Entamoeba histolytica: In Silico and In Vitro Oligomerization of EhHSTF5 Enhances Its Binding to the HSE of the EhPgp5 Gene Promoter.

Throughout its lifecycle, Entamoeba histolytica encounters a variety of stressful conditions. This parasite possesses Heat Shock Response Elements (HSEs) which are crucial for regulating the expression of various genes, aiding in its adaptation and survival. These HSEs are regulated by Heat Shock Transcription Factors (EhHSTFs). Our research has identified seven such factors in the parasite, designated as EhHSTF1 through to EhHSTF7. Significantly, under heat shock conditions and in the presence of the antiamoebic compound emetine, EhHSTF5, EhHSTF6, and EhHSTF7 show overexpression, highlighting their essential role in gene response to these stressors. Currently, only EhHSTF7 has been confirmed to recognize the HSE as a promoter of the EhPgp5 gene (HSE_EhPgp5), leaving the binding potential of the other EhHSTFs to HSEs yet to be explored. Consequently, our study aimed to examine, both in vitro and in silico, the oligomerization, and binding capabilities of the recombinant EhHSTF5 protein (rEhHSTF5) to HSE_EhPgp5. The in vitro results indicate that the oligomerization of rEhHSTF5 is concentration-dependent, with its dimeric conformation showing a higher affinity for HSE_EhPgp5 than its monomeric state. In silico analysis suggests that the alpha 3 α-helix (α3-helix) of the DNA-binding domain (DBD5) of EhHSTF5 is crucial in binding to the major groove of HSE, primarily through hydrogen bonding and salt-bridge interactions. In summary, our results highlight the importance of oligomerization in enhancing the affinity of rEhHSTF5 for HSE_EhPgp5 and demonstrate its ability to specifically recognize structural motifs within HSE_EhPgp5. These insights significantly contribute to our understanding of one of the potential molecular mechanisms employed by this parasite to efficiently respond to various stressors, thereby enabling successful adaptation and survival within its host environment.

Novel Aspects of cAMP-Response Element Modulator (CREM) Role in Spermatogenesis and Male Fertility.

Spermatogenesis is a very complex process with an intricate transcriptional regulation. The transition from the diploid to the haploid state requires the involvement of specialized genes in meiosis, among other specific functions for the formation of the spermatozoon. The transcription factor cAMP-response element modulator (CREM) is a key modulator that triggers the differentiation of the germ cell into the spermatozoon through the modification of gene expression. CREM has multiple repressor and activator isoforms whose expression is tissue-cell-type specific and tightly regulated by various factors at the transcriptional, post-transcriptional and post-translational level. The activator isoform CREMτ controls the expression of several relevant genes in post-meiotic stages of spermatogenesis. In addition, exposure to xenobiotics negatively affects CREMτ expression, which is linked to male infertility. On the other hand, antioxidants could have a positive effect on CREMτ expression and improve sperm parameters in idiopathically infertile men. Therefore, CREM expression could be used as a biomarker to detect and even counteract male infertility. This review examines the importance of CREM as a transcription factor for sperm production and its relevance in male fertility, infertility and the response to environmental xenobiotics that may affect CREMτ expression and the downstream regulation that alters male fertility. Also, some health disorders in which CREM expression is altered are discussed.

Genistein-mediated thermogenesis and white-to-beige adipocyte differentiation involve transcriptional activation of cAMP response elements in the Ucp1 promoter.

