[ES] El contexto de esta Tesis se enmarca en los sistemas de dos componentes (TCS) para comprender el mecanismo de transducción de la señal. Se analizó la especificidad en el reconocimiento de los TCS abarcando estudios a nivel funcional, estructural y evolutivo. Primero se utilizó el sistema HK853-RR468, que al estar previamente caracterizado nos permitió analizar específicamente las regiones de reconocimiento (HK-RR) correspondientes a los Lß3α3 y Lß4α4 de RR468 mutando residuos que determinaran la influencia en la transferencia del grupo fosfato. Los mutantes se caracterizaron de manera bioquímica y se hicieron aproximaciones estructurales pudiendo asignar la reacción de fosfotransferencia a una estructura formada por un complejo entre HK853 y RR468 mutante. Esta estructura nos permitió observar el carácter disociativo de dicha reacción que ha sido descrito previamente y la nula participación del dominio CA. Al mismo tiempo, se analizó la influencia del pH en los residuos catalíticos de la HK y el RR (His y Asp), utilizando un rango de pH de 5 a 8. Los ensayos bioquímicos generados en este rango nos mostraron como la His catalítica perdía su carácter nucleofílico cuando el pH se acercaba y disminuía de 6. Esto se relaciona con el pKa del anillo de imidazol presente en el residuo de His, que se se encuentra en torno a 6 y la pérdida de protonación. También se cristalizó el complejo HK-RR a diferentes pHs donde observamos que la His adquiría un rotámero gauche- que se asignaba a un estado inactivo o de reposo. Por otra parte, se analizó la influencia de la mutación G63V en el RR OmpR, que fue descrita como una mutación relacionada con la resistencia al antibiótico ertapenem. Para esto se generaron mutantes en OmpR en la posición G63, tanto en el dominio REC aislado como en la proteína completa. Los estudios bioquímicos de estas mutaciones demostraron como la mutación en esta posición disminuía la capacidad del RR para fosforilarse e incluso a dimerizar. Esto afectaba a la afinidad de este RR para interaccionar con su ADN correspondiente, las cajas ompF y ompC. Estos efectos se lograron evidenciar con la estructura de OmpRRECG63V, donde se observó como la mutación generaba un cambio conformacional al reducir el tamaño del Lßα3 y generaba un bolsillo hidrofóbico donde quedaba atrapada la cadena lateral de la Val. Finalmente se analizó el aspecto evolutivo de la señalización, para lo que se buscaron organismos endosimbiontes que presentaran una HK y uno o varios RRs. Estas características nos sugerían que la menor presión selectiva nos iba a permitir encontrar organismos con TCSs menos evolucionados cuya especificidad se haya visto reducida. Se analizaron los sistemas de Chlamydia trachomatis, Chlamydia psittaci, Simkania negevensis y Methanobrevibacter sp. Abm4. Solo pudo evidenciarse reacción de fosfotransferencia en el sistema perteneciente a Methanobrevibacter, el cual presenta una HK y 4 RRs. Sin embargo, esta fosfotransferencia presentaba una eficiencia diferenciada, siendo más rápida en RRMet572 y RRMet589-1 mientras que era nula en RRMet589-2. Por su parte las HKs de C. trachomatis y S. negevensis, fueron capaces de fosfotransferir, de manera no selectiva, a RR468, probablemente debido a la alta similitud que presenta la hélice α1 de las HKs con HK853. La aproximación estructural de estos sistemas permitió obtener las estructuras de los RRsMet589-1 y RRMet572, ambos en estado no fosforilado. Las dos estructuras presentaron grandes diferencias conformacionales a partir del Lßα4. Esto sugiere que sus mecanismos de reconocimiento con HKMet y de regulación son diferentes lo que apoya la selectividad diferenciada entre los RRs de este sistema. / [CA] Esta Tesi s'emmarca en l'estudi dels sistemes de dos components (TCS) amb la finalitat d'entendre el seu mecanisme de transducció de senyal basat en l'especificitat de reconeixement a nivell funcional, estructural i evolutiu. Utilitzant el TCS HK853-RR468, analitzarem les regions Lß3α3 y Lß4α4 del regulador de la resposta (RR) RR468, que prèviament s'havien mostrat importants en el reconeixement, generant mutants i determinant la influència en la transferència del grup fosforil. Els mutants foren caracteritzats bioquímicament, observant que afectaven a una reacció específica i permetent-nos captar la reacció de fosfotransferència en una estructura formada per un complex entre HK853 i RR468 mutant. Aquesta estructura va mostrar el caràcter dissociatiu d'aquesta reacció i la nul·la participació del domini CA de la HK. Al mateix tems, s'analitzà l'efecte del pH sobre la transducció del senyal utilitzant els TCS K853-RR468 i EnvZ-OmpR. Els assajos bioquímics generats dins del rang de pH entre 5 i 8 ens mostraren com la His de la HK catalítica perdia el seu caràcter nucleofílic quan el pH s'aproximava i disminuïa de 6, valor del pKa de l'anell d'imidazole de la cadena lateral del residu