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Methicillin-resistant Staphylococcus aureus infections are a global health problem. New control strategies, including fifth-generation cephalosporins such as ceftaroline, have been developed, however rare sporadic resistance has been reported. Our study aimed to determine whether disruption of two-component environmental signal systems detectably led to enhanced susceptibility to ceftaroline in S. aureus CA-MRSA strain MW2 at sub-MIC concentrations where cells normally continue to grow. A collection of sequential mutants in all fifteen S. aureus non-essential two-component systems (TCS) was first screened for ceftaroline sub-MIC susceptibility, using the spot population analysis profile method. We discovered a role for both ArlRS and VraSR TCS as determinants responsible for MW2 survival in the presence of sub-MIC ceftaroline. Subsequent analysis showed that dual disruption of both arlRS and vraSR resulted in a very strong ceftaroline hypersensitivity phenotype. Genetic complementation analysis confirmed these results and further revealed that arlRS and vraSR likely regulate some common pathway(s) yet to be determined. Our study shows that S. aureus uses particular TCS environmental sensing systems for this type of defense and illustrates the proof of principle that if these TCS were inhibited, the efficacy of certain antibiotics might be considerably enhanced.
Maite Villanueva; Melanie Roch; Iñigo Lasa; Adriana Renzoni; William Kelley. The Role of ArlRS and VraSR in Regulating Ceftaroline Hypersusceptibility in Methicillin-Resistant Staphylococcus aureus. Antibiotics 2021, 10, 821 .
AMA StyleMaite Villanueva, Melanie Roch, Iñigo Lasa, Adriana Renzoni, William Kelley. The Role of ArlRS and VraSR in Regulating Ceftaroline Hypersusceptibility in Methicillin-Resistant Staphylococcus aureus. Antibiotics. 2021; 10 (7):821.
Chicago/Turabian StyleMaite Villanueva; Melanie Roch; Iñigo Lasa; Adriana Renzoni; William Kelley. 2021. "The Role of ArlRS and VraSR in Regulating Ceftaroline Hypersusceptibility in Methicillin-Resistant Staphylococcus aureus." Antibiotics 10, no. 7: 821.
The control of dipteran pests is highly relevant to humans due to their involvement in the transmission of serious diseases including malaria, dengue fever, Chikungunya, yellow fever, zika, and filariasis; as well as their agronomic impact on numerous crops. Many bacteria are able to produce proteins that are active against insect species. These bacteria include Bacillus thuringiensis, the most widely-studied pesticidal bacterium, which synthesizes proteins that accumulate in crystals with insecticidal properties and which has been widely used in the biological control of insects from different orders, including Lepidoptera, Coleoptera, and Diptera. In this review, we summarize all the bacterial proteins, from B. thuringiensis and other entomopathogenic bacteria, which have described insecticidal activity against dipteran pests, including species of medical and agronomic importance.
Daniel Valtierra-De-Luis; Maite Villanueva; Colin Berry; Primitivo Caballero. Potential for Bacillus thuringiensis and Other Bacterial Toxins as Biological Control Agents to Combat Dipteran Pests of Medical and Agronomic Importance. Toxins 2020, 12, 773 .
AMA StyleDaniel Valtierra-De-Luis, Maite Villanueva, Colin Berry, Primitivo Caballero. Potential for Bacillus thuringiensis and Other Bacterial Toxins as Biological Control Agents to Combat Dipteran Pests of Medical and Agronomic Importance. Toxins. 2020; 12 (12):773.
Chicago/Turabian StyleDaniel Valtierra-De-Luis; Maite Villanueva; Colin Berry; Primitivo Caballero. 2020. "Potential for Bacillus thuringiensis and Other Bacterial Toxins as Biological Control Agents to Combat Dipteran Pests of Medical and Agronomic Importance." Toxins 12, no. 12: 773.
Bacillus thuringiensis is the most successful microbial insecticide agent and its proteins have been studied for many years due to its toxicity against insects mainly belonging to the orders Lepidoptera, Diptera and Coleoptera, which are pests of agro-forestry and medical-veterinary interest. However, studies on the interactions between this bacterium and the insect species classified in the order Coleoptera are more limited when compared to other insect orders. To date, 45 Cry proteins, 2 Cyt proteins, 11 Vip proteins, and 2 Sip proteins have been reported with activity against coleopteran species. A number of these proteins have been successfully used in some insecticidal formulations and in the construction of transgenic crops to provide protection against main beetle pests. In this review, we provide an update on the activity of Bt toxins against coleopteran insects, as well as specific information about the structure and mode of action of coleopteran Bt proteins.