Genistein is an isoflavone present in soybeans and is considered a bioactive compound due to its widely reported biological activity. We have previously shown that intraperitoneal genistein administration and diet supplementation activates the thermogenic program in rats and mice subcutaneous white adipose tissue (scWAT) under multiple environmental cues, including cold exposure and high-fat diet feeding. However, the mechanistic insights of this process were not previously unveiled. Uncoupling protein 1 (UCP1), a mitochondrial membrane polypeptide responsible for dissipating energy into heat, is considered the most relevant thermogenic marker; thus, we aimed to evaluate whether genistein regulates UCP1 transcription. Here we show that genistein administration to thermoneutral-housed mice leads to the appearance of beige adipocyte markers, including a sharp upregulation of UCP1 expression and protein abundance in scWAT. Reporter assays showed an increase in UCP1 promoter activity after genistein stimulation, and in silico analysis revealed the presence of estrogen (ERE) and cAMP (CRE) response elements as putative candidates of genistein activation. Mutation of the CRE but not the ERE reduced genistein-induced promoter activity by 51%. Additionally, in vitro and in vivo ChIP assays demonstrated the binding of CREB to the UCP1 promoter after acute genistein administration. Taken together, these data elucidate the mechanism of genistein-mediated UCP1 induction and confirm its potential applications in managing metabolic disorders.

Structural Characterization of the Interaction of Hypoxia Inducible Factor-1 with Its Hypoxia Responsive Element at the -964G > A Variation Site of the HLA-G Promoter Region.

Human Antigen Leukocyte-G (HLA-G) gene encodes an immune checkpoint molecule that has restricted tissue expression in physiological conditions; however, the gene may be induced in hypoxic conditions by the interaction with the hypoxia inducible factor-1 (HIF1). Hypoxia regulatory elements (HRE) located at the HLA-G promoter region and at exon 2 are the major HIF1 target sites. Since the G allele of the -964G > A transversion induces higher HLA-G expression when compared to the A allele in hypoxic conditions, here we analyzed HIF1-HRE complex interaction at the pair-atom level considering both -964G > A polymorphism alleles. Mouse HIF2 dimer crystal (Protein Data Bank ID: 4ZPK) was used as template to perform homology modelling of human HIF1 quaternary structure using MODELLER v9.14. Two 3D DNA structures were built from 5'GCRTG'3 HRE sequence containing the -964G/A alleles using x3DNA. Protein-DNA docking was performed using the HADDOCK v2.4 server, and non-covalent bonds were computed by DNAproDB server. Molecular dynamic simulation was carried out per 200 ns, using Gromacs v.2019. HIF1 binding in the HRE containing -964G allele results in more hydrogen bonds and van der Waals contact formation than HRE with -964A allele. Protein-DNA complex trajectory analysis revealed that HIF1-HRE-964G complex is more stable. In conclusion, HIF1 binds in a more stable and specific manner at the HRE with G allele.

Molecular mechanisms underlying AMH elevation in hyperoestrogenic states in males.

Anti-Müllerian hormone (AMH) is secreted by Sertoli cells of the testes from early fetal life until puberty, when it is downregulated by androgens. In conditions like complete androgen insensitivity syndrome (CAIS), AMH downregulation does not occur and AMH increases at puberty, due in part to follicle-stimulating hormone (FSH) effect. However, other conditions like Peutz-Jeghers syndrome (PJS), characterised by low FSH, also have increased AMH. Because both CAIS and PJS may present as hyperoestrogenic states, we tested the hypothesis that oestradiol (E2) upregulates AMH expression in peripubertal Sertoli cells and explored the molecular mechanisms potentially involved. The results showed that E2 is capable of inducing an upregulation of endogenous AMH and of the AMH promoter activity in the prepubertal Sertoli cell line SMAT1, signalling through ERα binding to a specific ERE sequence present on the hAMH promoter. A modest action was also mediated through the membrane oestrogen receptor GPER. Additionally, the existence of ERα expression in Sertoli cells in patients with CAIS was confirmed by immunohistochemistry. The evidence presented here provides biological plausibility to the hypothesis that testicular AMH production increases in clinical conditions in response to elevated oestrogen levels.

In silico analysis of putative metal response elements (MREs) in the zinc-responsive genes from Trichomonas vaginalis and the identification of novel palindromic MRE-like motif.