d'His, indicant que aquesta disminució en l'activitat es correlacionava amb el canvi en la protonació de l'anell. Un exhaustiu estudi estructural del complex HK853-RR468 a diferents pHs mostrà que la His catalítica sempre adquiria un rotàmer gauche- independentment del valor del pH, invalidant el model que proposava que el pH regulava l'activitat de les HKs de la família HisKA induint un canvi en el rotàmer de la His catalítica. D'altra banda, s'analitzà la influència de la mutació G63V en el RR OmpR, que fou descrita com una mutació relacionada amb la resistència a l'antibiòtic ertapenem. Amb aquesta finalitat, es generaren mutants a OmpR a la posició G63 tant al domini REC aïllat com a la proteïna completa. Els estudis bioquímics demostraren com la mutació en aquesta posició disminuïa la capacitat de OmpR per fosforilar-se i per dimeritzar, afectant a la capacitat d'interaccionar amb les seqüències d'ADN palindròmiques diana, corresponents a les caixes ompF i ompC. Aquests efectes es visualitzaren a nivell molecular al resoldre l'estructura del mutant G63V d'OmpRREC, on s'observava com la mutació induïa un canvi conformacional al reduir la mida del Lßα3 generant una butxaca hidrofòbica degut a la presència de la nova Val en posició 63. Aquests canvis es transmeten a la resta de l'estructura d'OmpR produint canvis en Lßα4 i α4 que impedeixen la formació d'una superfície de dimerització competent i impedint la seua interacció amb l'ADN. Finalment, s'analitzà l'aspecte evolutiu de l'especificitat HK-RR. Buscaren organismes endosimbionts que presentaren TCS aïllats consistent en una HK i un o diversos RRs, suggerint que la menor pressió selectiva permetria trobar TCS menys evolucionats, on l'especificitat s'haguera vist reduïda. S'analitzaren HKs i RRs presents en Chlamydia trachomatis, Chlamydia psittaci, Simkania negevensis i Methanobrevibacter sp. Abm4. La reacció de fosfotransferència es va detectar en Methanobrevibacter, que presenta una sola HK i 4 RRs. Aquesta HK mostrà eficiència diferenciada per a la reacció de fosfotransferència, presentant major velocitat per als RRs RRMet572, RRMet589-1 i nul·la per a RRMet589-2. Per la seua banda, les HKs de C. trachomatis i S. negevensis, foren capaces de transferir, de manera no selectiva, a RR468 de Thermotoga maritima, probablement degut a l'alta similitud que presenta l'hèlix α1 de les HKs amb HK853. L'aproximació estructural d'aquests sistemes ens va permetre resoldre les estructures dels RRsMet589-1 i RRMet572 en estat no fosforilat. Les dues estructures presentaren grans diferències conformacionals a partir del Lßα4, els que ens suggereix que els seus mecanismes de reconeixement amb HKMet i de regulació són diferents, cosa que suporta la selectivitat diferenciada entre els RRs d’aquest sistema. / [EN] The context of the Thesis is framed in the two component systems (TCS) to understand the signal transduction mechanism. The specificity in the recognition of TCS was analyzed covering studies at the functional, structural, and evolutionary level. First, the previously characterized HK853-RR468 was used, this allowed us to analyze specific recognition regions corresponding to Lß3α3 and Lß4α4 of RR468 and induce mutations in these regions and understand the recognition between HK and RR and determine the phosphate group transfer's influence. The mutants were characterized biochemically, and structural approximations were prepared, thus assigning the phosphotransfer reaction in a formed structure by an HK8536 and a mutant RR468 complex. This structure allowed us to observe the dissociative character of this reaction that has been previously described and the null participation of the CA domain. Simultaneously, the influence of pH on the catalytic residues of HK and RR (His and Asp) was analyzed, using a pH range of 5 to 8. The biochemical assays generated in this range showed how HK's catalytic His lost the nucleophilic characteristic when pH reached six or below. This is related to the pKa of the imidazole ring present in the His residue that if found around 6 and the loss of protonation. The HK853-RR468 complex was also crystallized at different pHs where we observed that His acquired a gauche- rotamer that was assigned an inactive or resting state. In addition, the influence of mutation G63V in the RR OmpR was analyzed. This mutation was associated with resistance to the antibiotic ertapenem in E. coli. For this, mutants in OmpR were generated in position G63 in both the isolated REC domain and in the whole protein. Biochemical studies of this mutations showed how the mutation in this position reduced the capacity of RR to phosphorylate and even to form a dimer. This affected the affinity of the RR to interact with it's corresponding DNA, the boxes ompF and ompR. These effects were shown with the structure of the REC domain of the OmpR protein mutant G63V. This mutation generated