Mikel Domínguez-Arrizabalaga; Maite Villanueva; Baltasar Escriche; Carmen Ancín-Azpilicueta; Primitivo Caballero. Insecticidal Activity of Bacillus thuringiensis Proteins Against Coleopteran Pests. Toxins 2020, 12, 430 .
AMA StyleMikel Domínguez-Arrizabalaga, Maite Villanueva, Baltasar Escriche, Carmen Ancín-Azpilicueta, Primitivo Caballero. Insecticidal Activity of Bacillus thuringiensis Proteins Against Coleopteran Pests. Toxins. 2020; 12 (7):430.
Chicago/Turabian StyleMikel Domínguez-Arrizabalaga; Maite Villanueva; Baltasar Escriche; Carmen Ancín-Azpilicueta; Primitivo Caballero. 2020. "Insecticidal Activity of Bacillus thuringiensis Proteins Against Coleopteran Pests." Toxins 12, no. 7: 430.
Bacillus thuringiensis (Bt)-based biological insecticides are used extensively to control insect pests and vectors of human diseases. Bt-based products provide greater specificity and biosafety than broad-spectrum synthetic insecticides. The biological activity of this bacterium resides in spores and crystals comprising complex mixtures of toxic proteins. We developed and validated a fast, accurate, and reproducible method for quantitative determination of the crystal components of Bt-based products. This method will find clear applications in the improvement of various aspects of the industrial production process of Bt. An important aspect of the production of Bt-based insecticides is its quality control. By specifically quantifying the relative proportion of each of the toxins that make up the crystal, our method represents the most consistent and repeatable evaluation procedure in the quality control of different batches produced in successive fermentations. This method can also contribute to the design of specific culture media and fermentation conditions that optimize Bt crystal composition across a range of Bt strains that target different pestiferous insects. Quantitative information on crystal composition should also prove valuable to phytosanitary product registration authorities that oversee the safety and efficacy of crop protection products.
Javier Caballero; Nerea Jiménez-Moreno; Irene Orera; Trevor Williams; Ana B. Fernández; Maite Villanueva; Juan Ferré; Primitivo Caballero; Carmen Ancín-Azpilicueta. Unraveling the Composition of Insecticidal Crystal Proteins in Bacillus thuringiensis: a Proteomics Approach. Applied and Environmental Microbiology 2020, 86, 1 .
AMA StyleJavier Caballero, Nerea Jiménez-Moreno, Irene Orera, Trevor Williams, Ana B. Fernández, Maite Villanueva, Juan Ferré, Primitivo Caballero, Carmen Ancín-Azpilicueta. Unraveling the Composition of Insecticidal Crystal Proteins in Bacillus thuringiensis: a Proteomics Approach. Applied and Environmental Microbiology. 2020; 86 (12):1.
Chicago/Turabian StyleJavier Caballero; Nerea Jiménez-Moreno; Irene Orera; Trevor Williams; Ana B. Fernández; Maite Villanueva; Juan Ferré; Primitivo Caballero; Carmen Ancín-Azpilicueta. 2020. "Unraveling the Composition of Insecticidal Crystal Proteins in Bacillus thuringiensis: a Proteomics Approach." Applied and Environmental Microbiology 86, no. 12: 1.
