Rozprawy doktorskie na temat „Mtb Infection”

Lymphoblastic leukemia 1 (Lyl1) is a well-studied transcription factor known to exhibit oncogenic potential during various forms of leukemia. Since its discovery in 1989, many reports have been published describing its relationship with cancer as well as demonstrating its function during hematopoiesis. Lyl1 has been shown to serve a significant role during thymopoiesis by contributing to T-cell development. However, it has been recently reported that irrespective of its significance during T-cell development, mature comparable single positive T-cells are observed in mouse models. The use of murine models has been crucial in identifying potential targets for host-directed therapies (HDT) which has been shown to provide great potential in treating tuberculosis (TB). It is evident that Mycobacterium tuberculosis (Mtb), the causative agent for TB, is capable of developing resistance to various treatments that target the bacterium itself. Therefore, by designing therapies that directly target host factors could assist in circumventing Mtb resistance. By analyzing Mtb-infected bone marrow-derived macrophages (BMDM) that have been subjected to genome-wide transcriptional deep sequencing of total RNA using a single molecule sequencer in conjunction with the cap analysis gene expression (CAGE) technique, various differentially expressed genes were identified, including the oncogenic transcription factor, Lyl1. With the use of murine models, we investigated whether Lyl1 is important for various immunological responses at steady state, the regulation of Lyl1 in response to various immune stimulants including LPS and whether this transcription factor is relevant in bacterial infections including Listeria monocytogenes (Lm) and Mtb. The data in this thesis demonstrate comparable immunological responses, including cellular recruitment by means of flow cytometry and cytokine responses by means of ELISA, between naïve littermate control and Lyl1-deficient mice. Further evaluation of Lyl1 regulation revealed the influence of MAPk and NFκB signaling on Lyl1 expression upon LPS stimulation by significantly downregulating this transcription factor in immune stimulated macrophages. A role for Lyl1 during bacterial infections was observed in Lm-infected mice whereby Lyl1-/- mice succumbed earlier to listeriosis compared to the littermate controls. We further established a functional role for this transcription factor during Mtb infection in vitro and in vivo. The early surrender of Lyl1-deficient mice to Mtb HN878 infection, accompanied by increased bacterial burden during chronic Mtb infection, demonstrated enhanced susceptibility in the absence of Lyl1. We show that Lyl1-deficient host susceptibility is a consequence of enhanced inflammatory responses and increased bacterial growth. This is demonstrated by increased neutrophilic inflammation, pro-inflammatory cytokine and chemokine secretion along with a reduction in anti-inflammatory cytokine release during chronic Mtb infection. Here, we demonstrate the first non-leukemia role for Lyl1 by suggesting a role and requirement for this transcription factor during bacterial infections. Given the significant role during Mtb infection, our studies suggest the use of Lyl1 associated pathways as a potential HDT target for TB.

Despite effective antibiotics, Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, still infects nearly one-third of the world's population. While key immune factors including CD4+ T cells and IFNg production have been identified, there are still many antimicrobial mechanisms yet to be explored. Here we characterized the role of invariant natural killer T (iNKT) cells and GM-CSF during Mtb infection.

Les cèl·lules presentadores d’antigen de llinatge mieloide tenen la capacitat de respondre a una infecció d’una manera ràpida i eficient coordinant respostes immunitàries innates i adaptatives. Malgrat això, en el cas de la infecció pel virus de la immunodeficiència humana de tipus 1 (VIH-1), aquestes cèl·lules poden contribuir a la patogènesi viral a través de la captura i la transmissió de partícules virals a les cèl·lules diana, un procés conegut com trans-infecció. Aquest mecanisme depèn de Siglec-1 (CD169), un receptor de membrana de les cèl·lules mieloides que reconeix gangliòsids sialilats presents a la membrana del virus. Per tal d’analitzar in vivo la contribució de la trans-infecció en la patogènesi del VIH-1, vam buscar individus SIGLEC1-deficients i vam identificar 85 individus heterozigots i 2 homozigots per una variant de pèrdua de funció que aboleix l’expressió de Siglec-1. De manera rellevant, les cèl·lules d’aquests individus mancaven de l’activitat de Siglec-1 en relació a la captura i la transmissió del VIH-1. Malgrat aquest fenotip, no vam observar diferències prominents pel què fa a la susceptibilitat a la infecció per VIH-1 ni a la progressió cap a la síndrome d’immunodeficiència adquirida (SIDA) en els individus portadors d’aquesta variant de SIGLEC1. Malgrat tot, l’anàlisi de l’efecte del truncament de Siglec-1 en la progressió a SIDA no va ser concloent degut a la mida limitada de la cohort, la manca d’una història clínica complerta amb informació sobre la data de seroconversió, la restricció d’estudiar només períodes sense tractament i la co-infecció amb patògens addicionals que podrien influenciar el fenotip observat en la direcció oposada al que s’esperava. De fet, aquesta darrera limitació ens va portar a investigar l’efecte de la variant SIGLEC1-deficient en les co-infeccions associades al VIH-1 i vam trobar una associació significativa entre aquesta variant i la disseminació extrapulmonar de Mycobacterium tuberculosis (Mtb) en dues cohorts clíniques que inclouen 6,256 individus. Quan vam analitzar l’absència de Siglec-1 en un model murí, els ratolins knockout per Siglec-1 van presentar una propagació local de bacteris al pulmó i malgrat tenir una càrrega bacil·lar similar, van desenvolupar lesions més extenses en comparació amb els ratolins salvatges. A més a més, vam demostrar que Siglec-1 és necessari per tal d’induir la presentació d’antígens a través de la captura de vesícules extracel·lulars. Proposem un model on l’absència de Siglec-1 endarrereix l’inici d’una immunitat que protegeix enfront el micobacteri limitant l’intercanvi d’antígens mitjançant vesícules extracel·lulars, permetent així una propagació local del micobacteri que incrementa el risc d’una disseminació extrapulmonar. En resum, al llarg d’aquesta tesi hem explorat el concepte d’antagonisme pleiotròpic en individus co-infectats portadors de la variant SIGLEC1-deficient, on l’alteració del control immunitari del micobacteri en absència de Siglec-1 podria influenciar el curs clínic dels individus infectats pel VIH-1, emmascarant així els beneficis esperats d’aquesta variant en retardar la progressió a SIDA.
Las células presentadoras de antígeno de linaje mieloide tienen la capacidad de responder a una infección de una manera rápida y eficiente coordinando respuestas inmunes innatas y adaptativas. Sin embargo, en el caso de la infección por el virus de la inmunodeficiencia humana de tipo 1 (VIH-1), estas células pueden contribuir en la patogénesis viral a través de la captura y la transmisión de partículas virales a las células diana, un proceso conocido como trans-infección. Este mecanismo depende de Siglec-1 (CD169), un receptor de membrana de las células mieloides que reconoce gangliósidos sialidados presentes en la membrana del virus. Para analizar in vivo la contribución de la trans-infección en la patogénesis del VIH-1, buscamos individuos SIGLEC1-deficientes e identificamos 85 individuos heterocigotos y 2 homocigotos para una variante de pérdida de función que suprime la expresión de Siglec-1. De manera relevante, las células de estos individuos carecían de la actividad de Siglec-1 en relación a la captura y la transmisión del VIH-1. A pesar de este fenotipo, no hemos observado diferencias prominentes con respecto a la susceptibilidad a la infección por VIH-1 ni a la progresión hacia el síndrome de inmunodeficiencia adquirida (SIDA) en los individuos portadores de esta variante de SIGLEC1. A pesar de ello, el análisis del efecto del truncamiento de Siglec-1 en la progresión a SIDA no resultó concluyente debido al tamaño limitado de la cohorte, la falta de una historia clínica completa con información sobre la fecha de seroconversión, la restricción de estudiar solamente períodos sin tratamiento y la co-infección con patógenos adicionales que podrían influenciar el fenotipo observado en la dirección opuesta a lo esperado. De hecho, esta última limitación nos llevó a investigar el efecto de la variante SIGLEC1-deficiente en las co-infecciones asociadas al VIH-1 y encontramos una asociación significativa entre esta variante y la diseminación extrapulmonar de Mycobacterium tuberculosis (Mtb) en dos cohortes clínicas que incluyen 6,256 individuos. Cuando analizamos la ausencia de Siglec-1 en un modelo murino, los ratones knockout para Siglec-1 presentaron una propagación local de bacterias en el pulmón y a pesar de tener una carga bacilar similar, desarrollaron lesiones más extensas en comparación con los ratones salvajes. Además, hemos demostrado que Siglec-1 es necesario para inducir la presentación de antígenos a través de la captura de vesículas extracelulares. Proponemos un modelo en el que la ausencia de Siglec-1 retrasa el inicio de una inmunidad que protege frente la micobacteria limitando el intercambio de antígenos mediante vesículas extracelulares, permitiendo así una propagación local de la micobacteria que incrementa el riesgo de una diseminación extrapulmonar. En resumen, a lo largo de esta tesis hemos explorado el concepto de antagonismo pleiotrópico en individuos co-infectados portadores de la variante SIGLEC1-deficiente, donde la alteración del control inmune de la micobacteria en ausencia de Siglec-1 podría influenciar el curso clínico de los individuos infectados por VIH-1, enmascarando así los beneficios esperados de esta variante en el retraso de la progresión a SIDA.
Antigen presenting cells of the myeloid lineage have the ability to respond rapidly and effectively to infection by coordinating innate and adaptive immune responses. However, in the case of human immunodeficiency virus type 1 (HIV-1) infection, these cells might contribute to viral pathogenesis through the capture and transmission of infectious viral particles to target cells, a process known as trans-infection. This mechanism depends on Siglec-1 (CD169), a myeloid-cell surface receptor that recognizes sialylated gangliosides present on the viral membrane. To dissect the contribution of trans-infection in HIV-1 pathogenesis in vivo, we searched for SIGLEC1 null individuals and identified 85 heterozygous and 2 homozygous people with a loss-of-function variant that abrogates Siglec-1 expression. Importantly, cells from these individuals were defective for Siglec-1 activity in HIV-1 capture and transmission. Despite this phenotype, we did not observe prominent differences on HIV-1 susceptibility nor progression to acquired immunodeficiency syndrome (AIDS) in individuals harboring the SIGLEC1 null variant. Nonetheless, analysis of the effect of Siglec-1 truncation on progression to AIDS was not conclusive due to the limited cohort size, the lack of complete clinical records such as the seroconversion date, the restriction to study only off-therapy periods, and the co-infection with additional pathogens that might influence the observed phenotype in the opposite direction from what was expected. As a matter of fact, the latest limitation prompted us to investigate the effect of the SIGLEC1 null variant in HIV-1 co-infections and we found a significant association between this variant and extrapulmonary dissemination of Mycobacterium tuberculosis (Mtb) in two clinical cohorts comprising 6,256 individuals. When we analyzed the absence of Siglec-1 in a murine model, local spread of bacteria within the lung was apparent in Mtb-infected Siglec-1 knockout mice which, despite having similar bacterial load, developed more extensive lesions compared to wild type mice. Moreover, we demonstrated that Siglec-1 is necessary to induce antigen presentation through extracellular vesicle uptake. We postulate that lack of Siglec-1 delays the onset of protective immunity against Mtb by limiting antigen exchange via extracellular vesicles, allowing for an early local spread of mycobacteria that increases the risk for extrapulmonary dissemination. Overall, through this thesis we have explored the concept of antagonistic pleiotropy in co-infected individuals harboring the SIGLEC1 null variant, where the impaired immune control of Mtb in the absence of Siglec-1 could influence the clinical course of HIV-1 infected individuals, thus masking the expected benefits of this variant on delaying AIDS progression.
Universitat Autònoma de Barcelona. Programa de Doctorat en Bioquímica, Biologia Molecular i Biomedicina

