly6C^high CD14⁺ F4/80⁺ and Gr1^int CD11b⁺ FSC^low Ly6C^low CD14^low F4/80^low, compatible with a monocytic and granulocytic lineage, respectively. Results are expressed as the log mean cell number (± SEM, n=5) in the Lung. ***Student t test, * p<0.05, ** p<0.01, *** p<0.001.</p

Higher numbers of CD11b⁺ cells in the lungs of NOS2-/- KO mice.

<p>Flow cytometry analysis was performed on single cell suspensions obtained from lungs of WT C57/BL6 or NOS2-/- mice. In these mice increased numbers of CD11b⁺ cells but not CD11b⁺CD11c⁺ cells could be observed as the infection progressed (A, B, C). In contrast, differences for CD11c⁺ and CD4⁺ cells only became evident at later time points (B, D). No significant differences were observed for CD8⁺ cells (E). Results are expressed as the mean values of log mean cell number (± SEM, n=5) in the lung. Bacterial burden (F) was enumerated at different time points after a low dose aerosol infection with <i>M. tuberculosis</i>. As observed, bacterial burden in both murine strains is similar at early time points after infection and diverges after 30 days of infection. Results are expressed as the mean Log 10 CFUs (±SEM, n=5) in the lungs. Student t-test, * p<0.05, ** p<0.01, *** p<0.001.</p

Significant numbers of Gr1^int CD11b⁺ cells were also found in RAG2-/- and C3HeB/FeJ mice.

<p>The influx of Gr1^intCD11b⁺ cells was evaluated in immunocompromised RAG-/- (A), and immunocompetent C3HeB/FeJ mice (C) and NOS-/- (F). Again, large numbers of Gr1^intCD11b⁺ cells were observed in these three mouse strains and significant differences were observed in the Gr1⁺ MFI (B, D, G). Similar to NOS2 -/- mice, arginase activity was also increased in C3HeB/FeJ undergoing lung necrosis (E). In contrast, C3H/HeOuJ did not have major arginase activity. Similar to NOS2 -/- mice, arginase activity was also increased in C3HeB/FeJ undergoing lung necrosis (E). Results are expressed as the mean values of mean fluorescence intensity (MFI) or arginase activity (± SEM, n=5) in the Lung. **Student t-test, p<0.001. </p

Modelo de un simulador de la maniobra de la endoscopia gástrica basado en video

The following paper deals with a simple model of a gastric endoscopy procedure and a plastic model of the stomach and its distension. The correct use of imaging helps in the development of virtual reality systems, and provides a greater realism to the simulation itself. The goal is to experience the possibility of building patient simulator systems in Colombia, using locally available technology, at low costs and intended for the training of medical students.En el siguiente artículo se expone un modelo simple del procedimiento de endoscopia gástrica y un modelo plástico del estómago y de la distensión estomacal. El uso correcto de imágenes ayuda al desarrollo de sistemas de realidad virtual, y presenta más realismo a la simulación. El objetivo del trabajo consiste en experimentar la posibilidad de construir sistemas simuladores de pacientes en Colombia, utilizando la tecnología localmente disponible, a bajo costo y destinados para la formación de estudiantes de medicina

Gr1^int and Gr1^hi cells sorted from <i>M. tuberculosis</i> infected NOS-/- mice expressed arginase I and IL-17.

<p>Specific populations of Gr1^int or Gr1^hi cells were sorted from infected NOS-/- mice in an attempt to further characterize Gr1^int and Gr1^hi cell function during tuberculosis using flow cytometry. Panel A, demonstrates pre-sorted Gr1+CD11b+ cells which were then further characterized. Gr1^hi and Gr1^int sorted cells (B, C) both demonstrated high expression of arginase I and IL-17. Panel D shows the isotype controls for arginase-1 and IL-17A. </p