Bacillus thuringiensis ser. israelensis (Bti) has been widely used as microbial larvicide for the control of many species of mosquitoes and blackflies. The larvicidal activity of Bti resides in Cry and Cyt δ-endotoxins present in the parasporal crystal of this pathogen. The insecticidal activity of the crystal is higher than the activities of the individual toxins, which is likely due to synergistic interactions among the crystal component proteins, particularly those involving Cyt1Aa. In the present study, Cry10Aa and Cyt2Ba were cloned from the commercial larvicide VectoBac-12AS® and expressed in the acrystalliferous Bt strain BMB171 under the cyt1Aa strong promoter of the pSTAB vector. The LC50 values for Aedes aegypti second instar larvae estimated at 24 hpi for these two recombinant proteins (Cry10Aa and Cyt2Ba) were 299.62 and 279.37 ng/mL, respectively. Remarkable synergistic mosquitocidal activity was observed between Cry10Aa and Cyt2Ba (synergistic potentiation of 68.6-fold) when spore + crystal preparations, comprising a mixture of both recombinant strains in equal relative concentrations, were ingested by A. aegypti larvae. This synergistic activity is among the most powerful described so far with Bt toxins and is comparable to that reported for Cyt1A when interacting with Cry4Aa, Cry4Ba or Cry11Aa. Synergistic mosquitocidal activity was also observed between the recombinant proteins Cyt2Ba and Cry4Aa, but in this case, the synergistic potentiation was 4.6-fold. In conclusion, although Cry10Aa and Cyt2Ba are rarely detectable or appear as minor components in the crystals of Bti strains, they represent toxicity factors with a high potential for the control of mosquito populations.
Daniel Valtierra-De-Luis; Maite Villanueva; Liliana Lai; Trevor Williams; Primitivo Caballero. Potential of Cry10Aa and Cyt2Ba, Two Minority δ-endotoxins Produced by Bacillus thuringiensis ser. israelensis, for the Control of Aedes aegypti Larvae. Toxins 2020, 12, 355 .
AMA StyleDaniel Valtierra-De-Luis, Maite Villanueva, Liliana Lai, Trevor Williams, Primitivo Caballero. Potential of Cry10Aa and Cyt2Ba, Two Minority δ-endotoxins Produced by Bacillus thuringiensis ser. israelensis, for the Control of Aedes aegypti Larvae. Toxins. 2020; 12 (6):355.
Chicago/Turabian StyleDaniel Valtierra-De-Luis; Maite Villanueva; Liliana Lai; Trevor Williams; Primitivo Caballero. 2020. "Potential of Cry10Aa and Cyt2Ba, Two Minority δ-endotoxins Produced by Bacillus thuringiensis ser. israelensis, for the Control of Aedes aegypti Larvae." Toxins 12, no. 6: 355.
The genome of the Bacillus thuringiensis BM311.1 strain was sequenced and assembled in 359 contigs containing a total of 6,390,221 bp. The plasmidic ORF of a putative cry gene from this strain was identified as a potential novel Cry protein of 1138 amino acid residues with a 98% identity compared to Cry7Aa1 and a predicted molecular mass of 129.4 kDa. The primary structure of Cry7Aa2, which had eight conserved blocks and the classical structure of three domains, differed in 28 amino acid residues from that of Cry7Aa1. The cry7Aa2 gene was amplified by PCR and then expressed in the acrystalliferous strain BMB171. SDS-PAGE analysis confirmed the predicted molecular mass for the Cry7Aa2 protein and revealed that after in vitro trypsin incubation, the protein was degraded to a toxin of 62 kDa. However, when treated with digestive fluids from Leptinotarsa decemlineata larvae, one major proteinase-resistant fragment of slightly smaller size was produced. The spore and crystal mixture produced by the wild-type BM311.1 strain against L. decemlineata neonate larvae resulted in a LC50 value of 18.8 μg/mL, which was statistically similar to the estimated LC50 of 20.8 μg/mL for the recombinant BMB17-Cry7Aa2 strain. In addition, when this novel toxin was activated in vitro with commercial trypsin, the LC50 value was reduced 3.8-fold to LC50 = 4.9 μg/mL. The potential advantages of Cry7Aa2 protoxin compared to Cry7Aa1 protoxin when used in the control of insect pests are discussed.
Mikel Domínguez-Arrizabalaga; Maite Villanueva; Ana Beatriz Fernandez; Primitivo Caballero. A Strain of Bacillus thuringiensis Containing a Novel cry7Aa2 Gene that Is Toxic to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Insects 2019, 10, 259 .
AMA StyleMikel Domínguez-Arrizabalaga, Maite Villanueva, Ana Beatriz Fernandez, Primitivo Caballero. A Strain of Bacillus thuringiensis Containing a Novel cry7Aa2 Gene that Is Toxic to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae). Insects. 2019; 10 (9):259.