Tuberculosis (TB) is primarily a pulmonary disease caused by Mycobacterium tuberculosis (Mtb). Mtb is highly infectious, but studies have shown that only 5–15% of Mtb-infected individuals develop TB disease. The Bacille Calmette-Gu.rin (BCG) vaccine is the only commercially available Mtb vaccine, but its efficacy varies based on the strain used. The Mtb PhoPR-mutant variant, MTBVAC, has been tested as a possible attenuated live vaccine against Mtb. Although it has successfully conferred durable CD4+ T-cell responses in infants, it has also resulted in adverse effects. Our goal is to identify PhoPR-regulated gene(s) that mediate Mtb-induced burst size necrosis in infected cells. PhoPR is a two-component system in mycobacteria. PhoR responds to environmental cues, such as changes in pH, and phosphorylates the PhoP transcription factor, which then activates or suppresses the expression of approximately 40 Mtb genes. The Mtb PhoPR-mutant strain is able to replicate in infected macrophages, but it does not induce the horizontal spread of Mtb to other immune cells. Our lab has previously shown that virulent, cytopathic strains of Mtb, such as H37Rv, suppress early apoptosis, have faster replication rates in macrophages, and trigger cell death at a lethal load threshold of approximately 25 bacteria. Cell death of infected macrophages primarily occurs via necrosis, which involves nuclear pyknosis without DNA fragmentation and general disruption of lipid bilayer membranes. Viable bacilli are released to infect other macrophages and neutrophils recruited to the developing TB lesion. Here, we show that PhoP contributes to burst size necrosis in macrophages and that the PhoP-regulated genes, fadD21 and pks3, are potential drivers of this necrosis. FadD21 and pks3 are involved in the generation of diacyl trehalose/penta-acyl trehalose (DAT/PAT) for cell wall synthesis, suggesting that Mtb cell wall composition may determine virulence. Therefore, we have uncovered potential targets for early intervention or vaccinations to avoid granuloma formation or tissue damage in response to Mtb-induced macrophage necrosis.

Macrophages infected with a heavy burden of M.tb Erdman undergo a cell death that initially resembles apoptosis but quickly transitions to necrosis. Unlike the previously reported TNF dependent apoptosis induced by avirulent Mycobacterium [1], this form of macrophage cell death is not microbicidal [2]. Microbicidal effects are observed however, when the heavily infected macrophage encounters an uninfected naïve macrophage. My studies describe in part, the crosstalk between the uninfected and infected macrophage that results in the killing of the intracellular M.tb Cell contact between the two cell populations is not necessary for this killing of bacilli to occur and the soluble “signal” of communication between the two cell populations is transferrable, without naïve macrophages present, to newly infected cells also resulting in the reduced viability of the bacilli. We have found that when the IL-1 receptor is absent in the naïve macrophage population that the co-culture antimycobacterial effect is abrogated, suggesting that IL-1 released by the infected dying macrophage is critical for naïve macrophages to respond in a way that results in the decrease in mycobacterial viability. The signaling between the two cell population ultimately converges on activation of iNOS in the infected cell however ROS appears not to be involved.

Macrophages can be polarized into M1 and M2 macrophages based on the composition of the milieu. Human macrophages have been poorly characterized. In this study, various macrophage subsets were generated by treating monocyte-derived macrophages (MDMs) with IFNγ (M1), IL-4 (M2a), LPS and IL-1β (M2b) or IL-10 (M2c) which were characterized with respect to their cell surface marker profile and functional profile in the context of cytokine production, susceptibility to HIV infection and apoptosis. Each polarization state demonstrated a unique cell surface marker profile and cytokine profile. In addition M1 macrophages were shown to produce IFNγ post TLR stimulation. Moreover, M1 macrophages were highly sensitive to apoptosis following Smac mimetic treatment. Furthermore, M2a and M2c macrophages were resistant to apoptosis, induced by PI3K blockage and IAPs degradation respectively, and at the same time supported productive HIV infection unlike the other macrophage subsets. These findings might lead to better understanding of HIV reservoir formation and be used to develop therapies to eradicate it.

Mycobacterium abscessus, une mycobactérie à croissance rapide, est responsable d'infections pulmonaires chez les patients atteints de mucoviscidose. Le traitement recommandé comprend une phase initiale basée sur l'association d'un carbapénème (imipénème), d'un macrolide (azithromycine), d'un aminoside (amikacine) et d'une glycylcycline (tigécycline). L’équipe a étudié l’optimisation des traitements impliquant les β-lactamines et a démontré que l’avibactam, un inhibiteur de β-lactamases de 2ème génération appartenant à la famille du diazabicyclooctane, inhibe le β-lactamase BlaMab produit par M. abscessus et augmente de manière significative l’efficacité de l'imipénème à la fois in vitro, dans les macrophages et dans le modèle de poisson zèbre. L'expression du gène de la β-lactamase s'est avérée être induite dans les macrophages infectés. L'objectif du projet de thèse était d'évaluer l'efficacité de nouvelles combinaisons comprenant des d'inhibiteurs de β-lactamase et d'étudier la régulation de la β-lactamase dans les macrophages. Dans la première partie de la thèse, de nouvelles combinaisons d'antibiotiques ont été évaluées in vitro et dans des macrophages infectés par M. abscessus. La rifabutine, habituellement utilisée dans le traitement d'infections dues à d'autres mycobactéries, a montré une activité synergique avec l'imipénème in vitro, mais l'association n'était pas bactéricide. Dans les macrophages infectés, la rifabutine a renforcé l'activité de l'imipénème et l'ajout d'avibactam a entraîné une diminution du nombre de bactérie intracellulaire. Le tédizolide, développé pour le traitement des infections à staphylocoques, a montré une faible synergie in vitro mais pas d’activité bactéricide contre M. abscessus. Dans les macrophages, le tédizolide augmentait l'activité de l'imipénème et la quadruple combinaison imipénème-tédizolide-rifabutine-avibactam permettait de diminuer le nombre de bactéries intracellulaires de 91%. Enfin, l'association de l'imipénème avec le relebactam, un nouvel inhibiteur de β-lactamases développé en association avec l'imipénème, s'est révélée aussi active que la combinaison imipénème-avibactam à la fois in vitro et dans les macrophages. La deuxième partie de la thèse était focalisée sur l'identification du stress responsable de l'induction de la production de β-lactamase dans les macrophages. Dans ce but, M. abscessus a été cultivée in vitro dans différents milieux de culture mimant des conditions de stress aux quelles la bactérie est potentiellement confrontée dans les macrophages. L'activité spécifique de la β-lactamase a été déterminée en utilisant comme substrat une β-lactamine chromogène, la nitrocéfine. Aucune des conditions physicochimiques testées n'a conduit à une induction, incluant un pH acide, de fortes concentrations de métaux, un stress oxydatif ou la présence de β-lactamines. Le dernier objectif était d’étudier l’impact du polymorphisme N versus G situé dans le motif conservé SDN des β-lactamases sur l’activité des combinaisons comprenant une β-lactamine et un inhibiteur de β-lactamase. BlaMab de M. abscessus contient le motif SDN alors que BlaC de M. tuberculosis contient le motif SDG, un polymorphisme qui détermine l'efficacité de l'inhibition de BlaMab par l'avibactam de BlaC par le clavulanate in vitro. Deux souches isogéniques de M. abscessus ont été construites par échange allélique. Comparativement à la souche sauvage, la souche produisant BlaMab avec la substitution N vers G était moins sensible aux combinaisons comprenant l’avibactam avec, en parallèle, un gain d’efficacité pour les combinaisons comprenant le clavulanate. Dans le contexte de BlaC, la substitution de G vers N a potentialisé l'inhibition par l'avibactam. Ces résultats établissent que le polymorphisme SDN/SDG détermine en partie l'efficacité des β-lactamines associées à l'avibactam ou au clavulanate, en accord avec les études cinétiques précédemment réalisées in vitro avec des β-lactamases purifiées
Mycobacterium abscessus, a rapidly growing mycobacteria, is responsible for pulmonary infections in cystic fibrosis patients. The recommended treatment consists in an initial phase with the combination of a carbapenem (imipenem), a macrolide (azithromycin), an aminoglycoside (amikacin), and a glycylcycline (tigecycline). The team has investigated the optimization of treatments involving β-lactams and have demonstrated that avibactam, a 2nd generation β-lactamase inhibitor belonging to the diazabicyclooctane (DBO) family, inhibits the β-lactamase BlaMab produced by M. abscessus and substantially increases the efficacy of imipenem both in vitro, intracellularly, and in a zebrafish model. Expression of the β-lactamase gene was found to be induced in infected macrophages. The aim of my PhD project was to evaluate the efficacy of new β-lactam-β-lactamase inhibitor combinations and to investigate β-lactamase regulation in macrophages. In the first part of the thesis, new antibiotic combinations were evaluated in vitro and in macrophages infected by M. abscessus. Rifabutin, usually used in the treatment of infections due to other mycobacteria, showed synergistic activity with imipenem in vitro but the combination was not bactericidal. In infected macrophages, rifabutin enhanced the activity of imipenem and the addition of avibactam led to increased killing. Tedizolid, developed for the treatment of staphylococcal infections, displayed weak synergy in vitro but no bactericidal activity against M. abscessus. In macrophages, tedizolid enhanced the activity of imipenem and the imipenem-tedizolid-rifabutin-avibactam quadruple combination afforded 91% intracellular killing. Finally, the association of imipenem with relebactam, a new β-lactamase inhibitor developed in combination with imipenem, was found to be as active as the imipenem-avibactam both in vitro and in macrophage model. The second part of the thesis was focused on the identification of the stressor triggering the induction of β-lactamase production in macrophages. M. abscessus was grown in vitro in different culture media mimicking stress conditions thought to prevail in macrophages. The β-lactamase specific activity was determined using a chromogenic β-lactam (nitrocefin) as the substrate. None of the physicochemical conditions that were tested led to induction, including acidic pH, high concentrations of metals, oxidative stress or β-lactams. The last objective was to study the impact of the N versus G polymorphism located in the conserved SDN motif of mycobacterial β-lactamases on activity of β-lactam-β-lactamase inhibitor combinations. BlaMab from M. abscessus contains motif SDN whereas BlaC from M. tuberculosis contains motif SDG, a polymorphism that determines efficacious inhibition by either avibactam of clavulanate, respectively. Two isogenic strains of M. abscessus were constructed by allelic exchange. In comparison to the wild-type enzyme, the strain producing BlaMab with the N to G substitution was less susceptible to the β-lactam-avibactam combinations but more efficaciously inhibited by combinations comprising clavulanate. In the context of BlaC, the G to N substitution potentiated inhibition by avibactam. These results establish that the SDN/SDG polymorphism determines the efficacy of combinations comprising a β-lactam and avibactam or clavulanate, as expected from previous kinetic studies performed with purified β-lactamases. N to G and G to N substitutions might be mechanisms of resistance acquisition in M. abscessus and M. tuberculosis, respectively

Neisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target. Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation. Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections. This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.

