Pleiotropic Clostridioides difficile Cyclophilin PpiB Controls Cysteine-Tolerance, Toxin Production, the Central Metabolism and Multiple Stress Responses

The Gram-positive pathogen Clostridioides difficile is the main bacterial agent of nosocomial antibiotic associated diarrhea. Bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) are well established modulators of virulence that influence the outcome of human pathologies during infections. Here, we present the first interactomic network of the sole cyclophilin-type PPIase of C. difficile (CdPpiB) and show that it has diverse interaction partners including major enzymes of the amino acid-dependent energy (LdhA, EtfAB, Had, Acd) and the glucose-derived (Fba, GapA, Pfo, Pyk, Pyc) central metabolism. Proteins of the general (UspA), oxidative (Rbr1,2,3, Dsr), alkaline (YloU, YphY) and cold shock (CspB) response were found bound to CdPpiB. The transcriptional (Lrp), translational (InfC, RFF) and folding (GroS, DnaK) control proteins were also found attached. For a crucial enzyme of cysteine metabolism, O-acetylserine sulfhydrylase (CysK), the global transcription regulator Lrp and the flagellar subunit FliC, these interactions were independently confirmed using a bacterial two hybrid system. The active site residues F50, F109, and F110 of CdPpiB were shown to be important for the interaction with the residue P87 of Lrp. CysK activity after heat denaturation was restored by interaction with CdPpiB. In accordance, tolerance toward cell wall stress caused by the exposure to amoxicillin was reduced. In the absence of CdPpiB, C. difficile was more susceptible toward L-cysteine. At the same time, the cysteinemediated suppression of toxin production ceased resulting in higher cytotoxicity. In summary, the cyclophilin-type PPIase of C. difficile (CdPpiB) coordinates major cellular processes via its interaction with major regulators of transcription, translation, protein folding, stress response and the central metabolism

Pleiotropic Clostridioides difficile Cyclophilin PpiB Controls Cysteine-Tolerance, Toxin Production, the Central Metabolism and Multiple Stress Responses

The Gram-positive pathogen Clostridioides difficile is the main bacterial agent of nosocomial antibiotic associated diarrhea. Bacterial peptidyl-prolyl-cis/trans-isomerases (PPIases) are well established modulators of virulence that influence the outcome of human pathologies during infections. Here, we present the first interactomic network of the sole cyclophilin-type PPIase of C. difficile (CdPpiB) and show that it has diverse interaction partners including major enzymes of the amino acid-dependent energy (LdhA, EtfAB, Had, Acd) and the glucose-derived (Fba, GapA, Pfo, Pyk, Pyc) central metabolism. Proteins of the general (UspA), oxidative (Rbr1,2,3, Dsr), alkaline (YloU, YphY) and cold shock (CspB) response were found bound to CdPpiB. The transcriptional (Lrp), translational (InfC, RFF) and folding (GroS, DnaK) control proteins were also found attached. For a crucial enzyme of cysteine metabolism, O-acetylserine sulfhydrylase (CysK), the global transcription regulator Lrp and the flagellar subunit FliC, these interactions were independently confirmed using a bacterial two hybrid system. The active site residues F50, F109, and F110 of CdPpiB were shown to be important for the interaction with the residue P87 of Lrp. CysK activity after heat denaturation was restored by interaction with CdPpiB. In accordance, tolerance toward cell wall stress caused by the exposure to amoxicillin was reduced. In the absence of CdPpiB, C. difficile was more susceptible toward L-cysteine. At the same time, the cysteine-mediated suppression of toxin production ceased resulting in higher cytotoxicity. In summary, the cyclophilin-type PPIase of C. difficile (CdPpiB) coordinates major cellular processes via its interaction with major regulators of transcription, translation, protein folding, stress response and the central metabolism

Predictive binding affinity of plant-derived natural products towards the protein kinase G enzyme of Mycobacterium tuberculosis (MtPknG)