Chicago/Turabian StyleMikel Domínguez-Arrizabalaga; Maite Villanueva; Ana Beatriz Fernandez; Primitivo Caballero. 2019. "A Strain of Bacillus thuringiensis Containing a Novel cry7Aa2 Gene that Is Toxic to Leptinotarsa decemlineata (Say) (Coleoptera: Chrysomelidae)." Insects 10, no. 9: 259.
Quantitative laboratory bioassay methods are required to evaluate the toxicity of novel insecticidal compounds for pest control and to determine the presence of resistance traits. We used a radioactive tracer based on 32P-ATP to estimate the volume of a droplet ingested by two dipteran pests: Ceratitis capitata (Tephritidae) and Drosophila suzukii (Drosophilidae). Using blue food dye it was possible to distinguish between individuals that ingested the solution from those that did not. The average volume ingested by C. capitata adults was 1.968 μl. Females ingested a ~20% greater volume of solution than males. Adults of D. suzukii ingested an average of 0.879 μl and females ingested ~30% greater volume than males. The droplet feeding method was validated using the naturally-derived insecticide spinosad as the active ingredient (a.i.). For C. capitata, the concentration-mortality response did not differ between the sexes or among three different batches of insects. Lethal dose values were calculated based on mean ingested volumes. For C. capitata LD50 values were 1.462 and 1.502 ng a.i./insect for males and females, respectively, equivalent to 0.274 and 0.271 ng a.i./mg for males and females, respectively, when sex-specific variation in body weight was considered. Using the same process for D. suzukii, the LD50 value was estimated at 2.927 ng a.i./insect, or 1.994 ng a.i./mg based on a mean body weight of 1.67 mg for both sexes together. We conclude that this technique could be readily employed for determination of the resistance status and dose-mortality responses of insecticidal compounds in many species of pestiferous Diptera.
Daniel Valtierra-De-Luis; Maite Villanueva; Javier Caballero; Isabel M. Matas; Trevor Williams; Primitivo Caballero. Quantification of dose-mortality responses in adult Diptera: Validation using Ceratitis capitata and Drosophila suzukii responses to spinosad. PLOS ONE 2019, 14, e0210545 .
AMA StyleDaniel Valtierra-De-Luis, Maite Villanueva, Javier Caballero, Isabel M. Matas, Trevor Williams, Primitivo Caballero. Quantification of dose-mortality responses in adult Diptera: Validation using Ceratitis capitata and Drosophila suzukii responses to spinosad. PLOS ONE. 2019; 14 (2):e0210545.
Chicago/Turabian StyleDaniel Valtierra-De-Luis; Maite Villanueva; Javier Caballero; Isabel M. Matas; Trevor Williams; Primitivo Caballero. 2019. "Quantification of dose-mortality responses in adult Diptera: Validation using Ceratitis capitata and Drosophila suzukii responses to spinosad." PLOS ONE 14, no. 2: e0210545.
Bacteria use two-component systems (TCSs) to sense and respond to environmental changes. The core genome of the major human pathogen Staphylococcus aureus encodes 16 TCSs, one of which (WalRK) is essential. Here we show that S. aureus can be deprived of its complete sensorial TCS network and still survive under growth arrest conditions similarly to wild-type bacteria. Under replicating conditions, however, the WalRK system is necessary and sufficient to maintain bacterial growth, indicating that sensing through TCSs is mostly dispensable for living under constant environmental conditions. Characterization of S. aureus derivatives containing individual TCSs reveals that each TCS appears to be autonomous and self-sufficient to sense and respond to specific environmental cues, although some level of cross-regulation between non-cognate sensor-response regulator pairs occurs in vivo. This organization, if confirmed in other bacterial species, may provide a general evolutionarily mechanism for flexible bacterial adaptation to life in new niches. Bacteria use two-component systems (TCSs) to sense and respond to environmental changes. Here, the authors show that Staphylococcus aureus can survive in the absence of all its 16 TCSs under growth arrest conditions, and each TCS seems to be sufficient to sense and respond to specific environmental clues.