Neisseria gonorrhoeae (Ng) which causes gonorrhea has become multidrug-resistant, necessitating the development of novel therapeutics and vaccines. mAb 2C7 which targets an epitope within an important virulence factor, the lipooligosaccharide (LOS), is a candidate therapeutic mAb. Ninety-four percent of clinical isolates express the 2C7-epitope which is also a vaccine target. Ng expresses multiple LOS(s) due to phase-variation (pv) of LOS glycosyltransferase (lgt) genes. mAb 2C7 reactivity requires a lactose extension from the LOS core Heptose (Hep) II (i.e. lgtG ‘ON’ [G+]). Pv results in HepI with: two (2-), three (3-), four (4-), or five (5-) hexoses (Hex). How HepI glycans impact Ng infectivity and mAb 2C7 function are unknown and form the bases of this dissertation. Using isogenic mutants, I demonstrate that HepI LOS glycans modulate mAb 2C7 binding. mAb 2C7 causes complement (C’)-dependent bacteriolysis of three (2-Hex/G+, 4-Hex/G+, and 5-Hex/G+) of the HepI mutants in vitro. The 3-Hex/G+ mutant (resistant to C’-dependent bacteriolysis) is killed by neutrophils in the presence of mAb and C’. In mice, 2- and 3-Hex/G+ infections are significantly shorter than 4- and 5-Hex/G+ infections. A chimeric mAb 2C7 that hyperactivates C’, attenuates only 4- and 5-Hex/G+ infections. This study enhances understanding of the role of HepI LOS pv in gonococcal infections and shows that longer HepI glycans are necessary for prolonged infections in vivo. This is the first study that predicts in vitro efficacy of mAb 2C7 against all four targetable HepI glycans thereby strengthening the rationale for development of 2C7-epitope based vaccines and therapeutics.

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In order to develop better therapeutics and treatment strategies for tuberculosis (TB) infection, it is imperative to understand interactions that occur in the host in response to the bacilli that contribute to disease progression. Modeling of TB in simple animal models such as mice and zebrafish is often incomplete as there are evolutionary differences, as well as structural issues, that reduce the faithfulness to human TB infection. The non-human primate model of TB infection provides the added benefit of providing a long-established model of SIV infection that recapitulates HIV infection in humans and has been expanded to model TB/HIV co-infection. Here, we have sought to identify correlates of protection in TB and TB/SIV co-infection using rhesus macaques. In the first experiment, we used two strains of Mycobacterium tuberculosis (Mtb), CDC1551 and Erdman, to investigate strain-specific mechanisms of virulence. As increased virulence of Mtb Erdman was associated with excess inflammatory responses, we sought to evaluate a host-directed therapeutic in a lethal challenge model of Mtb CDC1551 infection. We found that use of a type I interferon antagonist significantly improved host survival in the absence of antibiotic treatment and survival was associated with the presence of increased levels of granzyme B producing T cells. A major producer of granzyme B, mucosal-associated invariant T (MAIT) cells was investigated in Mtb/SIV co-infection, but was found to not contribute to protection in TB or TB/SIV. In order to further expand our model of Mtb/SIV co-infection, we co-infected latent Mtb-infected rhesus macaques with a non-pathogenic strain of SIV, SIVmac239ΔGY, and administered a CD4-depleting antibody, CD4R1, in place of SIVmac239 co-infection. Using SIVΔGY, we found that virulent viral replication was necessary for TB reactivation. Using both SIVΔGY and antibody-mediated CD4+ T cell depletion, we found that immune responses are disregulated in Mtb/SIV reactivators in a divergent manner, illustrating the presence of SIV-dependent factors that contribute to TB/SIV reactivation. Overall, these results indicate that immune mechanisms, especially those of inflammation, are significant in determining host outcomes. Developing ways to better control inflammation are necessary to supplement antibiotic treatment and cure TB.
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Allison Bucsan

Mycobacterium tuberculosis (Mtb) is an obligate intra-cellular pathogen that causes the disease tuberculosis (TB) in its human hosts. An estimated 1% of the world population is reported to get infected with the disease every year. The capacity of Mtb to tolerate multiple antibiotics, particularly within its host, represents a major problem in TB management. Moreover, patients co-infected with Mtb and another global pathogen human immunodeficiency virus (HIV) showed high rates of anti-TB therapy failure as compared to patients infected with only Mtb. The protracted therapy time for a cocktail of antibiotics often leads to non-adherence among patients resulting in ineffectiveness of the regimen. Also, prolonged exposure to antibiotics paves the way for the acquisition of mutations that generate genetically drug-resistant Mtb strains. Heterogeneity within Mtb populations has long been associated with refractoriness to antibiotic therapy during growth in vitro and inside host cells/tissues [Chapter 1]. It has been reported that the micro-environment faced by Mtb inside host phagocytes promotes tolerance towards clinically-relevant anti-TB drugs, possibly contributing to reduced clearance of Mtb from patient lesions. During chronic phase of infection, exposure to host immune pressures induces metabolic quiescence, which contributes to a drug-tolerant phenotype. Additionally, tolerance to antibiotics has recently also been attributed to replicating Mtb in unstimulated macrophages. Expansion of this drug-tolerant Mtb population within lesions could lead to dissemination of tolerant bacteria to new sites, ultimately reducing the efficacy of antibiotics in eradicating of Mtb. Association of host immune pressures in mobilizing drug tolerance indicates an active crosstalk between host and pathogen. In this regard, it is crucial that we identify host-specific cues and Mtb’s adaptation program in response to environmental signals for mechanistic dissection of phenotypic drug tolerance in replicating Mtb during infection. The major aim of this study was to characterize the cross-talk between host immune pressures and bacterial genetic determinants that sense these pressures to mediate realignment of bacterial metabolism for survival. Upon uptake by naïve macrophages, Mtb is exposed to host stresses such as limited acidification of the phagosome (~ pH 6.2) and superoxide (O2.) stress by recruitment of vacuolar ATPases (V-ATPases) and phagocyte oxidases (NOX2) on the phagosomal membrane, respectively. Since these stresses are known to induce redox imbalance, Mtb induces several protective mechanisms to maintain redox homeostasis for survival. A major technological advance in the redox field emerged by the development of a fluorescent biosensor (Mrx1-roGFP2) that facilitates real-time quantification of the redox potential of the major cytosolic antioxidant in Mtb (mycothiol/MSH; EMSH) during infection. Using this tool, it was reported that Mtb population localized within macrophages exhibits heterogeneity in EMSH as compared to uniform EMSH displayed by broth-grown bacteria. Further, redox-diverse bacterial sub-populations demonstrated variable susceptibility towards anti-TB drugs, suggesting a link between redox physiology and drug tolerance in Mtb. On this basis, we sought to comprehensively characterize redox-diverse fractions of intra-phagosomal Mtb to understand the underlying mechanism of phenotypic drug tolerance. Our RNAsequencing (RNA-seq) of redox-altered Mtb provided distinct transcriptional signatures, which aided in identification of bacterial determinants of antibiotic tolerance [Chapter 2]. Phagosomal acidification is possibly one of the earliest host stresses that Mtb faces upon internalization by macrophages. Previous reports suggest that maintenance of intrabacterial pH homeostasis, upon exposure to an acidified environment, could affect Mtb’s redox physiology. In this regard, we sought to dissect the link between phagosomal acidification, heterogeneity in EMSH and multi-class drug tolerance in Mtb during infection [Chapter 3]. We identified that redox-diverse Mtb sub-populations within macrophages faced different degrees of phagosomal acidification. Blocking phagosomal acidification using lysosomotropic agents subverted heterogeneity in EMSH and reversed drug tolerance to anti-TB drugs isoniazid (Inh) and rifampicin (Rif). We also show that the pH and redox-dependent drug tolerance of Mtb is significantly higher when the pathogen infects macrophages with actively replicating HIV-1, suggesting that it could contribute to high rates of TB therapy failure during HIV-TB coinfection. Our data emphasizes upon the crucial role played by host acidification in generating redox-based heterogeneity and drug tolerance in intra-macrophage Mtb. The role of the lysosomotropic agent chloroquine (CQ), in blocking acidification of sub-cellular compartments, is well-established in anti-malarial therapy as well as in autophagyrelated studies. Based on our results in Mtb-infected macrophages, where CQ consistently decreased tolerance to Inh and Rif, we attempted to assess the effectiveness of CQ in reversing Mtb drug tolerance in vivo [Chapter 4]. In chronically-infected BALB/c mice, coadministration of CQ with Inh or Rif dramatically reduced the fraction of drug-tolerant Mtb, ameliorated lung pathology, and reduced post-chemotherapeutic relapse. Similar decrease inbacterial lung burden and infection-mediated tissue damage was observed in Mtb-infected guinea pigs treated with a combination of CQ and Inh. The pharmacokinetic profile of CQ exhibited no significant drug-drug interaction with first line anti-TB drugs, making it a robust candidate to be considered for host-directed therapy in TB management. In summation, our data delineate a functional link between phagosomal pH, redox metabolism and multi-drug tolerance in replicating Mtb [Chapter 5]. We have attempted to investigate the mechanistic underpinnings of how intra-phagosomal Mtb senses acidification as a cue to generate phenotypic variants of redox state which consequently exhibit differential sensitivity to anti-TB drugs. We also propose the repositioning of CQ as host-directed therapy based on its role in blocking phagosomal acidification and redox heterogeneity to potentiate antibiotic efficacy. Our findings, along with the high oral bioavailability and long half-life in patient sera, makes CQ a promising candidate to be added to current treatment regimens for shortening TB therapy and achieving a relapse-free cure.