Zinc is an essential micronutrient that plays an important role as a co-factor to several proteins, including zinc-responsive transcription factors. Trichomonas vaginalis is able to survive in the presence of high zinc concentrations in the male urogenital tract. Several genes in T. vaginalis have been shown to respond to changes in zinc concentrations, however, the zinc-dependent mechanism remains undetermined. Recently, we identified in T. vaginalis the zinc finger protein, TvZNF1, which is an ortholog of the mammal metal transcription factor (MTF1). We searched for several of the zinc-responsive genes in T. vaginalis to determine whether if they contain metal response elements (MRE), cis-acting DNA elements that specifically bind MTF1. Six highly conserved over-represented sequence motifs (TvMREs), which share similarity with other eukaryotic MREs, were identified in the zinc-responsive genes in T. vaginalis. We also demonstrated that some of the TvMREs assemble as divalent complexes either as two closely spaced TvMREs or as two overlapping TvMREs forming a palindromic-like sequence: TGCC(N3)GGCA. Electrophoretic mobility shift assays were used to detect the zinc-dependent binding of TvZNF1 and nuclear proteins from T. vaginalis to this specific palindromic motif. Our results support a novel mechanism used by T. vaginalis for the transcriptional regulation of associated zinc-responsive genes through a MTF1/MRE-like system.

Crosstalk of BMP-4 and RA signaling pathways on Pomc gene regulation in corticotrophs.

Retinoic acid (RA), an active metabolite of Vitamin A, and bone morphogenetic protein 4 (BMP-4) pathways control the transcription of pro-opiomelanocortin (Pomc), the precursor of ACTH. We describe a novel mechanism by which RA and BMP-4 act together in the context of pituitary corticotroph tumoral cells to regulate Pomc transcription. BMP-4 and RA exert a potentiated inhibition on Pomc gene expression. This potentiation of the inhibitory action on Pomc transcription was blocked by the inhibitory SMADs of the BMP-4 pathway (SMAD6 and SMAD7), a negative regulator of BMP-4 signaling (TOB1) and a blocker of RA pathway (COUP-TFI). AtT-20 corticotrophinoma cells express RA receptors (RARB, RXRA and RXRG) which associate with factors of BMP-4 (SMAD4 and SMAD1) signaling cascade in transcriptional complexes that block Pomc transcription. COUP-TFI and TOB1 disrupt these complexes. Deletions and mutations of the Pomc promoter and a specific DNA-binding assay show that the complexes bind to the RARE site in the Pomc promoter. The enhanced inhibitory interaction between RA and BMP-4 pathways occurs also in another relevant corticotroph gene promoter, the corticotropin-releasing hormone receptor 1 (Crh-r1). The understanding of the molecules that participate in the control of corticotroph gene expression contribute to define more precise targets for the treatment of corticotrophinomas.

A novel salt-inducible CrGPDH3 promoter of the microalga Chlamydomonas reinhardtii for transgene overexpression.

The expression of transgenes in the nucleus is an attractive alternative for the expression of recombinant proteins in the green microalga Chlamydomonas reinhardtii. For this purpose, a strong inducible promoter that allows protein accumulation without possible negative effects on cell multiplication and biomass accumulation is desirable. A previous study at our laboratory identified that the CrGPDH3 gene from C. reinhardtii was inducible under NaCl treatments. In this study, we cloned and characterized a 3012 bp sequence upstream of the start codon of the CrGPDH3 gene, including the 285 bp 5' untranslated region. This region was identified as the full-length promoter and named PromA (- 2727 to + 285). Deletion analysis of PromA using GUSPlus as a reporter gene enabled us to identify PromC (- 653 to + 285) as the core promoter, displaying basal expression. A region named RIA1 (- 2727 to - 1672) was suggested to contain the NaCl response elements. Moreover, deletion analysis of RIA1 enabled us to identify a region of 577 bp named RIA3 (- 2727 to - 2150) that, when cloned upstream of PromC, was able to drive the expression of GUSPlus in response to 5 and 100 mM NaCl, and 100 mM KCl, similar to the native CrGPDH3 promoter. These results expand our understanding of the transcriptional mechanism of CrGPDH3 and clearly show that CrGPDH3 promoter and its chimeric forms are highly salt-inducible and can be used as inducible promoters for the overexpression of transgenes in C. reinhardtii.

Heme Oxygenase 1 Impairs Glucocorticoid Receptor Activity in Prostate Cancer.