Maite Villanueva; Begoña García; Jaione Valle; Beatriz Rapún; Igor Ruiz De Los Mozos; Cristina Solano; Miguel Martí; José R. Penadés; Alejandro Toledo-Arana; Iñigo Lasa. Sensory deprivation in Staphylococcus aureus. Nature Communications 2018, 9, 1 -12.
AMA StyleMaite Villanueva, Begoña García, Jaione Valle, Beatriz Rapún, Igor Ruiz De Los Mozos, Cristina Solano, Miguel Martí, José R. Penadés, Alejandro Toledo-Arana, Iñigo Lasa. Sensory deprivation in Staphylococcus aureus. Nature Communications. 2018; 9 (1):1-12.
Chicago/Turabian StyleMaite Villanueva; Begoña García; Jaione Valle; Beatriz Rapún; Igor Ruiz De Los Mozos; Cristina Solano; Miguel Martí; José R. Penadés; Alejandro Toledo-Arana; Iñigo Lasa. 2018. "Sensory deprivation in Staphylococcus aureus." Nature Communications 9, no. 1: 1-12.
RNA-binding proteins (RBPs) are essential to fine-tune gene expression. RBPs containing the cold-shock domain are RNA chaperones that have been extensively studied. However, the RNA targets and specific functions for many of them remain elusive. Here, combining comparative proteomics and RBP-immunoprecipitation-microarray profiling, we have determined the regulon of the RNA chaperone CspA of Staphylococcus aureus. Functional analysis revealed that proteins involved in carbohydrate and ribonucleotide metabolism, stress response and virulence gene expression were affected by cspA deletion. Stress-associated phenotypes such as increased bacterial aggregation and diminished resistance to oxidative-stress stood out. Integration of the proteome and targetome showed that CspA post-transcriptionally modulates both positively and negatively the expression of its targets, denoting additional functions to the previously proposed translation enhancement. One of these repressed targets was its own mRNA, indicating the presence of a negative post-transcriptional feedback loop. CspA bound the 5′UTR of its own mRNA disrupting a hairpin, which was previously described as an RNase III target. Thus, deletion of the cspA 5′UTR abrogated mRNA processing and auto-regulation. We propose that CspA interacts through a U-rich motif, which is located at the RNase III cleavage site, portraying CspA as a putative RNase III-antagonist.
Carlos J Caballero; Pilar Menendez-Gil; Arancha Catalan-Moreno; Marta Vergara-Irigaray; Begoña Garcia; Víctor Segura; Naiara Irurzun; Maite Villanueva; Igor Ruiz De Los Mozos; Cristina Solano; Iñigo Lasa; Alejandro Toledo-Arana. The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus. Nucleic Acids Research 2018, 46, 1345 -1361.
AMA StyleCarlos J Caballero, Pilar Menendez-Gil, Arancha Catalan-Moreno, Marta Vergara-Irigaray, Begoña Garcia, Víctor Segura, Naiara Irurzun, Maite Villanueva, Igor Ruiz De Los Mozos, Cristina Solano, Iñigo Lasa, Alejandro Toledo-Arana. The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus. Nucleic Acids Research. 2018; 46 (3):1345-1361.
Chicago/Turabian StyleCarlos J Caballero; Pilar Menendez-Gil; Arancha Catalan-Moreno; Marta Vergara-Irigaray; Begoña Garcia; Víctor Segura; Naiara Irurzun; Maite Villanueva; Igor Ruiz De Los Mozos; Cristina Solano; Iñigo Lasa; Alejandro Toledo-Arana. 2018. "The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus." Nucleic Acids Research 46, no. 3: 1345-1361.