Tese de mestrado, Ciências Biofarmacêuticas, Universidade de Lisboa, Faculdade de Farmácia, 2018
The human immunodeficiency virus (HIV) infection and tuberculosis are still major health problems. It is estimated that one third of the human population is latently infected with tuberculosis, with HIV infection being the major risk for reactivation. Eradication of HIV and Mycobacterium tuberculosis (Mtb) infections is challenging due to establishment of latent reservoirs, as is the case of macrophages (Mϕ) and dendritic cells (DC), and the emergence of drug resistant strains. This difficulty is aggravated during co-infection. Thus, there is the need to develop and establish an efficient treatment to eradicate these infections. The long-term goal of this work is the development of a host-directed strategy that, through the manipulation of lysosomal proteases, will boost the host cellular and humoral response against these microorganisms. In the first part of this thesis we performed a transcriptomic analysis of all cathepsins and their inhibitors cystatins during mono- or co-infections of Mϕ or DC. Upon co-infection with Mtb and HIV, DC and Mϕ have a differential profile of expression being the analysed genes upregulated and downregulated. Further, during co-infections, the gene expression was dominated by the HIV infection. In the second part we explored the role of cathepsin S and their manipulation as a strategy to control Mtb infection. Study of the involvement of microRNAs, revealed that miR-106b-5p is manipulated by Mtb and that it reduces cathepsin S protein expression. Through loss-of-function experiments, cathepsin S expression, Mtb killing within Mϕ and T cell activation increased. The decrease of Mtb survival was independent of apoptosis, necrosis and autophagy suggesting that miR-106b-5p action on cathepsin S enables Mtb to evade to the degradative activity of enzymes of the endocytic pathway. These results show a distinct expression profile between HIV Mtb co-infected DC and Mϕ and suggest cathepsin manipulation as a potential target for host directed therapy in Mtb infection.
O vírus da imunodeficiência humana (HIV) e a Mycobacterium tuberculosis (Mtb) são os patogenos causadores da síndrome da imunodeficiência adquirida e da tuberculose, respetivamente. É estimado que um terço da população mundial tem tuberculose latente, sendo a infeção por HIV o maior risco para a reactivação da tuberculose latente devido à extensa imunossupressão. A incidência da TB em pessoas infectadas por HIV teve um aumento de 40 % na Europa nos últimos 5 anos, sendo Portugal o terceiro país da Europa com a maior percentagem de pacientes com TB e infetados com HIV. Embora as doenças causadas por HIV e por Mtb tenham vindo a ser extensivamente estudadas, ainda não existe uma terapia capaz de eliminar estas infeções. Estas dificuldades são causadas em parte pelo aparecimento de estirpes resistentes aos antibióticos/antivirais utilizados e pela existência de reservatórios celulares latentes que contêm estes patogenos num estado de dormência, permitindo que o sistema imune não os ataque nem que estes sejam alvos dos diversos tratamentos. Além disso, quando o mesmo hospedeiro é infetado por HIV e por Mtb a dificuldade em tratar e erradicar ambas as infeções é ainda mais crítica, dado que estes patogenos exacerbaram a infeção um do outro. Deste modo, é necessário desenvolver e estabelecer novos tratamentos de forma a erradicar ou atenuar cada mono-infeção ou co-infeção. O objetivo deste projeto é o desenvolvimento de uma terapia direcionada ao hospedeiro, através da manipulação de proteases lisossomais, de forma a fortalecer a resposta celular e humoral do hospedeiro contra o HIV e o Mtb durante as mono-infeções e as co-infeções e potencialmente melhorar as terapias existentes. Para tal foi estabelecido um modelo de co-infeção que pretendia assemelhar-se à infeção por Mtb de um indíviduo previamente infectado por HIV e num estado de latência viral. Neste estudo foi também incluído um modelo de co-infeção por HIV-2 e Mtb. Desta forma foi estudada a infeção por HIV-2 que afecta um menor número de indivíduos comparativamente à infeção por HIV-1. Este vírus é também tido como um modelo de HIV com menor virulência. Numa primeira fase, foi determinada a expressão genética das catepsinas e dos seus inibidores naturais, as cistatinas, durante a mono-infeção por Mtb, HIV-1 e HIV-2 e durante a co-infeção Mtb-HIV em células dendríticas e macrófagos. Numa segunda fase foi determinado se a manipulação da catepsina S por Mtb via microRNAs (miRNAs) contribuía para a sobrevivência intracelular desta bactéria em macrófagos e para o escape à apresentação de antigénios a linfócitos T. De forma a caracterizar o modelo de infeção estabelecido foi determinada a carga viral e micobacteriana das células assim como a morte celular das mesmas por apototse e necrose. A carga micobacteriana da mono-infeção por Mtb era superior à da co-infeção HIV Mtb em células dendríticas e macrófagos aquando da medição da expressão genética das catepsinas e cistatinas. A morte celular por apoptose e necrose foi também medida e revelou que enquanto as células dendríticas encontram-se maioritariamente num estado inicial de apoptose, os macrófagos apresentam uma maior percentagem de células em fases mais avançadas da apoptose. Em ambos os tipos celulares a necrose ocorre em menos de 1 % das células. Tanto as mono-infeções por HIV-1 e HIV-2 como as co-infeções tiveram um aumento na percentagem de células apoptóticas comparativamente com as mono-infeções com micobactéria, sugerindo que é o efeito do HIV que impera durante as co-infeções. Neste trabalho foi elucidado o controlo da expressão genética resultante da infeção por Mtb, HIV-1 e HIV-2 durante a mono- e a co-infeção de células apresentadoras de antigénio. As células dendríticas e os macrófagos apresentam uma expressão genética diferencial entre si, observando-se um aumento de expressão geral nas células dendríticas relativamente a macrófagos. O perfil de expressão genética da mono-infeção por HIV-1 e por HIV-2 é idêntico em macrófagos, mas distinto em células dendríticas. Em ambos estes tipos celulares a expressão genética durante a co-infeção foi semelhante à da mono-infeção por HIV-1 e HIV-2. É de notar que as infeções bacterianas apresentam mais diferenças na expressão genética de catepsinas e cistatina durante a infeção de macrófagos. Em contraste, as infeções por HIV-1 e HIV-2 apresentam um maior número de genes diferencialmente expressos durante a infeção de células dendríticas. O estudo do envolvimento dos microRNAs (miRNA) na modulação da resposta celular do hospedeiro após a infeção por Mtb em Mϕ mostrou que o miR-106b-5p é manipulado por este microrganismo. Sendo que este miRNA tem como alvo a catepsina S, uma das catepsinas que participa na degradação lisossomal do Mtb e na apresentação de antigénios, foi determinado o seu efeito na expressão desta proteína. Os resultados obtidos indicam que o miR-106b-5p reduz a expressão proteica da catepsina S. A manipulação deste miRNA permitiu observar que ao aumentar a sua expressão, a expressão da catepsina S diminui e a sobrevivência intracelular do Mtb aumenta. Em contraste, ao diminuir o miR-106b-5p houve o aumento da expressão da catepsina S e a diminuição da sobrevivência intracelular do Mtb. Foi também demonstrado que a sobrevivência intracelular do Mtb é independente da apoptose, necrose e autofagia o que sugere que a ação do miR-106b-5p na catepsina S permite que o Mtb escape aos enzimas hidrolíticos das vias endocíticas. Ao inferir o impacto da manipulação do miR-106b-5p na apresentação de antigénios e consequentemente na ativação de células T, a inibição deste miRNA levou a um aumento da expressão de moléculas apresentadoras de antigénio à superfície da membrana celular dos Mϕ. Este aumento foi acompanhado por um aumento na ativação de células T após contacto com Mϕ infectados por Mtb. Estes resultados mostram que existe uma expressão diferencial dos genes das catepsinas e cistatinas entre DC e Mϕ co-infectados com HIV-1 ou HIV-2 e Mtb e que o miR-106b-5p é um potencial alvo para o desenvolvimento de uma terapia direcionada durante a infeção por Mtb.
This work was partially financed by ADEIM and by the project FCT PTDC/SAU-INF/28182/2017

M. tuberculosis (Mtb) senses and responds to changes in its environment primar-ily through two-component signalling systems (TCSs). Each TCS contains a trans-membrane histidine kinase (HK ) protein and a cytoplasmic response regulator (RR) protein. HK detects a stimulus and gets phosphorylated. It then binds and transfers the phosphoryl group to the RR of the same TCS. Activated RR then triggers gene ex-pression, including upregulation of the HK and RR involved, eliciting responses that are essential for the bacterium to adapt. Though di erent TCSs detect distinct stimuli, the binding regions of the HK s and RRs share signi cant similarity. This raises the possibil-ity of crosstalk, where HK s dissipate signals to RRs that do not belong to the same TCS. Studies have argued that such dissipation of signals impairs the fitness of the organism, as it decreases the output levels as well as triggers unwanted responses. In contrast, a recent experimental study has discovered that TCSs of Mtb share extensive crosstalk, violating the widely accepted specificity paradigm. In this study, we have attempted to unravel the evolutionary underpinnings of this extensive crosstalk observed in Mtb. We hypothesised that such crosstalk may be advantageous in programmed environments, where there are well-defined sequences of stimuli. In such situations, crosstalk can up-regulate HK s and RRs of non-cognate TCSs. This up-regulation primes the latter TCSs for upcoming signals, increasing their sensitivity. We constructed a mechanistic model of the functioning of TCSs and a fitness variable to qualitatively measure the response of a TCS to a signal, to test the hypothesis. We performed population genetics simulations of the evolution of phenotypes of different crosstalk patterns. We found that in a random environment, the phenotype without any crosstalk is selected over time, which is in agreement with prevalent arguments in favour of specificity of TCSs. But when the environment is programmed, the phenotype with a crosstalk pattern mirroring the pattern of stimuli dominates the population. Finally, we found evidence for the evolutionary preference to preserve crosstalk in gene sequences of HK s and RRs encoded in Mtb. We found that the binding domains of HK s and RRs, which were predicted to share crosstalk, are under greater pressure to be similar than those domains which do not crosstalk. Our study thus provides a plausible explanation of the unexpected presence of crosstalk in Mtb. Since these cross-interactions aid the pathogen to adapt in the host, inhibitors of such interactions are likely to have therapeutic potential.