Glucocorticoids are used during prostate cancer (PCa) treatment. However, they may also have the potential to drive castration resistant prostate cancer (CRPC) growth via the glucocorticoid receptor (GR). Given the association between inflammation and PCa, and the anti-inflammatory role of heme oxygenase 1 (HO-1), we aimed at identifying the molecular processes governed by the interaction between HO-1 and GR. PCa-derived cell lines were treated with Hemin, Dexamethasone (Dex), or both. We studied GR gene expression by RTqPCR, protein expression by Western Blot, transcriptional activity using reporter assays, and nuclear translocation by confocal microscopy. We also evaluated the expression of HO-1, FKBP51, and FKBP52 by Western Blot. Hemin pre-treatment reduced Dex-induced GR activity in PC3 cells. Protein levels of FKBP51, a cytoplasmic GR-binding immunophilin, were significantly increased in Hemin+Dex treated cells, possibly accounting for lower GR activity. We also evaluated these treatments in vivo using PC3 tumors growing as xenografts. We found non-significant differences in tumor growth among treatments. Immunohistochemistry analyses revealed strong nuclear GR staining in almost all groups. We did not observe HO-1 staining in tumor cells, but high HO-1 reactivity was detected in tumor infiltrating macrophages. Our results suggest an association and crossed modulation between HO-1 and GR pathways.

TyrR is involved in the transcriptional regulation of biofilm formation and D-alanine catabolism in Azospirillum brasilense Sp7.

Azospirillum brasilense is one of the most studied species of diverse agronomic plants worldwide. The benefits conferred to plants inoculated with Azospirillum have been primarily attributed to its capacity to fix atmospheric nitrogen and synthesize phytohormones, especially indole-3-acetic acid (IAA). The principal pathway for IAA synthesis involves the intermediate metabolite indole pyruvic acid. Successful colonization of plants by Azospirillum species is fundamental to the ability of these bacteria to promote the beneficial effects observed in plants. Biofilm formation is an essential step in this process and involves interactions with the host plant. In this study, the tyrR gene was cloned, and the translated product was observed to exhibit homology to TyrR protein, a NtrC/NifA-type activator. Structural studies of TyrR identified three putative domains, including a domain containing binding sites for aromatic amino acids in the N-terminus, a central AAA+ ATPase domain, and a helix-turn-helix DNA binding motif domain in the C-terminus, which binds DNA sequences in promoter-operator regions. In addition, a bioinformatic analysis of promoter sequences in A. brasilense Sp7 genome revealed that putative promoters encompass one to three TyrR boxes in genes predicted to be regulated by TyrR. To gain insight into the phenotypes regulated by TyrR, a tyrR-deficient strain derived from A. brasilense Sp7, named A. brasilense 2116 and a complemented 2116 strain harboring a plasmid carrying the tyrR gene were constructed. The observed phenotypes indicated that the putative transcriptional regulator TyrR is involved in biofilm production and is responsible for regulating the utilization of D-alanine as carbon source. In addition, TyrR was observed to be absolutely required for transcriptional regulation of the gene dadA encoding a D-amino acid dehydrogenase. The data suggested that TyrR may play a major role in the regulation of genes encoding a glucosyl transferase, essential signaling proteins, and amino acids transporters.

Wnt/ß-catenin signaling enhances transcription of the CX43 gene in murine Sertoli cells.

Sertoli cells provide the nutritional and metabolic support for germ cells. Wnt/ß-catenin signaling is important for the development of the seminiferous epithelium during embryonic age, although after birth this pathway is downregulated. Cx43 gene codes for a protein that is critical during testicular development. The Cx43 promoter contains TCF/ß-catenin binding elements (TBEs) that contribute CX43 expression in different cell types and which may also be regulating the expression of this gene in Sertoli cells. In this study, we demonstrate that 42GPA9 Sertoli cells respond to treatments that result in accumulation of ß-catenin within the nucleus and in upregulation of CX43 gene transcription. ß-Catenin binds to TBEs located both upstream and downstream of the transcriptional start site (TSS). Luciferase reporter experiments revealed that TBEs located upstream of the TSS are necessary for ß-catenin-mediated upregulation. Our results also indicate that the Wnt/ß-catenin-dependent upregulation of the Cx43 gene in Sertoli cells is accompanied by changes in epigenetic parameters that may be directly contributing to generating a chromatin environment that facilitates the establishment of the transcriptional machinery at this promoter.

