Comparative analysis of proteome of sweet orange and ponkan infected with Xylella fastidiosa

Orientadores: José Camilo Novello, Ione SalgadoDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: O sistema citrícola no Brasil representa um setor de grande importância econômica. O Estado de São Paulo é o principal produtor de citros, fazendo do país o maior exportador de suco de laranja concentrado congelado. Apesar do Brasil ocupar uma posição de destaque no cenário mundial de citricultura, o país não consegue aumentar a sua produtividade devido à ocorrência simultânea de pragas e doenças, sendo que a clorose variegada dos citros (CVC) se mostra como uma das mais limitantes sobre esta produção. Ela é causada pela bactéria Xylella fastidiosa, que é capaz de infectar todas as variedades de laranja doce (Citrus sinensis L. Osbeck), embora a tangerina Poncan (Citrus reticulata Blanco) seja considerada tolerante à sua infecção. Apesar de muitos estudos já tenham sidos realizados a fim de se compreender melhor os mecanismos da sua patogenicidade, questões ainda permanecem em aberto acerca dos mecanismos que controlam o seu processo de infecção e o desenvolvimento da doença. Desse modo, foi realizado um estudo comparativo do proteoma das folhas de laranja Pêra e de tangerina Poncan após 30 dias da inoculação com a X. fastidiosa e o dos obtidos de folhas não infectadas, empregando a técnica de eletroforese bidimensional (2DE) e espectrometria de massas (MS). Foram confeccionados mapas 2DE com o intuito de se verificar proteínas diferencialmente expressas que por ventura poderiam estar relacionadas aos mecanismos de defesa e resistência da planta. Entre as proteínas (spots) de laranja Pêra, separadas por eletroforese bidimensional, 60 spots foram considerados como estatisticamente relevantes, apresentando alteração de intensidade. Entre as proteínas de tangerina Poncan analisadas na mesma condição, 38 foram consideradas como estatisticamente relevantes. Confeccionou-se para a planta tangerina Poncan géis de poliacrilamida utilizando IPG com gradiente de pH linear de 4-7, visto que houve um grande número de proteína diferencialmente expressas nesta faixa. Como resultado, foram obtidos 45 spots com diferença de expressão. A identificação dessas proteínas foi feita por meio do seqüenciamento por espectrometria de massas através do sistema LC ESI-MS/MS ou MALDI-Q-TOF. O seqüenciamento por MS possibilitou a aquisição da seqüência de aminoácidos de 49,7% dos spots. Dentre eles, 76% dos spots foram identificados, enquanto que 24% não apresentaram homologia com nenhuma base de dados. Entre as proteínas identificadas quatro foram representadas por mais de um spot, podendo indicar a ocorrência de eventos provenientes do splicing alternativo, modificações pós-traducionais, variações alélicas de uma mesma proteína ou degradação da amostra. As proteínas identificadas foram relacionadas com a produção de energia, com o metabolismo primário, com mecanismo de defesa, proteínas de microrganismos e proteínas desconhecidas. Laranja Pêra apresentou uma diminuição da expressão de proteínas relacionadas à fotossíntese, o que coincide com os primeiros efeitos sentidos pelas plantas colonizadas pela bactéria. Em contrapartida, tangerina Poncan apresentou um aumento de expressão de proteínas relacionadas à resposta de defesa contra esse patógeno.Abstract: The citrus system in Brazil represents one of the most important economic sectors. The State of São Paulo is the main producer of citrus, settling the country as the biggest exporter of concentrated freezing orange juice. Besides holding the outstanding position in the worldwide citrus culture scene, the country cannot raise its productivity due to simultaneous occurrence of plagues and diseases, being the citrus variegated chlorosis (CVC) one of the most limiting diseases affecting the citrus production. This disease is caused by bacterium Xylella fastidiosa, that which is able to infect all sweet orange (Citrus sinensis L. Osbeck) varieties, however ponkan (Citrus reticulate Blanco) was considered resistant to it. Although, many studies have already been done in order to understand, in a better way, the mechanism of its pathogenicity, there are still queries about the mechanisms which control the process of its infection and the development of the disease. In this manner, we did a comparative proteomics study of leaves from sweet orange and ponkan after 30 days of the inoculation with X. fastidiosa versus leaves not infected with this bacterium (healthy plants), using two-dimensional gel electrophoresis (2DE) and mass spectrometry techniques. Comparative 2DE maps were done with the aim to verify differentially expressed proteins related with defense mechanism and the plant resistance. Among the proteins (spots) extracted from sweet orange, separated by two-dimensional gel electrophoresis, 60 spots were considered with statistical significance, showing intensity alteration. On the other hand, among the proteins (spots) extracted from ponkan and analyzed in the same condition, 38 spots were considered with statistical significance. Gels using linear pH gradient ranging from 4 to 7 were prepared for ponkan gels using, because there were a larger number of differentially expressed proteins in this area. As a result, we obtained 45 spots with difference in its expression. The identification of these proteins was done by sequencing using mass spectrometry like LC ESI-MS/MS or MALDI-Q-TOF. 143 spots were analyzed by mass spectrometry and were obtained amino acid sequence from 71 (49,7%) of the spots. Between them, 54 (76%) were identified, while 17 (24%) presented no homology in the database used. Overall, 4 proteins appeared as multiple spots and accounted for most of the protein found in the group. This observation may reflect post-translation modification, alternative splicing events, isozyme variation, allelic variation of the same protein, but also protein degradation. The identified proteins play a role in energy, primary metabolism, defense mechanism, unknown proteins and microorganism proteins. The sweet orange presented a decrease in expression of photosynthesis related protein, indicating a possible lower photosynthetic activity resulting from early effects of the bacterial colonization in affected plants. On the other hand, ponkan showed an increase in defense-related proteins response against this pathogen.MestradoBioquimicaMestre em Biologia Funcional e Molecula