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a growing public health concern worldwide, especially with the emerging challenge of drug resistance to the current drugs. Efforts to discover and develop some novel, more effective and safer anti-TB drugs are urgently needed. Products from natural sources, such as medicinal plants, have long played an important role in traditional medicine and continue to provide some inspiring templates for the design of new drugs. Protein kinase G, produced by M. tuberculosis (MtPKnG), is a serine/threonine kinase that has been reported to prevent phagosome-lysosome fusion and help prolong M. tuberculosis survival within the host’s macrophages. Here, we used an in silico target-based approach (docking) to predict the interactions between MtPknG and 84 chemical constituents from two medicinal plants (Pelargonium reniforme and Pelargonium sidoides) that have a well-documented historical use as natural remedies for TB. Docking scores for ligands towards the target protein were calculated using AutoDock Vina as the predicted binding free energies. Ten flavonoids present in the aerial parts of P. reniforme and/or P. sidoides showed docking scores ranging from -11.1 to -13.2 kcal/mol. Upon calculation of all ligand efficiency indices, we observed that the (- G/MW) ligand efficiency index for flavonoids (4), (5) and (7) was similar to the one obtained for the AX20017 control. When taking all compounds into account, we observed that the best (- G/MW) efficiency index was obtained for coumaric acid, coumaraldehyde, p-hydroxyphenyl acetic acid and p-hydroxybenzyl alcohol. We found that methyl gallate and myricetin had ligand efficiency indices superior and equal to the AX20017 control efficiency, respectively. It remains to be seen if any of the compounds screened in this study exert an effect in M. tuberculosis-infected macrophages

Tuberculosis transcriptomics: host protection and immune evasion mechanisms

Mycobacterium tuberculosis (Mtb) is the leading cause of death from an infectious disease. The success of the pathogen lies in its ability to subvert hostile intracellular macrophage environment. We performed genome-wide transcriptional deep sequencing on total RNA in murine bone marrow-derived macrophages (BMDM) infected with hypervirulent Beijing strain (HN878) in an extensive time kinetic manner using single molecule sequencer and cap analysis gene expression (CAGE) technique. CAGE analysis revealed nearly 36000 unique RNA transcripts with approximately 16000 are not unannotated to a specific gene. This thesis addressed global changes in RNA expression levels in macrophages infected with Mtb in a time kinetic manner to pinpoint novel host protection and immune evasion genes and elucidate the role of these genes in vitro macrophage assays and in vivo knockout mouse studies. The data in this thesis showed that basic leucine zipper transcription factor 2 (Batf2) was an important factor that regulates inflammatory responses in Mtb infection. Deletion of Batf2 led to the survival of mice with reduced lung inflammation and histopathology due to reduced recruitment of inflammatory macrophages. We also showed that Batf2 was highly expressed in peripheral blood from adolescents who progressed from infection to tuberculosis disease and a predictive human biomarker for tuberculosis disease. In contrast to Batf2, we showed that Protein Kinase C-delta (PKC-δ) deficient mice are highly susceptible to tuberculosis and human lung proteomics dataset revealed that PKC-δ was highly upregulated in the necrotic and cavitory regions of human granulomas in multi-drug resistant subjects. PKC-δ deficient mice had a significant reduction in alveolar macrophages and dendritic cells, reduced accumulation of lipid bodies and serum fatty acids. In vitro experiments showed that PKCδ was required for optimal killing effector functions which were independent of phagosome maturation and autophagy in primary murine macrophages. Our studies suggested that these novel genes play a role in the immune response to Mtb and should be studied more thoroughly to evaluate their potential in possible TB interventions

Study of crosstalk between G-protein coupled receptor-mediated signals and the Nuclear Factor-κB signal transduction cascade