a conformational change by reducing the Lßα3 and generating a hydrophobic pocket that trapped Val's lateral chain. Finally, the evolutive aspect of signaling was analyzed. For this, endosymbiotic organisms that had one HK or many RRs were identified. These characteristics suggested that lower selective pressure would allow us to find organisms with TCSs that showed lower KH-RR specificity. The systems of Chlamydia trachomatis, Chlamydia psittaci, Simkania negevensis, and Methanobrevibacter sp. Abm4 were analyzed. The phosphotransference reaction was only evident in the the Methanobrevigbacter system. This system presents only one KH and four RRs. This HK shows differentiated efficiency in the phosphotranference. It has a higher speed in RRMet572, RRMet589-1 and it is null in RRMet589-2. On the other hand, the HKs of C. trachomatis y S. negevensis were able to transfer in a nonselective manner the RR468 of T. maritima. This is due to the similarity between the α1 helix of the HKs with HK853. The structural approach of there systems allowed us to obtain the structure of RRMet589-1 y RRMet572, both in a non-phosphorylated state. The two structures presented large conformational differences from Lßα4. This suggests that the recognition mechanisms with KHMet and regulation are different. This supports differentiated selectivity between the RRs in this system. / Mideros Mora, C. (2021). Bases moleculares de la especificidad en el mecanismo de transducción de señal en los sistemas de dos componentes bacterianos [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/161920 / TESIS

Os sistemas de sinalização de dois componentes são sistemas prevalentes em bactérias, permitindo a adaptação a diferentes condições ambientais. O sistema de dois componentes classicamente possui uma proteína histidina quinase, o primeiro componente, capaz de reconhecer o estímulo ambiental e fosforilar o regulador de resposta, o segundo componente. Pseudomonas aeruginosa é uma proteobactéria ubíqua, capaz de infectar hospedeiros filogeneticamente distintos. Esse patógeno oportunista apresenta um dos maiores conjuntos de sistemas de dois componentes em bactérias, que permite que ela sobreviva numa grande gama de ambientes, incluindo humanos. P. aeruginosa UCBPP-PA14 apresenta pelo menos 64 histidina quinases e 76 reguladores de resposta codificados em seu genoma. Diversos sistemas de dois componentes já foram correlacionados com a virulência, sendo o sistema GacSA o exemplo melhor caracterizado. Há poucos estudos sistemáticos sobre o envolvimendo dos reguladores de resposta na virulência de P. aeruginosa e os sinais que induzem a ativação dos reguladores de resposta precisam ser encontrados. Para identificar novos reguladores de resposta envolvidos na patogenicidade, infecções in vitro em macrófagos e in vivo em Drosophila melanogaster foram realizadas neste trabalho. Os macrófagos foram infectados com cada mutante dos reguladores de resposta ou com a linhagem selvagem, e a produção da citocina pró-inflamatória TNF-α e o clearance bacteriano foram determinados. Alternativamente, as moscas foram infectadas utilizando-se a estratégia de feeding e a sobrevivência foi verificada. Utilizando-se essas abordagens, a identificação de diversos reguladores de resposta com papel na virulência foi alcançada, além de se corfirmar o papel de reguladores de resposta já estudados. Um dos novos genes envolvidos em virulência, PA14_26570 (nomeado neste trabalho de atvR), codifica um regulador de resposta atípico com substituição no aspartato fosforilável para glutamato, o que usualmente induz um estado sempre ativo. Um mutante não polar em atvR foi construído e macrófagos infectados com a linhagem ΔatvR confirmaram um maior clearance bacteriano e maior produção de TNF-α em comparação aos macrófagos infectados com a linhagem selvagem. Para comprovar a participação de AtvR durante a patogênese, um modelo de pneumonia aguda em camundongos foi utilizado. Camundongos infectados com a linhagem ΔatvR apresentaram uma maior sobrevivência em comparação aos camundongos infectados com a linhagem selvagem. Além disso, os camundongos infectados com ΔatvR apresentaram menor carga bacteriana, aumento no recrutamento de neutrófilos ativados e aumento na produção de citocinas pró-inflamatórias (TNF-α e IFN-γ). Utilizando-se uma abordagem transcritômica (RNA-Seq), foi determindo diversos genes são regulados positivamente na linhagem superexpressando AtvR em relação à linhagem controle. Dentre esses, os clusters de respiração anaeróbia nar, nir, nor e nos estão incluídos. Esse resultado foi confirmado por qRT-PCR e análises fenotípicas, em que a linhagem ΔatvR apresentou menor crescimento e expressão da nitrato redutase durante condições de hipóxia em comparação à linhagem selvagem. Em suma, neste trabalho foi demonstrado que diversos reguladores de resposta são importantes para a virulência de