Staphylococcus aureus is capable of causing a remarkable spectrum of disease, ranging from mild skin eruptions to life-threatening infections. The survival and pathogenic potential of S. aureus depend partly on its ability to sense and respond to changes in its environment. Spx is a thiol/oxidative stress sensor that interacts with the C-terminal domain of the RNA polymerase RpoA subunit, leading to changes in gene expression that help sustain viability under various conditions. Using genetic and deep-sequencing methods, we show that spx is essential in S. aureus and that a previously reported Δ spx strain harbored suppressor mutations that allowed it to grow without spx . One of these mutations is a single missense mutation in rpoB (a P-to-L change at position 519 encoded by rpoB [ rpoB -P519L]) that conferred high-level resistance to rifampin. This mutation alone was found to be sufficient to bypass the requirement for spx . The generation of rifampin resistance libraries led to the discovery of an additional rpoB mutation, R484H, which supported strains with the spx disruption. Other rifampin resistance mutations either failed to support the Δ spx mutant or were recovered at unexpectedly low frequencies in genetic transduction experiments. The amino acid residues encoded by rpoB -P519L and -R484H map in close spatial proximity and comprise a highly conserved region of RpoB. We also discovered that multicopy expression of either trxA (encoding thioredoxin) or trxB (encoding thioredoxin reductase) supports strains with the deletion of spx . Our results reveal intriguing properties, especially of RNA polymerase, that compensate for the loss of an essential gene that is a key mediator of diverse processes in S. aureus , including redox and thiol homeostasis, antibiotic resistance, growth, and metabolism. IMPORTANCE The survival and pathogenicity of S. aureus depend on complex genetic programs. An objective for combating this insidious organism entails dissecting genetic regulatory circuits and discovering promising new targets for therapeutic intervention. In this study, we discovered that Spx, an RNA polymerase-interacting stress regulator implicated in many stress responses in S. aureus , including responses to oxidative and cell wall antibiotics, is essential. We describe two mechanisms that suppress the lethality of spx disruption. One mechanism highlights how only certain rifampin resistance-encoding alleles of RpoB confer new properties on RNA polymerase, with important mechanistic implications. We describe additional stress conditions where the loss of spx is deleterious, thereby highlighting Spx as a multifaceted regulator and attractive drug discovery target.
Maite Villanueva; Ambre Jousselin; Kristoffer T. Baek; Julien Prados; Diego O. Andrey; Adriana Renzoni; Hanne Ingmer; Dorte Frees; William L. Kelley. Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus. Journal of Bacteriology 2016, 198, 2719 -2731.
AMA StyleMaite Villanueva, Ambre Jousselin, Kristoffer T. Baek, Julien Prados, Diego O. Andrey, Adriana Renzoni, Hanne Ingmer, Dorte Frees, William L. Kelley. Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus. Journal of Bacteriology. 2016; 198 (19):2719-2731.
Chicago/Turabian StyleMaite Villanueva; Ambre Jousselin; Kristoffer T. Baek; Julien Prados; Diego O. Andrey; Adriana Renzoni; Hanne Ingmer; Dorte Frees; William L. Kelley. 2016. "Rifampin Resistance rpoB Alleles or Multicopy Thioredoxin/Thioredoxin Reductase Suppresses the Lethality of Disruption of the Global Stress Regulator spx in Staphylococcus aureus." Journal of Bacteriology 198, no. 19: 2719-2731.
Staphylococcus aureus is an important pathogen manifesting virulence through diverse disease forms, ranging from acute skin infections to life-threatening bacteremia or systemic toxic shock syndromes. In the latter case, the prototypical superantigen is TSST-1 (Toxic Shock Syndrome Toxin 1), encoded by tst(H), and carried on a mobile genetic element that is not present in all S. aureus strains. Transcriptional regulation of tst is only partially understood. In this study, we dissected the role of sarA, sarS (sarH1), RNAIII, rot, and the alternative stress sigma factor sigB (σB). By examining tst promoter regulation predominantly in the context of its native sequence within the SaPI1 pathogenicity island of strain RN4282, we discovered that σB emerged as a particularly important tst regulator. We did not detect a consensus σB site within the tst promoter, and thus the effect of σB is likely indirect. We found that σB strongly repressed the expression of the toxin via at least two distinct regulatory pathways dependent upon sarA and agr. Furthermore rot, a member of SarA family, was shown to repress tst expression when overexpressed, although its deletion had no consistent measurable effect. We could not find any detectable effect of sarS, either by deletion or overexpression, suggesting that this regulator plays a minimal role in TSST-1 expression except when combined with disruption of sarA. Collectively, our results extend our understanding of complex multifactorial regulation of tst, revealing several layers of negative regulation. In addition to environmental stimuli thought to impact TSST-1 production, these findings support a model whereby sporadic mutation in a few key negative regulators can profoundly affect and enhance TSST-1 expression.