M. tuberculosis (Mtb) senses and responds to changes in its environment primar-ily through two-component signalling systems (TCSs). Each TCS contains a trans-membrane histidine kinase (HK ) protein and a cytoplasmic response regulator (RR) protein. HK detects a stimulus and gets phosphorylated. It then binds and transfers the phosphoryl group to the RR of the same TCS. Activated RR then triggers gene ex-pression, including upregulation of the HK and RR involved, eliciting responses that are essential for the bacterium to adapt. Though di erent TCSs detect distinct stimuli, the binding regions of the HK s and RRs share signi cant similarity. This raises the possibil-ity of crosstalk, where HK s dissipate signals to RRs that do not belong to the same TCS. Studies have argued that such dissipation of signals impairs the fitness of the organism, as it decreases the output levels as well as triggers unwanted responses. In contrast, a recent experimental study has discovered that TCSs of Mtb share extensive crosstalk, violating the widely accepted specificity paradigm. In this study, we have attempted to unravel the evolutionary underpinnings of this extensive crosstalk observed in Mtb. We hypothesised that such crosstalk may be advantageous in programmed environments, where there are well-defined sequences of stimuli. In such situations, crosstalk can up-regulate HK s and RRs of non-cognate TCSs. This up-regulation primes the latter TCSs for upcoming signals, increasing their sensitivity. We constructed a mechanistic model of the functioning of TCSs and a fitness variable to qualitatively measure the response of a TCS to a signal, to test the hypothesis. We performed population genetics simulations of the evolution of phenotypes of different crosstalk patterns. We found that in a random environment, the phenotype without any crosstalk is selected over time, which is in agreement with prevalent arguments in favour of specificity of TCSs. But when the environment is programmed, the phenotype with a crosstalk pattern mirroring the pattern of stimuli dominates the population. Finally, we found evidence for the evolutionary preference to preserve crosstalk in gene sequences of HK s and RRs encoded in Mtb. We found that the binding domains of HK s and RRs, which were predicted to share crosstalk, are under greater pressure to be similar than those domains which do not crosstalk. Our study thus provides a plausible explanation of the unexpected presence of crosstalk in Mtb. Since these cross-interactions aid the pathogen to adapt in the host, inhibitors of such interactions are likely to have therapeutic potential.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, has adapted with the host environment and evolved to survive in harsh conditions in the host. The pathogen has successfully evolved strategies not only to evade the host immune system but also to thrive within the host cells. Upon infection, the pathogen is either cleared due to the host immune response, or it survives and causes active tuberculosis (TB) infection. In a number of cases however, the pathogen is neither killed nor does it actively proliferate, but it remains dormant in the host until the environment becomes favorable. This dormant state of pathogen is responsible for latent TB infection (LTBI). WHO reports indicated that as much as a third of the whole world’s population is exposed to the pathogen, of which a significant proportion could be latently infected (WHO report, 2015). These individuals do not show symptoms of active TB infection and hence are difficult to detect. The latent TB infected (LTBI) individuals serve as a reservoir for the pathogen, which can lead to epidemics when the conditions change. Hence, it is necessary to understand the host -pathogen interactions during LTBI, as this might provide clues to developing new strategies to detect and curb a latent infection. Host-pathogen interactions are multifaceted, in which both species attempt to recognize and respond to each other, all of these through specific molecules making distinct interactions with the other species. The outcome of the infection is thus decided by a complex set of host-pathogen interactions. The complexity arises since a large number of molecular components are involved, also multiplicity of interactions among these components and due to several feedback, feed forwards or other regulatory or influential loops within the system. The complexity of biological systems makes modeling and simulation an essential and critical part of systems– level studies. Systems biology studies provide an integrated framework to analyze and understand the function of biological systems. This work addresses some of these issues with an unbiased systems-level analysis so as to identify and understand the important global changes both in the host and in the pathogen during LTBI. The broad objectives of the work was to identify the key processes that vary in the host during latent infection, the set of metabolic reactions in the host which can be modulated to control the reactivation of infection, global adaptation in Mycobacterium tuberculosis (Mtb) and then to utilize this knowledge to identify strategies for tackling latent infection. A review of literature of the current understanding of latency from the pathogen and the host perspective is described in chapter 1. From this, it is clear that most available studies have focused on the role of individual molecules and individual biological processes such as granuloma formation, toll-like receptor signaling, T cell responses as well as cytokine signaling, in either initiating or maintaining a latent infection, but there is no report till date about whether and how these processes are connected with each other. While transcriptome based studies have identified lists of differentially expressed genes in LTBI as compared to healthy controls, no further understanding is currently available for many of them, regarding the processes they may be involved in and what interactions they make, which may be important for understanding LTBI. The first part of the work is a systematic meta-analysis of genome-scale protein interaction networks rendered condition-specific with transcriptome data of patients with LTBI, which has provided a global unbiased picture of the transcriptional and metabolic variations in the host and in the pathogen during the latent infection. To start with, publicly available gene expression data related to LTBI, active TB and healthy controls were considered. In all, 183 datasets summing up to 105 LTBI, 41 active TB and 37 healthy control samples were analyzed. (Chapter 2). Standard analysis of the transcriptome profiles of these datasets indicated that there was zero overlap among them and that not a single gene was seen in common among all datasets for the same condition. An extensive human protein-protein interaction network was constructed using information available from multiple resources that comprehensively contained structural or physical interactions and genetic interactions or functional influences. Nodes in this network represented individual proteins and edges represented interactions between pairs of nodes. The identity of each node and the nature of interaction of each edge along with the type of evidence that was used as the basis for drawing the edge, was collated for the network. The gene expression data was integrated into the human protein-protein interaction (PPI) network for each condition, which essentially had weighted nodes and directed edges, specific to that condition, from which specific comparative networks were derived. The highest ranked perturbations in LTBI were identified through a network mining protocol previously established in the laboratory. This involved computing all versus all shortest paths on the comparative network, scoring the paths based on connectedness and various centrality measures of the nodes and the edges and finally ranking the paths based on the cumulative path scores. Intriguingly, the top-ranked set of perturbations were found to form a connected sub-network by themselves, referred to as a top perturbed sub-network (top-net), indicating that they were functionally linked or perhaps even orchestrated in some sense. Th17 signaling appears to be dominant. About 40 genes were identified in the unique set of LTBI condition as compared to the active TB condition, and these genes showed enrichment for processes such as apoptosis, cell cycle as well as natural killer cell mediated toxicity. Construction and analysis of a miRNA network indicated that 32 of these have strong associations with miRNA explaining the role of the latter in controlling LTBI. 3 other genes from the top-net are already established drug targets for different diseases with known drugs associated with them, which are BCL2, HSP90AA1 and NR3C1. These 3 proteins can be explored further as drug targets in LTBI whose manipulation using existing drugs may result in inhibiting the underlying biological process and thereby result in disturbing the state of latency. As a second objective, global variations in the host transcriptome were identified during ascorbic acid induced dormancy (Chapter 3). Ascorbic acid or Vitamin C is a nutrient supplement required in the diet. This organic compound has a known antioxidant property, as it is known to scavenge the free radicals. In a recent study, Taneja et al, demonstrated that Vitamin C could induce dormancy in Mtb. On similar lines, experiments were done in THP-1 cells infected with Mtb to determine the host responses during ascorbic acid (AA) induced dormancy. The raw gene expression data was provided by our collaborator Prof. Jaya Tyagi that included 0 hour, 4 days and 6 days time points with infection and vitamin C versus infection alone or vitamin C alone as controls. The transcriptome data was normalized and integrated into the human PPI network as described for the meta-analyses. It was experimentally determined that ascorbic acid induces dormancy in 4 days post infection. The top-ranked paths of perturbation were analyzed and compared for three different conditions: (i) uninfected condition, (ii) AA treated and infected condition, and (iii) AA, isoniazid and infected condition. The dormant pathogen is known to be drug-tolerant and thus as a marker for the state of dormancy, the lack of effect of isoniazid is also monitored in the infected host cells. The analysis revealed that there were some broad similarities as compared to LTBI from patient samples but AA induced dormancy in cell lines stood out a separate group indicating that there were significant differences such as involving Interferon Induced Transmembrane Proteins (IFITMs), vacuolar ATPase as well as GDF15, which belongs to TGF-beta signaling pathway. The highest ranked perturbed paths contained genes involved in innate immune responses of which ISG15, IFITMs, HLAs and ATPases emerge as the most altered in the dormant condition. CCR7 emerges as a key discriminator, which is subdued in the latent samples but highly induced in infection conditions. Pathway-based analysis of different conditions showed that oxidative stress, glutathione metabolism, proteasome degradation as well as type II interferon signaling are significantly up-regulated in AA induced dormancy. The dormant bacteria reside in the host cells and are known to modulate the host metabolism for their own benefit. So, the third objective was to understand the metabolic variations in the host during LTBI (Chapter 4). A genome-scale metabolic (GSM) model of alveolar macrophage was used in this study. The metabolic model contains information of the reactions, metabolites and the genes encoding enzymes that catalyze a particular reaction. Flux balance analysis (FBA), a constraint-based metabolic modeling method, is used for analyzing the alterations in the metabolism under different infection conditions. In order to mimic the physiological condition, gene expression data was used for constraining the bounds of the reactions in the model. Two different expression studies were used for analysis: GSE25534 (from Chapter 2) and ascorbic acid induced dormancy (Chapter 3). The analysis was carried out for latent TB versus healthy control and latent TB versus active TB to identify the most altered metabolic processes in LTBI. Differences in fluxes between the two conditions were calculated. A new classification scheme was devised to categorize the reactions on the basis of flux differences. In this chapter, higher fluxes in LTBI condition were identified for reactions involved in transport of small metabolites as well as amino acids. Solute carrier proteins responsible for the transport of the metabolites were identified and their biological significance is discussed. Reduced glutathione (GSH), arachidonic acid, prostaglandins, pantothenate were identified as important metabolites in LTBI condition and their physiological role has been described. Sub-system analysis for different conditions shows differential regulation for arachidonic acid metabolism, fatty acid metabolism, folate metabolism, pyruvate metabolism, glutathione metabolism, ROS detoxification, triacylglycerol synthesis and transport as well as tryptophan metabolism. From the study, transporter proteins and reactions altered during LTBI were identified, which again provide clues for understanding the molecular basis of establishing a latent infection. Mycoabcterium tuberculosis is known to undergo dormancy during stress conditions. In this chapter, the main objective was to identify the global variations in the dormant Mtb (Chapter 5). To carry out the analysis, the Mtb PPI network was constructed using information from available resources. Gene expression data of two different dormancy models, Wayne growth model and multiple-stress model, were used for the study. To identify the key players involved in reversal of dormancy, the transcriptome data of reaeration condition was also used. In this study, the Max-flow algorithm was implemented to identify the feasible paths or flows in different condition. The flows with higher scores indicate that more information is traversed by the path, and hence is important for the study. From the analysis of Wayne growth model (hypoxia model), important transcriptional regulators such as SigB, SigE, SigH, regulators in the two-component system such as MprA, MtrA, PhoP, RegX3 and TrcR were identified in stress condition. Multiple-stress model studied the growth of bacteria in low oxygen concentration, high carbon dioxide levels, low pH and nutrient starvation. The gene expression data was integrated in the Mtb PPI network and implementation of Max-flow algorithm showed that MprA, part of the MprA-MprB two-component system, is involved in the regulation of persistent condition. WhiB1 also features in the paths of dormant condition and its role in persistence can be explored. In reaeration model, WhiB1 and WhiB4 are present in the top flows of this condition indicating that the redox state is perturbed in the pathogen and the interactions of these proteins are important to understand the reversal of dormant condition. From the study, Rv2034, Rv2035, HigA, Rv1989, Rv1990 and Rv0837 proteins belonging to toxin-antitoxin systems were also identified in the dormant bacteria, indicating their role in adaptation during stress condition. The role of Rv2034 has been studied in persistence, but the function of other proteins can be analyzed to provide new testable hypotheses about the role of these proteins in dormancy. Thus, the flows or paths perturbed during dormancy were identified in this study. To get a better understanding of the metabolic network active in mycobacteria under different conditions, experiments were performed in Mycobacterium smegmatis MC2 155. The non-pathogenic strain of genus Mycobacteria, Mycobacterium smegmatis, is used as a surrogate to carry out molecular biology studies of Mtb. Mycobacterium smegmatis MC2 155 (Msm) is the commonly used laboratory strain for experimental purpose. In order to obtain a clear understanding of how comparable are the metabolic networks between the virulent M. tuberculosis H37Rv and the model system Msm, the latter model is first studied systematically. In Chapter 6, first the functional annotation of the Msm genome was carried out and the genes were categorized into different Tuberculist classes based on homology with the Mtb genome. A high-throughput growth characterization was carried out to characterize the strain systematically in terms of different carbon, nitrogen or other sources that promoted growth and thus served as nutrients and those that did not, together yielding a genome-phenome correlation in Msm. Gene expression was measured and used for explaining the observed phenotypic behavior of the organism. Together with the genome sequence, the transcriptome and phenome analysis, a set of about 257 different metabolic pathways were identified to be feasible in wild-type Msm. About 284 different carbon, nitrogen source and nutrient supplements were tested in this experiment and 167 of them supported growth of Msm. This indicates that the compounds enter the cells and are metabolized efficiently, thus yielding similar phenotypes. The expressed genes and metabolites supporting growth were mapped to the metabolic network of Msm, thus helping in the identification of feasible metabolic routes in Msm. A comparative study between Msm and Mtb revealed that these organisms share similarity in the nutrient sources that are utilized for growth. The study provides experimental proof to identify the feasible metabolic routes in Msm, and this can be used for understanding the metabolic capability in the two organisms under different conditions providing a basis to understand adaptations during dormancy. In the last part of the work presented in this thesis, the metabolic shift in the pathogen was studied using a genome-scale metabolic model of Mtb (Chapter 7). The model contains information of the reactions, metabolites and genes involved in the reactions. Flux balance analysis (FBA) was carried out by integrating normalized gene expression data (Wayne model and multiple-stress model transcriptome considered in Chapter 5) to identify the set of reactions, which have a higher flux in the dormant condition as compared to the control replicating condition. Glutamate metabolism along with propionyl CoA metabolism emerge as major up-regulated processes in dormant Mtb. Next, with an objective of identifying essential genes in dormant Mtb, a systematic in silico single gene knock-out analysis was carried out where each gene and it's associated reaction was knocked out of the model, one at a time and the ability of the model to reach its objective function assessed. About 168 common genes in Wayne model and multiple-stress model were identified as important in Mtb after the knockout analysis. Essentiality is in essence a systems property and requires to be probed through multiple angles. Towards this, essential genes were identified in Mtb using a multi-level multi-scale systems biology approach. About 283 genes were identified as essential on the basis of combined analysis of transcriptome data, FBA, network analysis and phyletic retention studies in Mtb. 168 genes identified as important in dormant Mtb were compared with 283 essential genes and about 91 genes were found to be essential. Finally, among the set of essential genes, those that satisfy other criteria for a drug target were analyzed using the list of high-confidence drug targets of Mtb available in the laboratory along with their associated drug or drug-like molecules. 38 out of the 168 important genes in Mtb were found to have one or more drugs associated with them from the DrugBank database. Colchicin-Rv1655, Raloxifene-Rv1653, Bexarotene-Rv3804, Rosiglitazone-Rv3804 are top-scoring drug-target pairs that can be explored for killing dormant bacilli. The study has thus been useful in identifying important proteins, reactions and drug targets in dormant Mtb. In summary, the thesis presents a comprehensive systems-level understanding of various aspects of host responses and pathogen adaptation during latent TB infection. Key host and pathogen factors involved in LTBI are identified that serve as useful pointers for deriving strategies for tackling a latent infection.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, has adapted with the host environment and evolved to survive in harsh conditions in the host. The pathogen has successfully evolved strategies not only to evade the host immune system but also to thrive within the host cells. Upon infection, the pathogen is either cleared due to the host immune response, or it survives and causes active tuberculosis (TB) infection. In a number of cases however, the pathogen is neither killed nor does it actively proliferate, but it remains dormant in the host until the environment becomes favorable. This dormant state of pathogen is responsible for latent TB infection (LTBI). WHO reports indicated that as much as a third of the whole world’s population is exposed to the pathogen, of which a significant proportion could be latently infected (WHO report, 2015). These individuals do not show symptoms of active TB infection and hence are difficult to detect. The latent TB infected (LTBI) individuals serve as a reservoir for the pathogen, which can lead to epidemics when the conditions change. Hence, it is necessary to understand the host -pathogen interactions during LTBI, as this might provide clues to developing new strategies to detect and curb a latent infection. Host-pathogen interactions are multifaceted, in which both species attempt to recognize and respond to each other, all of these through specific molecules making distinct interactions with the other species. The outcome of the infection is thus decided by a complex set of host-pathogen interactions. The complexity arises since a large number of molecular components are involved, also multiplicity of interactions among these components and due to several feedback, feed forwards or other regulatory or influential loops within the system. The complexity of biological systems makes modeling and simulation an essential and critical part of systems– level studies. Systems biology studies provide an integrated framework to analyze and understand the function of biological systems. This work addresses some of these issues with an unbiased systems-level analysis so as to identify and understand the important global changes both in the host and in the pathogen during LTBI. The broad objectives of the work was to identify the key processes that vary in the host during latent infection, the set of metabolic reactions in the host which can be modulated to control the reactivation of infection, global adaptation in Mycobacterium tuberculosis (Mtb) and then to utilize this knowledge to identify strategies for tackling latent infection. A review of literature of the current understanding of latency from the pathogen and the host perspective is described in chapter 1. From this, it is clear that most available studies have focused on the role of individual molecules and individual biological processes such as granuloma formation, toll-like receptor signaling, T cell responses as well as cytokine signaling, in either initiating or maintaining a latent infection, but there is no report till date about whether and how these processes are connected with each other. While transcriptome based studies have identified lists of differentially expressed genes in LTBI as compared to healthy controls, no further understanding is currently available for many of them, regarding the processes they may be involved in and what interactions they make, which may be important for understanding LTBI. The first part of the work is a systematic meta-analysis of genome-scale protein interaction networks rendered condition-specific with transcriptome data of patients with LTBI, which has provided a global unbiased picture of the transcriptional and metabolic variations in the host and in the pathogen during the latent infection. To start with, publicly available gene expression data related to LTBI, active TB and healthy controls were considered. In all, 183 datasets summing up to 105 LTBI, 41 active TB and 37 healthy control samples were analyzed. (Chapter 2). Standard analysis of the transcriptome profiles of these datasets indicated that there was zero overlap among them and that not a single gene was seen in common among all datasets for the same condition. An extensive human protein-protein interaction network was constructed using information available from multiple resources that comprehensively contained structural or physical interactions and genetic interactions or functional influences. Nodes in this network represented individual proteins and edges represented interactions between pairs of nodes. The identity of each node and the nature of interaction of each edge along with the type of evidence that was used as the basis for drawing the edge, was collated for the network. The gene expression data was integrated into the human protein-protein interaction (PPI) network for each condition, which essentially had weighted nodes and directed edges, specific to that condition, from which specific comparative networks were derived. The highest ranked perturbations in LTBI were identified through a network mining protocol previously established in the laboratory. This involved computing all versus all shortest paths on the comparative network, scoring the paths based on connectedness and various centrality measures of the nodes and the edges and finally ranking the paths based on the cumulative path scores. Intriguingly, the top-ranked set of perturbations were found to form a connected sub-network by themselves, referred to as a top perturbed sub-network (top-net), indicating that they were functionally linked or perhaps even orchestrated in some sense. Th17 signaling appears to be dominant. About 40 genes were identified in the unique set of LTBI condition as compared to the active TB condition, and these genes showed enrichment for processes such as apoptosis, cell cycle as well as natural killer cell mediated toxicity. Construction and analysis of a miRNA network indicated that 32 of these have strong associations with miRNA explaining the role of the latter in controlling LTBI. 3 other genes from the top-net are already established drug targets for different diseases with known drugs associated with them, which are BCL2, HSP90AA1 and NR3C1. These 3 proteins can be explored further as drug targets in LTBI whose manipulation using existing drugs may result in inhibiting the underlying biological process and thereby result in disturbing the state of latency. As a second objective, global variations in the host transcriptome were identified during ascorbic acid induced dormancy (Chapter 3). Ascorbic acid or Vitamin C is a nutrient supplement required in the diet. This organic compound has a known antioxidant property, as it is known to scavenge the free radicals. In a recent study, Taneja et al, demonstrated that Vitamin C could induce dormancy in Mtb. On similar lines, experiments were done in THP-1 cells infected with Mtb to determine the host responses during ascorbic acid (AA) induced dormancy. The raw gene expression data was provided by our collaborator Prof. Jaya Tyagi that included 0 hour, 4 days and 6 days time points with infection and vitamin C versus infection alone or vitamin C alone as controls. The transcriptome data was normalized and integrated into the human PPI network as described for the meta-analyses. It was experimentally determined that ascorbic acid induces dormancy in 4 days post infection. The top-ranked paths of perturbation were analyzed and compared for three different conditions: (i) uninfected condition, (ii) AA treated and infected condition, and (iii) AA, isoniazid and infected condition. The dormant pathogen is known to be drug-tolerant and thus as a marker for the state of dormancy, the lack of effect of isoniazid is also monitored in the infected host cells. The analysis revealed that there were some broad similarities as compared to LTBI from patient samples but AA induced dormancy in cell lines stood out a separate group indicating that there were significant differences such as involving Interferon Induced Transmembrane Proteins (IFITMs), vacuolar ATPase as well as GDF15, which belongs to TGF-beta signaling pathway. The highest ranked perturbed paths contained genes involved in innate immune responses of which ISG15, IFITMs, HLAs and ATPases emerge as the most altered in the dormant condition. CCR7 emerges as a key discriminator, which is subdued in the latent samples but highly induced in infection conditions. Pathway-based analysis of different conditions showed that oxidative stress, glutathione metabolism, proteasome degradation as well as type II interferon signaling are significantly up-regulated in AA induced dormancy. The dormant bacteria reside in the host cells and are known to modulate the host metabolism for their own benefit. So, the third objective was to understand the metabolic variations in the host during LTBI (Chapter 4). A genome-scale metabolic (GSM) model of alveolar macrophage was used in this study. The metabolic model contains information of the reactions, metabolites and the genes encoding enzymes that catalyze a particular reaction. Flux balance analysis (FBA), a constraint-based metabolic modeling method, is used for analyzing the alterations in the metabolism under different infection conditions. In order to mimic the physiological condition, gene expression data was used for constraining the bounds of the reactions in the model. Two different expression studies were used for analysis: GSE25534 (from Chapter 2) and ascorbic acid induced dormancy (Chapter 3). The analysis was carried out for latent TB versus healthy control and latent TB versus active TB to identify the most altered metabolic processes in LTBI. Differences in fluxes between the two conditions were calculated. A new classification scheme was devised to categorize the reactions on the basis of flux differences. In this chapter, higher fluxes in LTBI condition were identified for reactions involved in transport of small metabolites as well as amino acids. Solute carrier proteins responsible for the transport of the metabolites were identified and their biological significance is discussed. Reduced glutathione (GSH), arachidonic acid, prostaglandins, pantothenate were identified as important metabolites in LTBI condition and their physiological role has been described. Sub-system analysis for different conditions shows differential regulation for arachidonic acid metabolism, fatty acid metabolism, folate metabolism, pyruvate metabolism, glutathione metabolism, ROS detoxification, triacylglycerol synthesis and transport as well as tryptophan metabolism. From the study, transporter proteins and reactions altered during LTBI were identified, which again provide clues for understanding the molecular basis of establishing a latent infection. Mycoabcterium tuberculosis is known to undergo dormancy during stress conditions. In this chapter, the main objective was to identify the global variations in the dormant Mtb (Chapter 5). To carry out the analysis, the Mtb PPI network was constructed using information from available resources. Gene expression data of two different dormancy models, Wayne growth model and multiple-stress model, were used for the study. To identify the key players involved in reversal of dormancy, the transcriptome data of reaeration condition was also used. In this study, the Max-flow algorithm was implemented to identify the feasible paths or flows in different condition. The flows with higher scores indicate that more information is traversed by the path, and hence is important for the study. From the analysis of Wayne growth model (hypoxia model), important transcriptional regulators such as SigB, SigE, SigH, regulators in the two-component system such as MprA, MtrA, PhoP, RegX3 and TrcR were identified in stress condition. Multiple-stress model studied the growth of bacteria in low oxygen concentration, high carbon dioxide levels, low pH and nutrient starvation. The gene expression data was integrated in the Mtb PPI network and implementation of Max-flow algorithm showed that MprA, part of the MprA-MprB two-component system, is involved in the regulation of persistent condition. WhiB1 also features in the paths of dormant condition and its role in persistence can be explored. In reaeration model, WhiB1 and WhiB4 are present in the top flows of this condition indicating that the redox state is perturbed in the pathogen and the interactions of these proteins are important to understand the reversal of dormant condition. From the study, Rv2034, Rv2035, HigA, Rv1989, Rv1990 and Rv0837 proteins belonging to toxin-antitoxin systems were also identified in the dormant bacteria, indicating their role in adaptation during stress condition. The role of Rv2034 has been studied in persistence, but the function of other proteins can be analyzed to provide new testable hypotheses about the role of these proteins in dormancy. Thus, the flows or paths perturbed during dormancy were identified in this study. To get a better understanding of the metabolic network active in mycobacteria under different conditions, experiments were performed in Mycobacterium smegmatis MC2 155. The non-pathogenic strain of genus Mycobacteria, Mycobacterium smegmatis, is used as a surrogate to carry out molecular biology studies of Mtb. Mycobacterium smegmatis MC2 155 (Msm) is the commonly used laboratory strain for experimental purpose. In order to obtain a clear understanding of how comparable are the metabolic networks between the virulent M. tuberculosis H37Rv and the model system Msm, the latter model is first studied systematically. In Chapter 6, first the functional annotation of the Msm genome was carried out and the genes were categorized into different Tuberculist classes based on homology with the Mtb genome. A high-throughput growth characterization was carried out to characterize the strain systematically in terms of different carbon, nitrogen or other sources that promoted growth and thus served as nutrients and those that did not, together yielding a genome-phenome correlation in Msm. Gene expression was measured and used for explaining the observed phenotypic behavior of the organism. Together with the genome sequence, the transcriptome and phenome analysis, a set of about 257 different metabolic pathways were identified to be feasible in wild-type Msm. About 284 different carbon, nitrogen source and nutrient supplements were tested in this experiment and 167 of them supported growth of Msm. This indicates that the compounds enter the cells and are metabolized efficiently, thus yielding similar phenotypes. The expressed genes and metabolites supporting growth were mapped to the metabolic network of Msm, thus helping in the identification of feasible metabolic routes in Msm. A comparative study between Msm and Mtb revealed that these organisms share similarity in the nutrient sources that are utilized for growth. The study provides experimental proof to identify the feasible metabolic routes in Msm, and this can be used for understanding the metabolic capability in the two organisms under different conditions providing a basis to understand adaptations during dormancy. In the last part of the work presented in this thesis, the metabolic shift in the pathogen was studied using a genome-scale metabolic model of Mtb (Chapter 7). The model contains information of the reactions, metabolites and genes involved in the reactions. Flux balance analysis (FBA) was carried out by integrating normalized gene expression data (Wayne model and multiple-stress model transcriptome considered in Chapter 5) to identify the set of reactions, which have a higher flux in the dormant condition as compared to the control replicating condition. Glutamate metabolism along with propionyl CoA metabolism emerge as major up-regulated processes in dormant Mtb. Next, with an objective of identifying essential genes in dormant Mtb, a systematic in silico single gene knock-out analysis was carried out where each gene and it's associated reaction was knocked out of the model, one at a time and the ability of the model to reach its objective function assessed. About 168 common genes in Wayne model and multiple-stress model were identified as important in Mtb after the knockout analysis. Essentiality is in essence a systems property and requires to be probed through multiple angles. Towards this, essential genes were identified in Mtb using a multi-level multi-scale systems biology approach. About 283 genes were identified as essential on the basis of combined analysis of transcriptome data, FBA, network analysis and phyletic retention studies in Mtb. 168 genes identified as important in dormant Mtb were compared with 283 essential genes and about 91 genes were found to be essential. Finally, among the set of essential genes, those that satisfy other criteria for a drug target were analyzed using the list of high-confidence drug targets of Mtb available in the laboratory along with their associated drug or drug-like molecules. 38 out of the 168 important genes in Mtb were found to have one or more drugs associated with them from the DrugBank database. Colchicin-Rv1655, Raloxifene-Rv1653, Bexarotene-Rv3804, Rosiglitazone-Rv3804 are top-scoring drug-target pairs that can be explored for killing dormant bacilli. The study has thus been useful in identifying important proteins, reactions and drug targets in dormant Mtb. In summary, the thesis presents a comprehensive systems-level understanding of various aspects of host responses and pathogen adaptation during latent TB infection. Key host and pathogen factors involved in LTBI are identified that serve as useful pointers for deriving strategies for tackling a latent infection.