P. aeruginosa em macrófagos in vitro e in vivo em Drosophila, além de caracterizar o regulador de resposta atípico AtvR, que regula a respiração anaeróbica por desnitrificação, permitindo que P. aeruginosa possa infectar e colonizar o hospedeiro com maior eficiência. / Two-component systems are widespread in bacteria, allowing the adaptation to environmental changes. A two-component system is classically composed by a sensor kinase that phosphorylates a cognate response regulator. Pseudomonas aeruginosa is a ubiquitous proteobacterium able to cause disease in several hosts. This opportunistic pathogen presents one of the largest sets of two-component systems known in bacteria, which certainly contributes to its ability to thrive in a wide range of environmental settings, including humans. P. aeruginosa UCBPP-PA14 genome codes for at least 64 sensor kinases and 76 response regulators. Some response regulators are already known to be related to virulence, with the GacSA system as the best characterized. There are no systematic studies about the involvement of P. aeruginosa response regulators in virulence. Moreover, the input signal that triggers the response regulator activation is yet to be uncovered for most systems. To find new response regulators involved in virulence, in vitro infections werecarried out using macrophages. Briefly, the macrophages were infected with each response regulator mutant or the wild-type strain, the pro-inflammatory cytokine production (TNF-α) and the bacterial clearance were evaluated. Using this approach, we identified several response regulators involved in virulence, and we also confirmed the involvement of known response regulators in this process. One of the novel virulence-related response regulators, PA14_26570 (named here as AtvR), is an atypical response regulator with a substitution in the phosphorylable aspartate to glutamate, that usually leads to an always-on state. A non-polar mutant was constructed, and macrophage infection with ΔatvR confirmed an increased bacterial clearance as well as a higher TNF-α production as compared to the wild-type strain. To ascertain the role of AtvR during the pathogenic process, an acute pneumonia model was used. Mice infected with ΔatvR showed an increased survival as compared to mice infected with the wildtype strain. In addition, ΔatvR infected mice showed reduced bacterial burden, increased neutrophil recruitment and activation, as well as increased pro-inflammatory cytokine production (TNF-α and IFN-γ). Also, using a transcriptomic approach (RNASeq), we showed that several genes were upregulated in the strain overexpressing AtvR. These genes include the anaerobic respiration clusters nar, nir, nor and nos. This result was confirmed by qRT-PCR and phenotypic analysis, in which ΔatvR showed reduced growth and nitrate reductase expression during hypoxic conditions as compared to the wild-type strain. In conclusion, we have demonstrated that several response regulators are important for P. aeruginosa virulence in vitro. In addition, we further characterized the atypical response regulator AtvR, which regulates anaerobic respiration via denitrification, allowing this bacterium to infect and colonize the host more efficiently.

Os sistemas de sinalização de dois componentes são sistemas prevalentes em bactérias, permitindo a adaptação a diferentes condições ambientais. O sistema de dois componentes classicamente possui uma proteína histidina quinase, o primeiro componente, capaz de reconhecer o estímulo ambiental e fosforilar o regulador de resposta, o segundo componente. Pseudomonas aeruginosa é uma proteobactéria ubíqua, capaz de infectar hospedeiros filogeneticamente distintos. Esse patógeno oportunista apresenta um dos maiores conjuntos de sistemas de dois componentes em bactérias, que permite que ela sobreviva numa grande gama de ambientes, incluindo humanos. P. aeruginosa UCBPP-PA14 apresenta pelo menos 64 histidina quinases e 76 reguladores de resposta codificados em seu genoma. Diversos sistemas de dois componentes já foram correlacionados com a virulência, sendo o sistema GacSA o exemplo melhor caracterizado. Há poucos estudos sistemáticos sobre o envolvimendo dos reguladores de resposta na virulência de P. aeruginosa e os sinais que induzem a ativação dos reguladores de resposta precisam ser encontrados. Para identificar novos reguladores de resposta envolvidos na patogenicidade, infecções in vitro em macrófagos e in vivo em Drosophila melanogaster foram realizadas neste trabalho. Os macrófagos foram infectados com cada mutante dos reguladores de resposta ou com a linhagem selvagem, e a produção da citocina pró-inflamatória TNF-α e o clearance bacteriano foram determinados. Alternativamente, as moscas foram infectadas utilizando-se a estratégia de feeding e a sobrevivência foi verificada. Utilizando-se essas abordagens, a identificação de diversos