CFTR function is impaired in a subset of patients with pancreatitis carrying rare CFTR variants

Background: Many affected by pancreatitis harbor rare variants of the cystic fibrosis (CF) gene, CFTR, which encodes an epithelial chloride/bicarbonate channel. We investigated CFTR function and the effect of CFTR modulator drugs in pancreatitis patients carrying CFTR variants. Methods: Next-generation sequencing was performed to identify CFTR variants. Sweat tests and nasal potential difference (NPD) assays were performed to assess CFTR function in vivo. Intestinal current measurement (ICM) was performed on rectal biopsies. Patient-derived intestinal epithelial monolayers were used to evaluate chloride and bicarbonate transport and the effects of a CFTR modulator combination: elexacaftor, tezacaftor and ivacaftor (ETI). Results: Of 32 pancreatitis patients carrying CFTR variants, three had CF-causing mutations on both alleles and yielded CF-typical sweat test, NPD and ICM results. Fourteen subjects showed a more modest elevation in sweat chloride levels, including three that were provisionally diagnosed with CF. ICM indicated impaired CFTR function in nine out of 17 non-CF subjects tested. This group of nine included five carrying a wild type CFTR allele. In epithelial monolayers, a reduction in CFTR-dependent chloride transport was found in six out of 14 subjects tested, whereas bicarbonate secretion was reduced in only one individual. In epithelial monolayers of four of these six subjects, ETI improved CFTR function. Conclusions: CFTR function is impaired in a subset of pancreatitis patients carrying CFTR variants. Mutations outside the CFTR locus may contribute to the anion transport defect. Bioassays on patient-derived intestinal tissue and organoids can be used to detect such defects and to assess the effect of CFTR modulators

In silico analysis and theratyping of an ultra-rare CFTR genotype (W57G/A234D) in primary human rectal and nasal epithelial cells

Mutation targeted therapy in cystic fibrosis (CF) is still not eligible for all CF subjects, especially for cases carrying rare variants such as the CFTR genotype W57G/A234D (c.169T>G/c.701C>A). We performed in silico analysis of the effects of these variants on protein stability, which we functionally characterized using colonoids and reprogrammed nasal epithelial cells. The effect of mutations on cystic fibrosis transmembrane conductance regulator (CFTR) protein was analyzed by western blotting, forskolin-induced swelling (FIS), and Ussing chamber analysis. We detected a residual CFTR function that increases following treatment with the CFTR modulators VX661±VX445±VX770, correlates among models, and is associated with increased CFTR protein levels following treatment with CFTR correctors. In vivo treatment with VX770 reduced sweat chloride concentration to non-CF levels, increased the number of CFTR-dependent sweat droplets, and induced a 6% absolute increase in predicted FEV1% after 27 weeks of treatment indicating the relevance of theratyping with patient-derived cells in CF