Renovación contextual de la distribución temporal del responder / Contextual Renewal of Temporal Patterns of Responding

Resumen Se ha reportado que en ciertos fenómenos (recuperación espontánea, readquisición, resurgencia y renovación contextual) es posible observar la recuperación de una respuesta previamente extinguida. La renovación contextual, se refiere a la reaparición de una respuesta extinguida que resulta de un cambio de contexto entre la fase de extinción y prueba. El propósito del presente trabajo consistió en evaluar la renovación contextual, atendiendo tanto a la distribución temporal del responder, como a la tasa de respuesta. Se encontró una recuperación de la tasa de respuesta, siendo un resultado consistente con los reportados en el área de renovación contextual. Además, se pudo identificar una renovación de la distribución temporal de respuesta. Los resultados se discuten reconociendo que la tasa y la distribución temporal constituyen dos dimensiones de la ejecución que son moduladas por los cambios de contexto.

Abstract It has been reported that in certain phenomena (spontaneous recovery, reacquisition, resurgence and contextual renewal) it is possible to observe the recovery of a previously extinguished response. Contextual renewal refers to the reappearance of an extinguished response that result from a change of context between the extinction and testing phase. The purpose of the present study was to evaluate contextual renewal, considering both the temporal distribution of the responses and the response rate. A recovery of the response rate was found, being a result consistent with those reported in the area of ​​contextual renewal. In addition, a renewal of the temporal distribution of the responses could be identified. The results are discussed by recognizing that rate and temporal distribution are two behavioral dimensions that are modulated by contextual changes.

Alterations in System xc- Expression in the Retina of Type 1 Diabetic Rats and the Role of Nrf2.

Nrf2 (nuclear factor erythroid 2-related factor 2), a transcription factor that controls expression of several proteins that are related to cellular antioxidant capacity, such as the subunit xCT of the system xc-, is dysregulated in diabetes. Recently, it was described that system xc- is decreased in the retina after 3 weeks of diabetes. So, in the present work, the temporal relationship between xCT and Nrf2 in the retina of diabetic animals was investigated. Diabetes was induced in male Wistar rats (200 g) by a single injection of streptozotocin, and retinas were collected after 1, 2, and 6 months of diabetes induction. Expression of xCT, Nrf2 activity, and binding to antioxidant-responsive element (ARE) sequence were evaluated. Glutathione and reactive oxygen species (ROS) levels were also assessed. After 1 month of diabetes, Nrf2 activity, xCT expression, and glutathione levels were reduced whereas ROS were increased. Although glutathione and ROS levels remain unchanged until later stages, Nrf2 activity and xCT expression returned to normal levels after 2 months. However, they were decreased again at 6 months of diabetes. Accordingly, Nrf2 binding to xCT ARE sequence followed the same pattern of Nrf2 activity and xCT expression. These data showed that retinal xCT expression is regulated by Nrf2 in diabetic condition. The results also demonstrated a temporal relationship between Nrf2 and system xc- which could be implicated in the initiation of oxidative stress in retina in diabetes.

Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus.