reguladores de resposta com papel na virulência foi alcançada, além de se corfirmar o papel de reguladores de resposta já estudados. Um dos novos genes envolvidos em virulência, PA14_26570 (nomeado neste trabalho de atvR), codifica um regulador de resposta atípico com substituição no aspartato fosforilável para glutamato, o que usualmente induz um estado sempre ativo. Um mutante não polar em atvR foi construído e macrófagos infectados com a linhagem ΔatvR confirmaram um maior clearance bacteriano e maior produção de TNF-α em comparação aos macrófagos infectados com a linhagem selvagem. Para comprovar a participação de AtvR durante a patogênese, um modelo de pneumonia aguda em camundongos foi utilizado. Camundongos infectados com a linhagem ΔatvR apresentaram uma maior sobrevivência em comparação aos camundongos infectados com a linhagem selvagem. Além disso, os camundongos infectados com ΔatvR apresentaram menor carga bacteriana, aumento no recrutamento de neutrófilos ativados e aumento na produção de citocinas pró-inflamatórias (TNF-α e IFN-γ). Utilizando-se uma abordagem transcritômica (RNA-Seq), foi determindo diversos genes são regulados positivamente na linhagem superexpressando AtvR em relação à linhagem controle. Dentre esses, os clusters de respiração anaeróbia nar, nir, nor e nos estão incluídos. Esse resultado foi confirmado por qRT-PCR e análises fenotípicas, em que a linhagem ΔatvR apresentou menor crescimento e expressão da nitrato redutase durante condições de hipóxia em comparação à linhagem selvagem. Em suma, neste trabalho foi demonstrado que diversos reguladores de resposta são importantes para a virulência de P. aeruginosa em macrófagos in vitro e in vivo em Drosophila, além de caracterizar o regulador de resposta atípico AtvR, que regula a respiração anaeróbica por desnitrificação, permitindo que P. aeruginosa possa infectar e colonizar o hospedeiro com maior eficiência. / Two-component systems are widespread in bacteria, allowing the adaptation to environmental changes. A two-component system is classically composed by a sensor kinase that phosphorylates a cognate response regulator. Pseudomonas aeruginosa is a ubiquitous proteobacterium able to cause disease in several hosts. This opportunistic pathogen presents one of the largest sets of two-component systems known in bacteria, which certainly contributes to its ability to thrive in a wide range of environmental settings, including humans. P. aeruginosa UCBPP-PA14 genome codes for at least 64 sensor kinases and 76 response regulators. Some response regulators are already known to be related to virulence, with the GacSA system as the best characterized. There are no systematic studies about the involvement of P. aeruginosa response regulators in virulence. Moreover, the input signal that triggers the response regulator activation is yet to be uncovered for most systems. To find new response regulators involved in virulence, in vitro infections werecarried out using macrophages. Briefly, the macrophages were infected with each response regulator mutant or the wild-type strain, the pro-inflammatory cytokine production (TNF-α) and the bacterial clearance were evaluated. Using this approach, we identified several response regulators involved in virulence, and we also confirmed the involvement of known response regulators in this process. One of the novel virulence-related response regulators, PA14_26570 (named here as AtvR), is an atypical response regulator with a substitution in the phosphorylable aspartate to glutamate, that usually leads to an always-on state. A non-polar mutant was constructed, and macrophage infection with ΔatvR confirmed an increased bacterial clearance as well as a higher TNF-α production as compared to the wild-type strain. To ascertain the role of AtvR during the pathogenic process, an acute pneumonia model was used. Mice infected with ΔatvR showed an increased survival as compared to mice infected with the wildtype strain. In addition, ΔatvR infected mice showed reduced bacterial burden, increased neutrophil recruitment and activation, as well as increased pro-inflammatory cytokine production (TNF-α and IFN-γ). Also, using a transcriptomic approach (RNASeq), we showed that several genes were upregulated in the strain overexpressing AtvR. These genes include the anaerobic respiration clusters nar, nir, nor and nos. This result was confirmed by qRT-PCR and phenotypic analysis, in which ΔatvR showed reduced growth and nitrate reductase expression during hypoxic conditions as compared to the wild-type strain. In conclusion, we have demonstrated that several response regulators are important for P. aeruginosa virulence in vitro. In addition, we further characterized the atypical response regulator AtvR, which regulates anaerobic respiration via denitrification, allowing this bacterium to infect and colonize the host more efficiently.