Nuclear Factor-κB (NF-κB) is an ubiquitously expressed transcription factor that is activated in response to a broad spectrum of inflammatory stimuli, including the proinflammatory cytokine Tumour Necrosis Factor-α (TNF-α). Whereas NF-κB is pivotal for coordination of the immune/inflammatory response, its excessive activation is associated with the onset and propagation of multiple disease processes. NF-κB activity is mostly studied in cells subjected to proinflammatory stimuli, but in "real life" cells are simultaneously exposed to a plethora of signalling molecules that can modulate NF-κB activity. It has been known for many decades that sympathetic stress modulates immunity and inflammation, yet the molecular bases are not completely understood. Therefore, in this thesis, we focused on the activity of the β2- adrenergic receptor (β2-AR), one of the key mediators of the stress response, as a modulator of NF-κB function. In line with other reports describing the anti-inflammatory action of β2-AR agonists (β-agonists), we observed that cotreatment of human astrocytes with TNF-α and a β- agonist, inhibited the expression of several NF-κB-driven genes. However, we found that at the same time it potently enhanced the expression of other prototypical NF-κB target genes, including the proinflammatory cytokine Interleukin-6 (IL-6). We found that the IL-6 synergy, depended on the formation of an enhanceosome structure, and hypothesized that the IL-6 promoter acted as a "coincidence" detector, which requires input from multiple signalling cascades for maximal activation. Our previous research was limited to the study of β2-AR/NF-κB crosstalk in the central nervous system, using astrocytes as a cellular model system. In this thesis, we have extended our previous research to skeletal muscle cells. In addition, we have attempted to further unravel the molecular details of the very strong transcriptional synergy apparent at the IL-6 gene using a proteomics approach. Firstly, we have investigated signalling in response to TNF-α/β-agonist cotreatment in C2C12 cells, a murine skeletal muscle model, representing a physiologically relevant cell type to study β2-AR/NF-κB crosstalk. We observed many similarities in the outcome of β2-AR/NF-κB crosstalk in skeletal muscle cells as compared to astrocytes, although cell-type specific differences in the signalling cascades induced by β- agonists/TNF-α were also apparent. In particular, the very potent synergy at the IL-6 promoter was also detected in skeletal muscle cells. In addition, we found that the expression of several chemokines, influencing the migration potential of undifferentiated skeletal muscle cells, was upregulated upon TNF-α/β-agonist costimulation At the molecular level, we demonstrated that β-agonist-induced potentiation of NF-κB-dependent transcription of the IL-6 gene was associated with histone modifications, chromatin relaxation and formation of an enhanceosome structure. Secondly, using an unbiased proteomics approach, combining DNA-affinity purification and mass spectrometric analysis, we identified Transcription Enhancer Factor 1 (TEF-1) as a novel interactor of the IL-6 promoter. We found that TEF-1 recruitment to the IL-6 promoter was induced upon TNF-α/β-agonist costimulation and that it acted as a transcriptional repressor. Our results furthermore indicate that TEF-1 modulates the transcriptional activity of CREB, but not NF-κB, and that this is associated with altered accessibility of the IL-6 promoter to transcriptional regulators. Importantly, TEF-1 modulated NF-κB-dependent transcription in a gene selective manner. As the effects of β-agonists appear to be highly gene-selective, further elucidation of its molecular basis might lead to the identification of novel targets for the development of selective NF-κB inhibitors. In conclusion, these findings indicate that β2-AR/NF-κB crosstalk promotes potent transcriptional synergy for a subset of NF-κB target genes, including IL-6 and several chemokines. This synergy is apparent in multiple relevant cell types, suggesting it might have general significance. As IL-6 has been attributed with devastating properties in inflammatory disease, and as β-agonists are mainstream therapy for respiratory disease, our data warrant further investigation into the outcome of β2- AR/NF-κB crosstalk in vivo

Modification of the animal immune system by feeding probiotics

The objective of this study was to examine immune effects of feeding novel probiotic Lactobacillus acidophilus strain NP51 to specific pathogen-free Balb/c mice challenged with Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne\u27s disease (JD). We hypothesized that feeding the NP51 would activate the adaptive immunity and impede the development of MAP infection in murine model of JD. Thus, Balb/c mice were randomized to treatment groups in a factorial design including mice that either fed the heat-killed or viable NP51 (HNP51 or VNP51, respectively) and challenged with either a heat-killed or a viable MAP (HMAP or VMAP, respectively). Mice were fed 1 × 106 CFU of either HNP51 or VNP51*mice-1*day-1 mixed with standard mouse chow until the end of the study. Subsequently, mice were challenged with 1 × 108 CFU of HMAP or VMAP injected intraperitonealy on day 45 of the study. Ten mice from each group were euthanized on days 45, 90, 135, and 180. Spleens were excised and used for an in vitro splenocytes cell cultures that were either stimulated with sonicated MAP antigen or concanavalin A and examined for the cytokine secretion pattern and frequency of T lymphocyte subpopulations. Blood was withdrawn by cardiac puncture and used for examination of immunoglobulins production. HNP51 and VNP51 differentially stimulated the adaptive immunity and decrease MAP tissue burden. With VMAP as the inoculum, both VNP51 and HNP51 stimulated CD8α+ immune cell-mediated immunity and decreased the humoral immunity. When HMAP was used as the inoculum, VNP51 stimulated the CD8α+ immune cells-mediated and the humoral immunity. In contrast, HNP51 feeding induced CD8α+ immune cells-mediated immunity only as verified by the differential cytokines and immunoglobulins secretion pattern. The data provide persuasive evidence that NP51 has the potency to prevent JD infection in murine model of JD