Diego Andrey; Ambre Jousselin; Maite Villanueva; Adriana Renzoni; Antoinette Monod; Christine Barras; Natalia Rodriguez; William L. Kelley. Impact of the Regulators SigB, Rot, SarA and sarS on the Toxic Shock Tst Promoter and TSST-1 Expression in Staphylococcus aureus. PLOS ONE 2015, 10, e0135579 .
AMA StyleDiego Andrey, Ambre Jousselin, Maite Villanueva, Adriana Renzoni, Antoinette Monod, Christine Barras, Natalia Rodriguez, William L. Kelley. Impact of the Regulators SigB, Rot, SarA and sarS on the Toxic Shock Tst Promoter and TSST-1 Expression in Staphylococcus aureus. PLOS ONE. 2015; 10 (8):e0135579.
Chicago/Turabian StyleDiego Andrey; Ambre Jousselin; Maite Villanueva; Adriana Renzoni; Antoinette Monod; Christine Barras; Natalia Rodriguez; William L. Kelley. 2015. "Impact of the Regulators SigB, Rot, SarA and sarS on the Toxic Shock Tst Promoter and TSST-1 Expression in Staphylococcus aureus." PLOS ONE 10, no. 8: e0135579.
The precise understanding of the biology of a living cell requires the identification and quantification of the molecular components necessary to sustain life. One such element is RNA. Two independent high-throughput strategies are available to identify the entire collection of RNA molecules produced by a cell population, which is currently known as the transcriptome. One technique relies on microarray technology (tiling arrays), whereas the second one relies on sequencing the RNA pool (RNA-seq) (1). Both techniques offer the advantage that the identification of the RNA content is not biased by protein-based genome annotation. The application of these methods to the transcriptome analysis in bacteria has uncovered the existence of a large amount of RNA molecules that overlap at least in some portion with protein-encoding RNA transcripts, generating perfect sense/antisense RNA duplexes (2⇓⇓–5).Peer reviewe
I. Lasa; Maite Villanueva. Overlapping transcription and bacterial RNA removal. Proceedings of the National Academy of Sciences 2014, 111, 2868 -2869.
AMA StyleI. Lasa, Maite Villanueva. Overlapping transcription and bacterial RNA removal. Proceedings of the National Academy of Sciences. 2014; 111 (8):2868-2869.
Chicago/Turabian StyleI. Lasa; Maite Villanueva. 2014. "Overlapping transcription and bacterial RNA removal." Proceedings of the National Academy of Sciences 111, no. 8: 2868-2869.
The presence of regulatory sequences in the 3′ untranslated region (3′-UTR) of eukaryotic mRNAs controlling RNA stability and translation efficiency is widely recognized. In contrast, the relevance of 3′-UTRs in bacterial mRNA functionality has been disregarded. Here, we report evidences showing that around one-third of the mapped mRNAs of the major human pathogen Staphylococcus aureus carry 3′-UTRs longer than 100-nt and thus, potential regulatory functions. We selected the long 3′-UTR of icaR, which codes for the repressor of the main exopolysaccharidic compound of the S. aureus biofilm matrix, to evaluate the role that 3′-UTRs may play in controlling mRNA expression. We showed that base pairing between the 3′-UTR and the Shine-Dalgarno (SD) region of icaR mRNA interferes with the translation initiation complex and generates a double-stranded substrate for RNase III. Deletion or substitution of the motif (UCCCCUG) within icaR 3′-UTR was sufficient to abolish this interaction and resulted in the accumulation of IcaR repressor and inhibition of biofilm development. Our findings provide a singular example of a new potential post-transcriptional regulatory mechanism to modulate bacterial gene expression through the interaction of a 3′-UTR with the 5′-UTR of the same mRNA. At both sides of the protein-coding region, the mRNA molecule contains sequences that are not translated to protein. In eukaryotes, the untranslated 3′ region (3′-UTR), which comprises from the last codon used in translation to the 3′ end of the mRNA, controls mRNA stability, location and translation efficiency. In contrast, knowledge about the functions of 3′-UTRs in bacterial physiology is scarce. Here, we demonstrate that bacterial 3′-UTRs might play regulatory functions that might resemble those already described in eukaryotes. Transcriptome analysis of the human pathogen Staphylococcus aureus revealed that at least 30% of mRNAs contain long 3′-UTRs. Using the 3′-UTR of the mRNA encoding the main biofilm repressor IcaR as a model, we show that the 3′-UTR interferes with the translation initiation complex and promotes mRNA decay through base pairing with the ribosome binding site. This event contributes to adjusting IcaR level and modulating exopolysaccharide production and biofilm development in S. aureus. Our data illustrate that bacterial 3′-UTRs can provide strategies for fine-tuning control of gene expression.