Salmonella enterica is an intracellular bacterial pathogen of humans and the causative agent of the acute gastrointestinal disease, salmonellosis, and the chronic systemic infection, typhoid fever. Sensor proteins convert environmental signals, including signals detected within the host environment, into biochemical signals to control cellular responses. It has been previously established that the two component regulatory system SsrA-SsrB, consisting of the integral membrane sensor kinase protein SsrA and the cytoplasmic DNA-binding response regulator SsrB are essential for regulation of bacterial factors during systemic intracellular infection. The first chapter of this thesis describes characterization of the sensor kinase SsrA. The structure of the periplasmic sensor domain is modeled and evidence is presented that it is involved in enhancing signaling activity in response to environmental acidification encountered within the intracellular environment. A mechanism whereby protonation of histidine residues within this region in response to acidification drives conformational strain and thereby signaling is proposed. The second chapter describes identification of the DNA-binding motif of SsrB within regulated promoters as well as its regulon. Integration of experimental data with comparative genomics data resulted in identification of the palindromic heptameric DNA recognition motif of SsrB as well as identification of novel SsrB-regulated promoters. In addition, a DNA microarray analysis is described wherein the complete SsrB regulon is identified. Finally, the third chapter describes regulatory input of SsrB to the S. enterica type VI secretion system. This chapter also describes the contribution of this system to systemic dissemination of S. enterica during host infection. Altogether, these data advance understanding of how Salmonella controls factors essential for disease in response to the host environment during infection. / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

Les relais de phosphorylation de type histidine/aspartate constituent des voies de signalisation impliquées dans la perception et la transduction des signaux jusqu’à la mise en place de réponses spécifiques. Ils mettent en jeu un récepteur ou Histidine aspartate Kinase (HK), des protéines navettes en charge de la transmission du phosphate (HPt) et des Régulateurs de Réponse (RR). L’implication d’un tel système dans la transduction du signal de la contrainte osmotique est avérée chez la levure et fortement suspectée chez Arabidopsis. Ce travail de thèse visait d’une part à caractériser l’implication de cette voie de transduction de la contrainte osmotique chez le peuplier, avec l’identification de partenaires HPt et RR en aval du récepteur HK1 et d’autre part à caractériser le mode de fonctionnement d’un RR de type-B. HK1, un osmosenseur membranaire détecterait le signal et le transmettrait à trois HPt préférentielles. De plus, un partenariat d’interaction se dégagerait entre ces trois HPt et certains RR-B. La régulation transcriptionnelle observée lors d’une contrainte osmotique pour deux des représentants des RR-B témoigne d’une possible implication de ces RR dans cette voie. Ces protéines sont des facteurs de transcription dont la fonction a été confirmée in planta pour l’un d’entre eux. La dimérisation du domaine receveur du RR et son interaction avec le domaine de fixation à l’ADN ou domaine GARP apparaissent comme des points de contrôle clés dans la régulation de l’activité effectrice des RR-B. De plus, la capacité d’un RR-B à se fixer sur ses motifs de reconnaissance (boîtes AGAT) a pu être vérifiée in vitro et la présence de ces séquences a d’ailleurs été retrouvée dans des gènes régulés par la contrainte osmotique. Ce travail prospectif ouvre des perspectives concernant l’implication des RR-B dans la voie de transduction du signal de la contrainte osmotique, et propose notamment des mécanismes fins pour l’élaboration d’une réponse hautement spécifique. / Multistep His-to-Asp phosphorelay systems are signaling pathways devoted to signal perception and transduction for establishment of specific responses. These systems are composed of three successive partners: Histidine-aspartate Kinases (HKs), Histidine-containing Phosphotransfer proteins (HPts), and Response Regulators (RRs). One of the best characterized corresponding systems is the osmo-responsive pathway in yeast. Such systems are also suspected in Arabidopsis. This work aimed to characterize the involvement of an osmosensing pathway in Populus by identifying HPt and RR elements downstream of HK1 and to reveal the underlying mechanisms for the activity of a RR-B. HK1, membrane osmosensor, is expected to be responsible for signal detection and propagation by triggering the activation of three preferential HPt. Furthermore, an interacting partnership between those HPts and particular B-type RRs was observed. Two of them appear to be regulated by an osmotic stress, suggesting their possible involvement in this pathway. The B-type RR members, the final output elements of the pathway, act as transcription factors, as shown for at least for one of them in planta. Taken together, the dimerization of the RR receiver domain and its interaction with its DNA binding domain (GARP), are likely key checkpoints in the regulation of RR-B activity. Besides, the ability of one RR-B to bind its cognate specific DNA sequences (AGAT boxes) was confirmed in vitro and those were found in promoters of osmotic response genes. This work opens up prospects for the involvement of RR-B in the osmotic stress signaling pathway and suggests mechanisms tuning induction of specific responses.