Peroxissomas e infeções virais: para além da defesa antiviral

Peroxisomes are multifunctional intracellular organelles, crucial for different physiological and pathological processes. Recently, MAVS (mitochondrial antiviral signaling), the mitochondrial adaptor protein essential for the RLR (retinoic acid inducible gene I-like receptors)-mediated antiviral defense was identified at peroxisomes. Upon infection, viral RNA is recognized by RLRs which induce a signaling cascade that initiates with MAVS activation at both mitochondria and peroxisomes. This culminates with the production of antiviral effectors, such as type I IFNs (interferons) and ISGs (IFN-stimulated genes) that prevent viral replication and dissemination. It has been demonstrated that peroxisomal and mitochondrial MAVS act together in a complementing manner: peroxisomal MAVS induces a rapid but short-term response, while mitochondrial MAVS leads to a delayed but long-lasting antiviral response. The aim of this work was to understand the importance of peroxisomes in the cellular antiviral defense and in viral infections. The results presented in this thesis prove that HCV (hepatitis C virus) NS3-4A and HCMV (human cytomegalovirus) vMIA target peroxisomes to inhibit peroxisomal MAVSdependent antiviral signaling, impairing the production of ISGs. We show that NS3-4A inhibits peroxisomal-dependent signaling through the cleavage of peroxisomal MAVS cytosolic domain. We also show that vMIA interacts with peroxisomal MAVS, inhibiting its oligomerization and impairing the downstream signaling. Additionally, vMIA induces peroxisomal fragmentation, which we prove to be independent of the vMIA-mediated peroxisomal MAVS inhibition. Moreover, we show that vMIA is dependent of MFF, an adaptor protein of peroxisomal fission machinery, and we demonstrate that MFF mediates the interaction between vMIA and peroxisomal MAVS. Finally, we present an interactome of protein-protein interactions between human viruses and peroxisomes, revealing that distinct viruses target peroxisomal proteins. A detailed analysis of the identified interaction revealed that lipid metabolism may be the main peroxisomal function exploited by viruses, possibly to enhance viral infection, or for cellular host defense. Altogether, these results enforce the role of peroxisomes as platforms for RLR signaling and, moreover, suggest that their importance for viral infection may go beyond the antiviral defense. Further studies are proposed to better disclose the role of peroxisomes in viral infection, which can ultimately lead to the discovery of novel targets for the development antiviral therapeutics.Os peroxissomas são organelos intracelulares multifuncionais, cruciais para diferentes processos fisiológicos e patológicos. Recentemente, a MAVS, proteína adaptadora mitocondrial essencial para a defesa antiviral mediada pelos recetores RLR, foi identificada nos peroxissomas. Após infeção, o ácido ribonucleico viral é reconhecido pelos recetores RLRs que induzem uma cascata de sinalização que se inicia com a ativação da MAVS, tanto nas mitocôndrias como nos peroxissomas, culminando com a produção de efetores antivirais, tais como interferões do tipo I e genes induzidos pelos interferões (ISGs), que impedem a replicação e disseminação viral. Foi demonstrado que as MAVS peroxissomais e mitocondriais atuam de forma complementar: a MAVS peroxissomal induz uma resposta rápida, mas de curto prazo, enquanto que a MAVS mitocondrial leva a uma resposta antiviral tardia, porém duradoura. O objetivo deste trabalho foi compreender a importância dos peroxissomas na defesa antiviral celular e nas infeções virais. Os resultados apresentados nesta tese provam que a NS3-4A do vírus da hepatite C (HCV) e a vMIA do citomegalovírus humano (HCMV) exploram os peroxissomas para inibir a sinalização antiviral dependente da MAVS, impedindo a produção dos ISGs. Mostramos que a NS3-4A inibe a sinalização dependente dos peroxissomas através da clivagem do domínio citosólico da MAVS e também mostramos que a vMIA interage com a MAVS peroxissomal, inibindo a sua oligomerização e impedindo a sinalização a jusante. Para além disso, a vMIA induz a fragmentação peroxissomal, que provamos ser independente da inibição da sinalização antiviral. Mostramos também que a vMIA é dependente da MFF, uma proteína adaptadora da fissão peroxissomal, e demonstramos que a MFF medeia a interação entre a vMIA e a MAVS peroxissomal. Finalmente, apresentamos um interatoma das interações proteína-proteína entre vírus humanos e peroxissomas, revelando que vírus distintos interagem com diferentes proteínas peroxissomais. Uma análise detalhada das interações identificadas revelou que o metabolismo lipídico pode ser a principal função peroxissomal explorada pelos vírus, possivelmente para aumentar a infeção viral, ou para a defesa do hospedeiro celular. Em conjunto, estes resultados reforçam o papel dos peroxissomas como plataformas para a sinalização dos RLRs e, além disso, sugerem que a sua importância para a infeção viral pode ir além da defesa antiviral. Novos estudos são propostos para compreender melhor o papel dos peroxissomas na infeção viral, o que pode levar à descoberta de novos alvos para o desenvolvimento de terapias antivirais.Programa Doutoral em Biomedicin