Innate immunity refers to the first line of defence system of the host that comes into play immediately or within hours of appearance of invading pathogens like bacteria, viruses and fungi. Cells of the innate immune system such as macrophages and dendritic cells are equipped with several cell surface pattern recognition receptors (PRRs) like TLR1, TLR2, TLR4, TLR5, TLR6, TLR11, DECTIN 1, DECTIN 2, DECTIN3, MINCLE while TLR3, TLR7, TLR8, and TLR9 are expressed in endosome. Another cytosolic class of PRR include NOD2 and RIGI. These PRRs recognize pathogen associated molecular patterns (PAMs) carried by the foreign organisms but not present in the host. The type of PRRs involved upon appearance of foreign bodies depend on the type of PAMs displayed by the invading micro-organisms. Activation of PRRs by invading pathogens are essential to turn on the host innate immune system for early containment of the pathogen. For example, upon triggering of innate immune system various effectors molecules required to control the infection such as TNF-α, IL-1β, IL-12, IL-6, IL-18, chemokines, IFNβ etc. are upregulated. Tuberculosis; an infectious disease caused by Mycobacterium tuberculosis (Mtb) is one of the leading causes of death worldwide. According to World Health Organization, about one third of the world’s population is latently infected by Mtb. Mtb is inhaled as droplet from the atmosphere. Alveolar macrophages phagocytosed the Mtb and induces localized pro-inflammatory responses that lead to the recruitment of mononuclear cells; the building block of granuloma from neighbouring blood vessels. Granuloma consists of a kernel of infected macrophages, surrounded by foamy giant cells and macrophages with a mantle of lymphocytes delineating the periphery of the structure. At this stage Mtb is contained and the individuals do not show overt signs of the disease. However, the containment of Mtb fails after a change in the immune status of the host, which is usually a consequence of old age, malnutrition, or HIV-coinfection. Under such circumstances, the centre of the granuloma undergoes caseation and spills viable, infectious bacilli into the airways. This leads to development of a productive cough that facilitates aerosol spread of infectious bacilli. The host innate immune and adaptive immune system plays crucial role in containment of the Mtb. The predominant effector molecules that are crucial for controlling Mtb such as IFN-γ, TNF-α, IL-12, IL-6, IL-1β, chemokines, prostaglandins, reactive oxygen and nitrite species are being produced during infection. These effector molecules trigger series of events that are essential for the containment of the infection. Despite the robust immune responses elicited by Mtb is highly successful pathogen and still survive within the host. The success of Mtb to survive within the host amidst robust immune response can be attributed in its ability to modulate host defensive operations. For example, as a successful pathogen Mtb can induce production of anti-inflammatory effector molecules, downregulate MHC class II expression, inhibit fusion of phagosome with lysosome and inhibit apoptosis. Mtb is also known to modulate of epigenetic factors and micro RNAs of the host for its benefit. Increasing number of studies has shown that modulation of host cellular signalling cascades is one of the strategies employed by Mtb to significantly reprogram the innate immune cells for its benefit. Although, certain signalling cascades of host are also essential for controlling Mtb infection. In this context, understanding the modulation of host signalling cascades during mycobacterial infection of primary responder of innate immune system like macrophages and dendritic cells is crucial to get deeper insight into the pathogenesis of Mtb. In this regard, the role for PRRs in orchestrating host innate immune responses attain prime importance because the PRRs play significant role in shaping the immune response to mycobacterial infection. TLR2 is the major receptor that recognizes the Mtb. Recognition of Mtb by TLR2 leads to the activation of downstream signalling cascades which eventually lead to the containment or elimination of the pathogen. Mtb contains complex lipid rich cell which can serve as ligands for PRRs. TLR2 ligands of Mtb includes phosphatidyl Inositol Mannosidase (PIM), 19-kDa antigen, Lipoarabinomannan, ESTAT6 etc. Studies have also shown that some of these TLR2 ligands such as LpqH (Rv3763) and ESTAT6 can inhibit TLR2 driven responses. However, during mycobacterial infection besides, TLR2 mediate signalling networks several other signalling cascades takes place as an effector cascade of TLR to execute specific functions in divergent context. Recent studies from our laboratory has reported that several developmental specific signalling pathways like Wnt, Notch and Sonic-hedgehog are reprogrammed during mycobacterial infection to regulate specific functions in a TLR2 dependent manner. One such developmental specific pathway garnering importance is Hippo pathway. The current study primarily focuses on the role of Hippo signalling in regulating specific functions of the host during infection with Mtb and during TLR4 stimulation by its agonist lipopolysaccharide (LPS). Hippo signalling was originally identified in Drosophila as a significant regulator of apoptosis and cell proliferation. However, various studies had shown that Hippo signalling pathway also regulates wide ranges of biological processes including T cell development, autophagy etc. Mice lacking Hippo pathway component MST1 were reported to have reduced CD4+, CD8+ T cells and neutropenia. Very recent studies had also shown the implication of Hippo signalling in regulating reactive oxygen species production and also in regulating viral infection. However, the role of Hippo signalling in modulating host responses during mycobacterial infection is not known. The current investigation demonstrates that during mycobacterial infection Hippo signalling pathway is activated in vitro as well as in vivo to regulate host immune responses. The activation of Hippo pathway was dependent on TLR2 pathway. Core component of TLR2 signalling such as MyD88, IRAK1 and IRAK4 were essential for activation of Hippo signalling during mycobacterial infection of host. We identified IRAK1 and IRAK4 as novel interacting partners of core component of Hippo pathway MST1/2. Further, mechanistic insights led us to the identification of transcription factor interferon regulatory factor (IRF-3) to be activated by MST1/2 to orchestrate the production of chemokines like CXCL1 and CXCL2. Altogether, this study highlights the involvement of TLR2-IRAK1/4-MST1/2-IRF3 axis in Mtb-triggered modulation of chemokines CXCL1 and CXCL2 production and identifies MST1/2 as novel regulators of host-Mtb interactions. As mentioned, Mtb driven MST1/2 orchestrated the expression of CXCL1 and CXCL2. Conventionally, these chemokines are well known for their crucial role in directing the recruitment of immune cells to the site of immunity breach. However, mounting evidences suggest the involvement of chemokines in regulating cellular processes like cytokine production, T cell activation and reactive oxygen species. One of the prime pathways that regulates inflammation is the inflammasome pathway. Inflammasome is a multiprotein complex activated in response to invading foreign organisms and also by endogenous signals like ATP, Ca2+, MSU, LTB4, ROS etc to generate active inflammatory cytokine IL-1β. Because chemokines CXCL1 and CXCL2 also modulate inflammatory responses the modulation of inflammasome by CXCL1 and CXCL2 was intriguing. In this context, for the first time we have unravelled interesting and novel role for CXCL1 and CXCL2 where the concerned chemokines driven by MST1/2 during mycobacterial infection activated selective signalling cascades involving PKCμ-ILK axis leading to the activation of one of the prime innate immune defence pathways called the “Inflammasome”. Activation of inflammasome is crucial for generation of bioactive IL-1β and is very much required by the host to defend against infections. Activation of inflammasome by CXCL1 and CXCL2 was dependent on their cognate receptor CXCR2; a G-protein coupled receptor (GPCR). We found that CXCR2 is expressed on macrophages. Interestingly GPCRs ligand such as LTB4 and extracellular Ca2+ were being shown to regulate inflammasome. The mechanistic study further, identified CXCR2 dependent activation of PKCμ and ILK in regulating the co-ordinated assembly of component of inflammasome in response to CXCL1. Altogether, the current study demonstrates for the first time that Mtb infection driven CXCL1/2-CXCR2-PKCμ-ILK-NLRP3-CASPASE1 axis orchestrate the generation of mature pro- inflammatory IL-1β. The results obtained so far demonstrated that Mtb specific TLR2 drives the Hippo pathway activation to enhance the production of inflammatory chemokines CXCL1 and CXCL2. Further, these CXCL1 and CXCL2 go on to evoke one of the prime inflammatory pathways “NLRP3 INFLAMMASOME” to regulate production of active IL-1β. With these perspectives, we further went on to elucidate the role of TLR4 in regulating the Hippo pathway. In this context, we found significant induction in the activation of Hippo upon stimulation of TLR with LPS. Epigenetic modifiers like histone methyl transferases, acetyl transferases, lysine demethylases are significant regulator of gene expression in various scenarios and work from our laboratory have significantly emphasized the crucial role of various epigenetic factors like ASHL2, JMJD3, EZH2, SIRTUINS etc. in regulating host responses during mycobacterial infection. With these perspectives, our screening experiments has led to the identification of one of epigenetic modulator known as Lysine demethylase 1 (LSD1) as novel regulator of inflammation upon stimulation of macrophages or mice with LPS in vitro as well as in vivo. We observed significant upregulation of LSD1 during inflammation and the upregulation of LSD1 was significantly dependent on Hippo pathway component MST1/2. LSD1 is known to remove gene repressive marks such as H3K9 or activating marks H3K4. Depending on the context, LSD1 is known to act as activator of gene expression or repressor of gene expression. Activation of TLR4 leads to the enhanced production of both pro-inflammatory as well as anti-inflammatory cytokines. Although, pro-inflammatory cytokines are crucial for mounting an effective immune response to restrict infection, the overt and sustained productions of these cytokines are harmful to the host and hence, fine tuning of expression of pro-inflammatory cytokines are essential to maintain an immunological homeostasis. In this context, we observed that LSD1 surprisingly, inhibited the excessive production of pro-inflammatory cytokines like TNF-α and IL-12 during LPS induced inflammation. LSD1 was found to execute this phenomenon by inhibiting hyper- activation of MAPK pathway components including ERK1/2, JNK1/2 and P38 signalling. Further, deep into mechanistic details revealed that the enhanced expression of the dual specificity phosphatases DUSP4 and DUSP10 are positively regulated by LSD1 during LPS induced inflammation. DUSP4 and DUSP10 are known to dephosphorylate MAPK pathway components and thus, prevent their hyperactivation. Altogether, this study identified a previously unknown function of LSD1 as a feedback inhibitor of LPS induced pro-inflammatory cytokine production.