Mycobacterium tuberculosis, the causative organism of the disease tuberculosis (TB) in humans, leads to nearly two million deaths each year. This versatile pathogen can exist in highly distinct physiological states such as asymptomatic latent TB infection where bacilli lie dormant or as active TB disease in which the bacilli replicate in macrophages. The pathogenic lifestyle requires the tubercle bacillus to sense and respond to multiple environmental cues to ensure its survival. Such stimuli include hypoxia, nutrient limitation, presence of reactive oxygen and reactive nitrogen intermediates, pH alterations, and cell wall/ membrane stress. Two component systems (TCSs) form the primary apparatus for sensing and responding to environmental cues in bacteria. A prototypical TCS is composed of a sensory protein called sensor kinase (SK) and a response generating protein called response regulator (RR). M. tuberculosis encodes 11 genetically paired TCSs, 2 orphan sensor kinases and six orphan response regulator proteins. Studies of the TB bacilli using transcriptional profiling and gene knockouts have revealed that TCSs play an important role in facilitating successful adaptation to diverse environmental conditions encountered within the host. The mtrAB and prrAB genes encoding corresponding TCSs have been shown to be essential for survival, mprAB for persistence and devRS for hypoxic adaptation. Further, inactivation of the TCSs regX3-senX3, tcrXY, trcRS, phoPR or kdpDE was shown to affect the growth and/or virulence of M. tuberculosis in animal infection models. The SK and RR proteins of TCSs are modular and contain variable input and output domains coupled to conserved ‘transmitter’ and ‘receiver’ domains. Despite the modular nature and extensive homology of SK and RR proteins across TCSs, which may allow non-cognate interactions, it is believed that crosstalk across different TCSs is not favored and that individual pathways are generally well insulated. The existing profiling studies have been performed on the TCSs of bacterial species containing a relatively large number of TCSs. In those studies, specificity and insulation have been the norm and thus become the prevalent paradigm of TCS signaling. In vitro genome wide phosphotransfer profiling has revealed only a few cross- communication nodes in the TCSs of Escherichia coli (~3%), while none in Caulobacter crescentus (in 352 interactions tested, in short time duration) and Myxococcus xanthus (in 250 interactions tested). Yet, many instances of cross talk have been reported in literature. For example, E. coli TCSs PmrAB and EnvZ-OmpR show cross-communication with QseBC and ArcBA, and many more. In M. tuberculosis, indirect evidence of the existence of such cross regulation has originated from studies where mutations in phoPR have been shown to affect the expression of the TCS devRS and its regulon. It is thus interesting to examine the extent of crosstalk in the TCSs of M. tuberculosis, which has an exceptionally small number of TCS proteins compared to E. coli. As mentioned earlier, M. tuberculosis H37Rv has 11 cognate pairs of TCSs, 2 orphan sensor kinases and 6 orphan response regulators. To study the entire landscape, we aimed to study all 221 connections between SK and RR proteins including 12 cognate interactions. While 10 of the cognate TCS interactions were established in the literature, two putative systems KdpDE and NarSL and 5 orphan response regulators were still uncharacterized, therefore we initiated our work with the characterization of these TCSs. At the biochemical level, the KdpDE two component system of M. tuberculosis is not well studied, though one report showed interaction of the C-terminal domain of KdpD SK and KdpE RR using yeast two hybrid assay and another reported the interaction of the SK with LRP protein. Besides these associations, there is no evidence for the functionality of KdpDE system. Similarly, NarSL system also has not been characterized and it not known whether these putative two component proteins are functional. The initial part of the study includes the characterization of these two TCSs, NarS-NarL and KdpD-KdpE, at biochemical and physiological levels. In our studies we demonstrated that KdpDE system is a bonafide two component system of M. tuberculosis, and KdpD SK undergoes autophosphorylation at His642 residue in presence of Mg+2 ions and then it transfers phosphoryl group to a conserved Asp52 residue on the KdpE RR protein. The acid-base stability analysis revealed the nature of chemical bonds present in the KdpD and KdpE proteins, and further confirmed that KdpD and KdpE are typical SK and RR respectively. SPR analysis demonstrated that KdpD and KdpE proteins interact under basal non-phosphorylated conditions and the interaction affinity reduced when SK was phosphorylated. The reduction in the interaction affinity indicated towards a possible dissociation of SK and RR protein during phosphotransfer, which allows RRs to exert their regulatory effect. On the similar line, the phosphorylation defective SK (KdpDH642Q) had least affinity with KdpE suggesting that perhaps this mutant SK, fails to interact with the RR. We have also shown that both the kdpD and kdpE genes are in the same operon and are up regulated in potassium ions limitation and osmotic stress conditions. Overall, using the biochemical approaches, we have established that Rv1027c–Rv1028c operon of M. tuberculosis encodes a functional and a typical KdpDE two component signal transduction system. Using the similar biochemical and biophysical approaches, we have demonstrated that NarS-NarL proteins constitute a functional TCS and His241 