IDENTIFICATION AND CHARACTERIZATION OF ARABIDOPSIS MUTANT PLANTS IMPAIRED IN NO-MEDIATED HR-CELL DEATH

L'ossido nitrico (NO) \ue8 una molecola gassosa radicale e una molecola di segnalazione chiave coinvolto nella mediazione di varie condizioni di sviluppo e di risposta allo stress biotico e abiotico nelle piante. Nell'interazione pianta-patogeno, NO presenta un ruolo cruciale nella difesa contro i microbi biotrofici, lavorando in collaborazione con ROS per innescare la morte cellulare nella zona infetta; un meccanismo noto come risposta ipersensibile (HR). Tuttavia, i meccanismi molecolari di come NO coordina questo processo \ue8 ancora sconosciuta. Per identificare i geni coinvolti nella segnalazione NO durante il processo di morte cellulare ipersensibile, abbiamo effettuato uno screening genetico diretto, utilizzando un sistema di doppia selezione per identificare piante mutanti di Arabidopsis compromessi nella signaling di NO durante la morte ipersensibile. Per lo screening primario, una piattaforma di fumigazione di NO \ue8 stato sviluppato e in seguito \ue8 stato identificato condizioni per innescare la morte cellulare in piante wild-type di Arabidopsis indotta dal trattamento col NO. Fumigando in totale 25.600 M2 piante provenienti dal etil metano sulfonato (EMS) e della popolazione mutante con neutroni (FN), abbiamo identificato 19 linee mutanti compromessi nella morte cellulare indotta dal NO. Il secondo screening consisteva nell'esaminare la riduzione della HR indotta dal patogeno in questi mutanti pre-selezionati. Analizzando 13 linee mutanti, resistenti al\u2019NO, per una diminuzione della morte cellulare innescata dell'infezione con patogeni non virulenti, abbiamo identificato 7 linee mutanti che sono stati alterati in questo processo. Le caratterizzazione dei processi riguardo alla HR \ue8 stata eseguita in tre di questi mutanti selezionati. Analizzando il loro sistema antiossidante e la presenza di normale produzione di NO e ROS durante la risposta ipersensibile in queste mutanti \ue8 stato possibile allocare la loro mutazione in posizione diversa nella segnalazione di NO durante la risposta di resistenza delle piante. I nostri risultati hanno dimostrato che i tre mutanti mostravano alterazioni nel sistema antiossidante, ci\uf2 influenzato negativamente la produzioni di ROS in uno degli mutanti. Inoltre, la sotto regolazione dell'attivit\ue0 della catalase ha contribuito per il loro superiore livello di ROS. A quanto riguarda la produzione di NO, questo \ue8 stato ridotto invece in tutte le tre linee ma, solo una linea ha presentato una completamente compromessa produzione di NO. \uc8 interessante notare che, in tutti i tre mutanti \ue8 stato disturbato la modulazione dell'attivit\ue0 GSNOR durante l\u2019HR. Concludendo, i nostri dati indicano che non solo il livello di produzione di entrambe NO e ROS, ma anche i meccanismi per la loro rimozioni sono stati determinanti per avere il normale sviluppo della morte ipersensibile; e quindi, la strategia di selezione progettato potrebbe anche selezionare mutanti deteriorati in questi processi. In questo modo, questi risultati rinforzano l'importanza di avere biodisponibilit\ue0 equilibrata tra H2O2 e NO nell'esecuzione della morte cellulare programmata nella risposta di resistenza delle piante al patogeno. La caratterizzazione della produzione di NO e di ROS in altri mutanti identificati permetter\ue0 di selezionare mutanti pi\uf9 interessanti da sequenziare. La preparazione della popolazione di mappatura utilizzando piante mutanti M3 originali ad incroci (OC) si ha rivelato di essere negativo per il nostro studio. La presenza di genotipi polimorfici nella popolazione ibrido F2 OC ha prodotto una segregazione genetica distorta, precludendo per determinare in modo inequivocabile il gene mutato. Pertanto, strategia supplementare per l'identificazione della mutazione causale, come l'uso di mutanti F2 backcrossed per la creazione di una popolazione di mappatura, dovrebbe essere considerata una volta che saranno prescelti candidati mutanti migliori.Nitric oxide (NO) is a gaseous free radical and a key signaling molecule involved in the mediation of various developmental and stress-related conditions in plants. In plant-pathogen interaction, NO plays a crucial role in defense against biotrophic microbes, working together with ROS to trigger cell death in the infected area; a mechanism known as hypersensitive response (HR). However, the molecular mechanisms of how NO coordinates this process is still unknown. To identify candidate genes involved in NO signaling during the HR-cell death process, we carried out a forward genetic screen, using a double-selection system to identify Arabidopsis mutant plants compromised in NO-mediated HR-cell death. For the primary screening, an NO fumigation platform was developed and conditions inducing uniform cell death on wild-type Arabidopsis plants identified. Fumigating in total 25,600 M2 ethyl methane sulfonate (EMS) and fast neutron (FN) mutant plants, we identified 19 mutant lines as non-responsive for NO-induced cell death. The second screening step consisted then, in examining the reduction in pathogen induced HR-cell death in these pre-selected mutants. Evaluation of 13 NO resistant mutant lines for decreased cell death upon avirulent pathogen infection, we finally identified 7 mutant lines that were impaired in this process. An extensive characterization of HR-cell death process was performed in three of these selected mutants. Analyzing the antioxidant system and the presence of normal NO and ROS bursts in these lines, was possible to allocate their mutation in different position in NO signaling in plant resistance. Our results demonstrated that all three mutants showed alteration in the antioxidant system, what affected negatively the ROS burst in one of them. The down-regulation of catalase activity contributed for their higher endogenous level of ROS. Moreover, the NO burst was reduced instead in all three lines but, only one presented a fully compromised NO burst. Interestingly, in all three mutants the modulation of GSNOR activity during HR was disturbed. Concluding, our data indicated that not only the rate of NO and ROS production but also the mechanisms for their turnover were determinant for having normal development of HR-cell death; and therefore, the designed selection strategy also could select mutants impaired in these processes. Thus, these findings reinforce the importance of having balanced bioavailability between H2O2 and NO in the execution of cell death program in plant resistance response. Characterization of NO and ROS in other identified mutants will allow to select most interesting mutants to be sequenced. Mapping population obtained from original M3 mutant plants for outcrossing (OC) revealed to be not the best material for our study. The presence of polymorphic genotypes in the hybrid F2 OC population yielded a distorted genetic segregation and could preclude to determine unequivocally, the mutated gene. Therefore additional strategy for identification of the causal mutation, like the use of F2 backcrossed mutants for creating a mapping population, should be considered once best candidates will be finally selected