Ph.D.
Background: AIDS is characterized by a number of opportunistic infections and the immune depletion caused by HIV infection is the strongest risk factor for both reactivation of tuberculosis (TB) and progression of Mycobacterium tuberculosis (Mtb) infection to disease. Numerous studies have shown that concurrent infection of the same host cell by HIV,and M.tb stimulates replication of both pathogens. The interaction between the two is lethal. A synergistic relationship exists between Mtb and HIV. While HIV spurs the spread of TB, mycobacterial infection results in acceleration of HIV disease progression. The requirement for iron as a crucial factor for cellular processes has long been demonstrated. Excess iron leads to infections with harmful consequences such as cell death and function impairment. During infection, iron is required by both the host cell and the pathogens. Iron chelation is believed to modulate some of these effects. Objectives: Mtb, HIV and Fe-overload are common in sub-Saharan Africa and iron plays a major role in determining the outcome of several infections. In view of this, we wanted to (1) investigate the effect of excess iron on host cell defences during co-infection with the mentioned microorganisms, (2) evaluate the differences in both host and pathogen responses during acute and chronic infection in the presence of iron overload and (3) Determine the efficacy of iron chelation (with DFO) as a means of counteracting conditions associated with iron overload. Hypotheses: The combination of Fe-overload and co-infection of host cells with HIV and Mtb in an in vitro model should stimulate replication of the pathogens, which would ultimately result in host cell stress manifesting as lower viability or cell death and impaired immune defence functions. Also the detrimental effects of excess iron on host cell viability could be counteracted through the use of iron chelators. Methods: We analyzed the in vitro effect of Mtb in bothchronically and acutely HI V-infected cells (PBMC's and monocytes), exposed to 500 uM FeSO 4 and/or DFO for 4 days. Host cell viability, survival and death were assessed through viability assays (MIT and Alamar Blue) and flow cytometric analyses of apoptosis/necrosis (using Annexin V and propidium iodide). Secretion of IL- 6 and TNF-a and production of total nitrate were monitored as host immune/defence responses using specialized ELISAs. HIV replication was investigated by looking at core protein (p24) contents and reverse transcriptase (RT) activity. Mtb replication and growth was monitored using the microplate Alamar Blue assay (MABA) and quantitative culturing.Results: Co-infection caused a reduction of host cell viability (± 20% and 45% inhibition during chronic and acute infection respectively;, as measured by MTT), increases in the numbers of viral particles (2.3 times and 20% increases for chronic and acute infections respectively) and stimulation of both bacterial viability (36%) and host defence responses (30% increase in TNF-ct secretion). Excess iron further decreased viability with a marked increase in necrosis of cells and was found to enhance pathogen replication and growth (26% for HIV and 47% for Mtb). Chelation of iron with DFO abrogated the enhanced replication of the pathogens with a marginal restoration of host viability. Conclusion: The results obtained demonstrate the deleterious effect of excess iron during concurrent infection with both pathogens as well as its stimulating/enhancing properties on pathogens. On the other hand, DFO inhibited pathogen replication and host viability.