Glycogen synthase kinase 3 (GSK3) is a constitutive enzyme implicated in the regulation of cytokine expression and the inflammatory response during bacterial infections. Mammals have two GSK3 isoforms named GSK3α and GSK3ß that plays different but often overlapping functions. Although the role of GSK3ß in cytokine regulation during the inflammatory response caused by bacteria is well described, GSK3α has not been found to participate in this process. Therefore, we tested if GSK3α may act as a regulatory isoform in the cytokine expression by bovine endothelial cells infected with Staphylococcus aureus because this bacterium is one of the major pathogens that cause tissue damage associated with inflammatory dysfunction. Interestingly, although both isoforms were phosphorylated-inactivated, we consistently observed a higher phosphorylation of GSK3α at Ser21 than that of GSK3ß at Ser9 after bacterial challenge. During a temporal course of infection, we characterized a molecular switch from pro-inflammatory cytokine expression (IL-8), promoted by nuclear factor-kappa B (NF-κB), at an early stage (2 h) to an anti-inflammatory cytokine expression (IL-10), promoted by cAMP response element binding (CREB), at a later stage (6 h). We observed an indirect effect of GSK3α activity on NF-κB activation that resulted in a low phosphorylation of CREB at Ser133, a decreased interaction between CREB and the co-activator CREB-binding protein (CBP), and a lower expression level of IL-10. Gene silencing of GSK3α and GSK3ß with siRNA indicated that GSK3α knockout promoted the interaction between CREB and CBP that, in turn, increased the expression of IL-10, reduced the interaction of NF-κB with CBP, and reduced the expression of IL-8. These results indicate that GSK3α functions as the primary isoform that regulates the expression of IL-10 in endothelial cells infected with S. aureus.

DNA Methylation Variability in a Single Locus of the RXRα Promoter from Umbilical Vein Blood at Term Pregnancy.

DNA methylation status of RXRα gene promoter has been correlated with maternal diet during early pregnancy, and associated with offspring's adiposity and bone mineral content. In adult life, increased methylation of RXRα promoter region is associated with myocardium pathologies. Early growth response proteins (EGR) are zinc finger transcription factors associated with several cellular pathways such as inflammation, apoptosis, and cardiopathies. DNA-binding sequences of EGR proteins have been reported in the RXRα gene promoter using chromatin immunoprecipitation methods. Here, we used correlations between the maternal pre-pregnancy body mass index (p-BMI), gestational weight gain (GWG), and birth weight (BW) as indirect indicators of the maternal nutritional status as modifier of DNA methylation in the offspring. DNA methylation status from newborns' umbilical vein blood in full-term pregnancy was evaluated in a short sequence (116 pb) of the RXRα gene promoter that contains the elements of response sequence for EGR proteins. Fifty-three bisulfite-modified DNA samples were assessed through methyl-sensitive high-resolution melting (MS-HRM) analysis. To validate the results, we directly sequenced MS-HRM samples to confirm the presence of CpG-methylated positions. In addition, the RXRα protein levels in extracts of umbilical vein blood were evaluated by western blot. We found differential methylation in a specific locus of the RXRα promoter surrounding the EGR-binding sequence; however, no correlation was found with the level of RXRα protein expression. Variability in the methylation status of the RXRα promoter near the EGR transcription factor binding site in newborn cord blood provides controversial epigenetic insights into RXRα regulation via EGR proteins.

Emergent Properties in Complex Synthetic Bacterial Promoters.

Regulation of gene expression in bacteria results from the interplay between hundreds of transcriptional factors (TFs) at target promoters. However, how the arrangement of binding sites for TFs generates the regulatory logic of promoters is not well-known. Here, we generated and fully characterized a library of synthetic complex promoters for the global regulators, CRP and IHF, in Escherichia coli, which are formed by a weak -35/-10 consensus sequence preceded by four combinatorial binding sites for these two TFs. Using this approach, we found that while cis-elements for CRP preferentially activate promoters when located immediately upstream of the promoter consensus, binding sites for IHF mainly function as "UP" elements and stimulate transcription in several different architectures in the absence of this protein. However, the combination of CRP- and IHF-binding sites resulted in emergent properties in these complex promoters, where the activity of combinatorial promoters cannot be predicted from the individual behavior of its components. Taken together, the results presented here add to the information on architecture-logic of complex promoters in bacteria.

The CCAAT box in the proximal SERCA2 gene promoter regulates basal and stress-induced transcription in cardiomyocytes.