and Asp61 are the phosphorylatable residues. In contrast to KdpDE which shows typical behaviour of TCS, NarSL TCS showed atypical behaviour. Malhotra and group’s work on NarSL suggested that there is cross-regulation between NarS/NarL and DevS/DosT/DevR systems. We addressed this possibility on three separate levels, by examining (i) the cross-phosphorylation of DevR and NarL RRs by non-cognate sensor kinases NarS and DevS/DosT respectively, (ii) the interaction between DevR and NarL RR proteins, and (iii) examining the effect of DevR-NarL interactions on their DNA binding properties. Our studies ruled out the presence of any physiologically relevant phosphorylation mediated cross-talk between NarS/NarL and DevS/DosT/DevR. We identified that the cross talk between these TCSs could be explained on the basis of interaction between NarL and DevR RRs and their subsequent binding to the target gene promoter regions for concerted regulation of gene expression. We also identified that DevR activation is critical for cooperative action with NarL. This process comes out as a novel mechanism of gene regulation via heteromerization of RRs. We hypothesized that formation of NarL-DevR heteromers may arise because of high sequence similarities. Conclusively, our study provides insights into the functionality of M. tuberculosis NarL/NarS TCS and regulatory function of NarL protein which acts in concert with another RR, DevR. Overall, NarS-NarL system showed an atypical, novel mode of gene regulation involving RR heteromerization. Subsequent to the basic biochemical characterization of NarSL and KdpDE system, the genome wide phosphotransfer profiling was done to identify the cross-connections between TCSs. Remarkably, we found that specificity was the exception rather than the rule. While only three of the TCS pairs were completely specific, all the other nine TCS pairs exhibited crosstalk, including a few that were highly promiscuous. We classified the interactions as specific, one-to-many, and many-to-one signaling circuits. We also profiled all the RRs including the orphans for their ability to accept phosphoryl group from a low molecular weight donor, acetyl phosphate, and interestingly found that only two RRs DevR and NarL were capable of accepting phosphoryl group from such a donor. Interestingly, none of the orphan RRs accepted phosphoryl group from any donor, neither SKs nor low molecular weight phospho donors, warranting further analysis of their roles and presence in the M. tuberculosis genome. Our exhaustive map of the crosstalk between the TCSs of M. tuberculosis sets the stage for a renewed view of TCS signaling and proposes a dispersive-integrative landscape for TCS signaling rather than one of insulation. As an extension of our basic characterization work of NarSL TCS, we also attempted to understand the localization pattern of NarS sensor kinase in M. smegmatis cells using fluorescence approaches. It is known that many bacterial receptors including sensor kinases form clusters or show specific localization patterns inside the cell. We found that NarS shows distinct cellular localization pattern. However, the functional significance of this localization pattern is not obvious yet and warrants further investigations. We also developed a few non-radioactive methods to study interaction between two component systems to overcome the limitations associated with radioactive experiments in studying TCSs. We developed fluorescence resonance energy transfer (FRET) to study in vitro interaction between two component proteins which was sensitive to the phosphorylation status of the proteins. Using fluorescently tagged SKs and RRs, we determined a change in FRET for KdpDE and NarSL TCS pairs in vitro. Our study thus also provides an alternative approach to study TCS signaling, using an easier, non-radioactive and high throughput approach. In summary, our study presents the evidence of an alternative paradigm of bacterial signaling, where significant crosstalk between the underlying TCSs prevails. The new paradigm is expected to have important implications in our understanding of the virulence and pathogenesis of bacterial infections. Overall, our studies (i) allowed the establishment of functionality of all paired TCSs encoded in the genome of M. tuberculosis including NarSL and KdpDE TCSs, (ii) identified the novel mechanism of gene regulation by NarL RR and DevR, (iii) demonstrated the existence of TCS signaling which is contrary to the existing notion of specificity (iv) showed the distinct localization pattern of NarS and (v) developed non-radioactive approaches to study two component interactions.

Greater understanding of how we can use protein simulations and statistical characteristics of biomolecular interfaces as proxies for biological function will make manifest major advances in protein engineering. Here we show how to use calculated change in binding affinity and coevolutionary scores to predict the functional effect of mutations in the interface between a Histidine Kinase and a Response Regulator. These proteins participate in the Two-Component Regulatory system, a system for intracellular signalling found in bacteria. We find that both scores work as proxies for functional mutants and demonstrate a ~30 fold improvement in initial positive predictive value compared with choosing randomly from a sequence space of 160 000 variants in the top 20 mutants. We also demonstrate qualitative differences in the predictions of the two scores, primarily a tendency for the coevolutionary score to miss out on one class of functional mutants with enriched frequency of the amino acid threonine in one position.