Computing organoids’ volume in medical images: the case study of cystic fibrosis

We present CORVO (Computing Organoids VOlume in medical images), a tool for calculating the volume of complex time-changing 3D structures from medical videos. In order to identify anisotropies in volume variation over time, CORVO is equipped with a module implementing an advanced regression-based statistical analysis. We tested CORVO for the analysis of the variation of rectal organoids volume, whose anisotropic or missing expansion indicates a pathological state and a non-response to a pharmacological treatment, respectively

Theratyping of the Rare CFTR Genotype A559T in Rectal Organoids and Nasal Cells Reveals a Relevant Response to Elexacaftor (VX-445) and Tezacaftor (VX-661) Combination

Despite the promising results of new CFTR targeting drugs designed for the recovery of F508del- and class III variants activity, none of them have been approved for individuals with selected rare mutations, because uncharacterized CFTR variants lack information associated with the ability of these compounds in recovering their molecular defects. Here we used both rectal organoids (colonoids) and primary nasal brushed cells (hNEC) derived from a CF patient homozygous for A559T (c.1675G>A) variant to evaluate the responsiveness of this pathogenic variant to available CFTR targeted drugs that include VX-770, VX-809, VX-661 and VX-661 combined with VX-445. A559T is a rare mutation, found in African-Americans people with CF (PwCF) with only 85 patients registered in the CFTR2 database. At present, there is no treatment approved by FDA (U.S. Food and Drug Administration) for this genotype. Short-circuit current (Isc) measurements indicate that A559T-CFTR presents a minimal function. The acute addition of VX-770 following CFTR activation by forskolin had no significant increment of baseline level of anion transport in both colonoids and nasal cells. However, the combined treatment, VX-661-VX-445, significantly increases the chloride secretion in A559T-colonoids monolayers and hNEC, reaching approximately 10% of WT-CFTR function. These results were confirmed by forskolin-induced swelling assay and by western blotting in rectal organoids. Overall, our data show a relevant response to VX-661-VX-445 in rectal organoids and hNEC with CFTR genotype A559T/A559T. This could provide a strong rationale for treating patients carrying this variant with VX-661-VX-445-VX-770 combination