The cardiac sarco/endoplasmic reticulum Ca2+-ATPase-2a (SERCA2a) is vital for the correct handling of calcium concentration in cardiomyocytes. Recent studies showed that the induction of endoplasmic reticulum (ER) stress (ERS) with the SERCA2 inhibitor Thapsigargin (Tg) increases the mRNA and protein levels of SERCA2a. The SERCA2 gene promoter contains an ERS response element (ERSE) at position -78 bp that is conserved among species and might transcriptionally regulate SERCA2 gene expression. However, its involvement in SERCA2 basal and calcium-mediated transcriptional activation has not been elucidated. In this work, we show that in cellular cultures of neonatal rat ventricular myocytes, the treatment with Tg or the calcium ionophore A23187 increases the SERCA2a mRNA and protein abundance, as well as the transcriptional activity of two chimeric human SERCA2 gene constructs, containing -254 and -2579 bp of 5'-regulatory region cloned in the pGL3-basic vector and transiently transfected in cultured cardiomyocytes. We found that the ERSE present in the SERCA2 proximal promoter contains a CCAAT box that is involved in basal and ERS-mediated hSERCA2 transcriptional activation. The EMSA results showed that the CCAAT box present in the ERSE recruits the NF-Y transcription factor. Additionally, by ChIP assays, we confirmed in vivo binding of NF-Y and C/EBPß transcription factors to the SERCA2 gene proximal promoter.

Novos reguladores de resposta envolvidos na virulência de Pseudomonas aeruginosa / New response regulators involved in Pseudomonas aeruginosa virulence

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

TvZNF1 is a C2H2 zinc finger protein of Trichomonas vaginalis.

The zinc fingers proteins (ZNF) are the largest family of DNA binding proteins and can act as transcriptional factors in eukaryotes. ZNF are implicated in activation in response to environmental stimulus by biometals such as Zn2+. Many of these proteins have the classical C2H2 zinc finger motifs (C2H2-ZNFm) of approximately 30 amino acids, where a Zn2+ ion is coordinated by two cysteine and two histidine residues. Trichomonas vaginalis is a protozoan parasite than responds to environmental changes including Zn2+. Until now has not been described any ZNF that could be involved in the regulation of genic expression of T. vaginalis. Here, we characterized in silico and experimentally an annoted ZNF (TvZNF1) from T. vaginalis and isolated the gene, tvznf1 encoding it. TvZNF1 have eight C2H2-ZNFm with residues that maybe involved in the structural stability of DNA binding motifs. In this work we confirmed the Zn2+ upregulation expression of tvznf1 gene. Recombinant TvZNF1 was able to bind to specific DNA sequences according to EMSA assay. Additionally, we demonstrated that recombinant TvZNF1 bind to MRE signature in vitro, which strongly suggests its role in transcriptional regulation, similar to the one observed for mammalian MTF-1. This result suggested a conserved mechanism of genic regulation mediated by ZNFs in T. vaginalis.

Genetic basis of mycotoxin susceptibility differences between budding yeast isolates.

Micophenolic acid (MPA) is an immunosuppressant mycotoxin which impairs yeast cell growth to variable degrees depending on the genetic background. Such variation could have emerged from several phenomena, including MPA gene resistance mutations and variations in copy number and localisation of resistance genes. To test this, we evaluated MPA susceptibility in four S. cerevisiae isolates and genetically dissected variation through the identification of Quantitative Trait Loci. Via linkage analysis we identified six QTLs, majority of which were located within subtelomeres and co-localised with IMD2, an inosine monophosphate dehydrogenase previously identified underlying MPA drug resistance in yeast cells. From chromosome end disruption and bioinformatics analysis, it was found that the subtelomere localisation of IMD2 within chromosome ends is variable depending on the strain, demonstrating the influence of IMD2 on the natural variation in yeast MPA susceptibility. Furthermore, GxE gene expression analysis of strains exhibiting opposite phenotypes indicated that ribosome biogenesis, RNA transport, and purine biosynthesis were impaired in strains most susceptible to MPA toxicity. Our results demonstrate that natural variation can be exploited to better understand the molecular mechanisms underlying mycotoxin susceptibility in eukaryote cells and demonstrate the role of subtelomeric regions in mediating interactions with the environment.