Exploring mitochondrial quality control mechanisms and mitochondria-lipid droplet interactions in cardiac cell models

Mitochondria are the main energy producing units (organelles) in cardiac cells (cardiomyocytes). Cardiomyocytes have especially high mitochondrial content due to the heart’s continuous energy-intensive pumping. Studies of heart disease (such as heart failure) indicate that mitochondrial dysfunction is central to disease progression. There are many cellular mechanisms that protect mitochondria from harm and dysfunctional mitochondria can be removed. These mechanisms are the cell´s tools for quality control of mitochondrial function. How these quality control mechanisms function in the human heart is still not fully known. The preferred substrates consumed by the adult heart for sustaining beating are fatty acids, converted to energy by the mitochondria. Fatty acids can be stored within cells in lipid droplets for controlled use. An overabundance of lipid droplets is associated with cardiomyopathy in patients with diseases such as obesity or diabetes mellitus. The cellular response to, and mechanisms for resolving, lipid droplet overabundance in cardiac cells remain poorly understood. In the works constituting this thesis, we applied rat H9c2 cardiomyoblasts and human inducible pluripotent stem cell derived cardiomyocytes as cardiac cell models. In the cardiomyoblasts, we investigated mitophagy and mitochondrial derived vesicles, constituting different mitochondrial quality controls. Furthermore, we studied lipid droplet accumulation, degradation, and interaction with mitochondria in both cell models. For these purposes we utilized different advanced microscopy techniques. Our findings reveal that mitochondria in cells with increased mitochondrial respiration display elevated activity in the targeted quality control mechanisms. Furthermore, cells engaged in increased mitochondrial respiration accumulate less lipid droplets in response to lipid loading treatments. We also detected dynamic and close interactions between mitochondria and lipid droplets. Our work provides important insights and contributes to understanding mitochondria quality control mechanisms and the role of lipid droplets in the heart.Mitokondrier er hovedprodusentene for energi i hjerteceller (kardiomyocytter). Kardiomyocytter har spesielt høyt mitokondrielt innhold på grunn av hjertets kontinuerlige energikrevende pumping. Studier av hjertesykdommer (som hjertesvikt) indikerer at mitokondriell dysfunksjon er sentral for sykdomsprogresjonen. Det finnes mange cellulære mekanismer som beskytter mitokondrier mot skade, inkludert blant disse er mekanismer for fjerning av dysfunksjonelle mitokondrier. Disse mekanismene er cellens verktøy for kvalitetskontroll av mitokondriefunksjonen. Hvordan disse kvalitetskontrollmekanismene fungerer i det menneskelige hjertet er fortsatt ikke fullstendig forstått. Det foretrukne substratet som forbrukes av det voksne hjertet for å opprettholde hjerteslag er fettsyrer som blir konvertert til energi av mitokondrier. Fettsyrer kan lagres av celler i lipiddråper for kontrollert bruk. En overflod av lipiddråper er assosiert med kardiomyopati hos pasienter med sykdommer som fedme eller diabetes mellitus. Den cellulære responsen på, og mekanismene for å løse, overflod av lipiddråper i hjerteceller er fortsatt dårlig forstått. I arbeidene som utgjør denne avhandlingen brukte vi rotte H9c2 kardiomyoblaster og menneskelige kardiomyocytter avledet fra induserbare pluripotente stamceller som hjertecellemodeller. I kardiomyoblastene undersøkte vi mitofagi og mitokondrielle vesikler, som representerer forskjellige mitokondrielle kvalitetskontrollmekanismer. Videre studerte vi lipiddråpe akkumulering, nedbrytning og interaksjon med mitokondrier i begge cellemodellene. For disse formålene brukte vi forskjellige avanserte mikroskopiteknikker. Våre funn avslører at mitokondrier i celler med økt mitokondriell respirasjon viser økt aktivitet i de undersøkte kvalitetskontrollmekanismene. Videre akkumulerer celler som er engasjert i økt mitokondriell respirasjon mindre lipiddråper som respons på lipidbelastning. Vi observerte også dynamiske og nære interaksjoner mellom mitokondrier og lipiddråper. Vårt arbeid gir viktige innsikter og bidrar til å forstå mitokondrielle kvalitetskontrollmekanismer og rollen til lipiddråper i hjertet