Multilinear Regression Analysis of Sweat Secretion Volumes in Cystic Fibrosis Patients

Personalized medicine approach in cystic fibrosis (CF) is focusing on detection of cystic fibrosis transmembrane conductance regulator (CFTR) function in single patients and standardized outcomes for CFTR function in vivo. We applied Optical Sweat Rate Beta Adrenergic (OSRBA) test for measuring sweat rates in individual human sweat glands. The results were analyzed according to a multilinear regression model in non- CF, healthy carriers (HTZ), CF patients; two groups of these were tested during treatment with CFTR modulators such as lumacaftor/ivacaftor (Orkambi) or PTC 124 (Ataluren). We found that different sets of statistically significant coefficients of the multilinear regression of the volume of sweat secretory glands characterize different CFTR genotype as well as different responses to different pharmacological treatments

Optical Measurements of Sweat for in Vivo Quantification of CFTR Function in Individual Sweat Glands

Optical measurement of CFTR-dependent sweat secretion stimulated by a beta-adrenergic cocktail (C-phase) vs. CFTR-independent sweat secretion induced by methacholine (M-phase) can discriminate cystic fibrosis (CF) patientts from controls and healthy carriers by the ratio of sweat rate in the C-phase vs. the M-phase (C/M ratio). However, image analysis is experimentally demanding and time-consuming. Here, sweat droplet number (SDN) in the C-phase, corresponding to the number of sweat-secreting glands, was a statistically significant predictor for detecting the effects of CFTR-targeted therapy. We show that in 44 non-CF subjects and 110 CF patients, SDN in the C-phase provides a linear readout of CFTR function that is more sensitive than that using the C/M ratio. In CF patients, increased SDN in the C-phase during treatment with (LUMA/IVA) was associated with a trend toward improved lung function (FEV1). Our method is suitable for multicenter monitoring of the effects of CFTR modulators

CFTR Modulators Rescue the Activity of CFTR in Colonoids Expressing the Complex Allele p.[R74W;V201M;D1270N]/dele22_24

: An Italian, 46-year-old female patient carrying the complex allele p.[R74W;V201M;D1270N] in trans with CFTR dele22_24 was diagnosed at the Cystic Fibrosis (CF) Center of Verona as being affected by CF-pancreatic sufficient (CF-PS) in 2021. The variant V201M has unknown significance, while both of the other variants of this complex allele have variable clinical consequences, according to the CFTR2 database, with reported clinical benefits for treatment with ivacaftor + tezacaftor and ivacaftor + tezacaftor + elexacaftor in patients carrying the R74W-D1270N complex allele, which are currently approved (in USA, not yet in Italy). She was previously followed up by pneumologists in northern Italy because of frequent bronchitis, hemoptysis, recurrent rhinitis, Pseudomonas aeruginosa lung colonization, bronchiectasis/atelectasis, bronchial arterial embolization and moderately compromised lung function (FEV1: 62%). Following a sweat test with borderline results, she was referred to the Verona CF Center where she presented abnormal values in both optical beta-adrenergic sweat tests and intestinal current measurement (ICM). These results were consistent with a diagnosis of CF. CFTR function analyses were also performed in vitro by forskolin-induced swelling (FIS) assay and short-circuit currents (Isc) in the monolayers of the rectal organoids. Both of these assays showed significantly increased CFTR activity following treatment with the CFTR modulators. Western-blot analysis revealed increased fully glycosylated CFTR protein after treatment with correctors, in line with the functional analysis. Interestingly, tezacaftor, together with elexacaftor, rescued the total organoid area under steady-state conditions, even in the absence of the CFTR agonist forskolin. In conclusion, in ex vivo and in vitro assays, we measured a residual function that was significantly enhanced by in vitro incubation with CFTR modulators, especially by ivacaftor + tezacaftor + elexacaftor, suggesting this combination as a potentially optimal treatment for this case