碩士
國立中興大學
分子生物學研究所
101
Neisseria meningitidis (NM) is an encapsulated gram-negative diplococcus, which can be classified into 13 serogroups based on the antigenicity of their capsular polysaccharide (CPS). Most pathogenic strains belong to serogroup A, B, C, Y, W135 and X. In addition to the variation on the CPS, many outer membrane proteins of these organisms are also subjected to antigenic and/or phase variation consequently limiting the effectiveness of single antigen vaccines. One way to obtain a broadly protective vaccine against NM infection is to use multi-component vaccines. The meningococcal lipoprotein Ag473 as a vaccine candidate has previously been demonstrated. In this study, the effectiveness of an Ag473-based multi-component vaccine (MP) including four minor outer membrane proteins (mOMPs), Ag5407, Ag-lyi, P64k(F) and Ag-IIC3(F) to protect mice against group B meningococcal disease was evaluated. Groups of mice were immunized with Ag473, Ag473 plus mOMPs (MP), or PBS. After confirming the presence of anti-NM specific antibodies in Ag473 and MP-immunized mice by ELISA, mice were challenged with the serogroup B strain Nm22209-6R3. With 300 CFU challenge dose, the survival rates for PBS-, Ag473-, and MP-immunized mice were 20%, 40%, and 80%, respectively. When increase the challenge dose to 1500 CFU, the survival rate of the Ag473-immunized group reduced to 20% while 80% of the MP-immunized mice remained to be protected. The results show that adding of the minor proteins, although each alone does not exhibit protection, to the Ag473 can significantly improve the protection effectiveness. In addition, the epitope on Ag5407, the target antigen of the monoclonal antibody (mAb) 54-07 which binds to the meningococcal surface, was defined by gene fragmentation. The results indicate that the functional epitope lies within the C-terminal 67 amino acid residues. Deletion of five residues from either N- or C-terminus of this region destroyed the antigenicity completely suggesting that the mAb 54-07 recognizes a conformational epitope. Because Ag5407 is a ribosomal subunit and detected only on a small portion of meningococcal surfaces suggesting that this protein may have other functions. To address this question, plasmid pEN11-LS5407 encoding a lipidated Ag5407 was introduced into group B MC58 and W135 NM1996-020 strains. The surface expression of the lipidated Ag5407 in the transformants grown in the presence of IPTG was confirmed by flow cytometry. Functional analysis suggested that the surface expression of Ag5407 enhances the adhesion of meningococci to human epithelial cells but does not affect the serum sensitivity of meningococcal strains. Finally, this study accidently discovered that chloramphenycol acetyltransferase, the selectable marker on the plasmid pEN11, may use the capsular polysaccharide of W135 as the acetyl group acceptor in the absence of chloramphenical.