Igor Ruiz De Los Mozos; Marta Vergara-Irigaray; Victor Segura; Maite Villanueva; Nerea Bitarte; Margarida Saramago; Susana Domingues; Cecilia Arraiano; Pierre Fechter; Pascale Romby; Jaione Valle; Cristina Solano; Iñigo Lasa; Alejandro Toledo-Arana. Base Pairing Interaction between 5′- and 3′-UTRs Controls icaR mRNA Translation in Staphylococcus aureus. PLoS Genetics 2013, 9, e1004001 .
AMA StyleIgor Ruiz De Los Mozos, Marta Vergara-Irigaray, Victor Segura, Maite Villanueva, Nerea Bitarte, Margarida Saramago, Susana Domingues, Cecilia Arraiano, Pierre Fechter, Pascale Romby, Jaione Valle, Cristina Solano, Iñigo Lasa, Alejandro Toledo-Arana. Base Pairing Interaction between 5′- and 3′-UTRs Controls icaR mRNA Translation in Staphylococcus aureus. PLoS Genetics. 2013; 9 (12):e1004001.
Chicago/Turabian StyleIgor Ruiz De Los Mozos; Marta Vergara-Irigaray; Victor Segura; Maite Villanueva; Nerea Bitarte; Margarida Saramago; Susana Domingues; Cecilia Arraiano; Pierre Fechter; Pascale Romby; Jaione Valle; Cristina Solano; Iñigo Lasa; Alejandro Toledo-Arana. 2013. "Base Pairing Interaction between 5′- and 3′-UTRs Controls icaR mRNA Translation in Staphylococcus aureus." PLoS Genetics 9, no. 12: e1004001.
Demyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage. To monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage. The model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases.
Alessandra Di Penta; Beatriz Moreno; Stephanie Reix; Begoña Fernandez-Diez; Maite Villanueva; Oihana Errea; Nagore Escala; Koen Vandenbroeck; Joan Comella; Pablo Villoslada. Oxidative Stress and Proinflammatory Cytokines Contribute to Demyelination and Axonal Damage in a Cerebellar Culture Model of Neuroinflammation. PLoS ONE 2013, 8, e54722 .
AMA StyleAlessandra Di Penta, Beatriz Moreno, Stephanie Reix, Begoña Fernandez-Diez, Maite Villanueva, Oihana Errea, Nagore Escala, Koen Vandenbroeck, Joan Comella, Pablo Villoslada. Oxidative Stress and Proinflammatory Cytokines Contribute to Demyelination and Axonal Damage in a Cerebellar Culture Model of Neuroinflammation. PLoS ONE. 2013; 8 (2):e54722.
Chicago/Turabian StyleAlessandra Di Penta; Beatriz Moreno; Stephanie Reix; Begoña Fernandez-Diez; Maite Villanueva; Oihana Errea; Nagore Escala; Koen Vandenbroeck; Joan Comella; Pablo Villoslada. 2013. "Oxidative Stress and Proinflammatory Cytokines Contribute to Demyelination and Axonal Damage in a Cerebellar Culture Model of Neuroinflammation." PLoS ONE 8, no. 2: e54722.
RNA deep sequencing technologies are revealing unexpected levels of complexity in bacterial transcriptomes with the discovery of abundant noncoding RNAs, antisense RNAs, long 5′ and 3′ untranslated regions, and alternative operon structures. Here, by applying deep RNA sequencing to both the long and short RNA fractions (<50 nucleotides) obtained from the major human pathogen Staphylococcus aureus, we have detected a collection of short RNAs that is generated genome-wide through the digestion of overlapping sense/antisense transcripts by RNase III endoribonuclease. At least 75% of sense RNAs from annotated genes are subject to this mechanism of antisense processing. Removal of RNase III activity reduces the amount of short RNAs and is accompanied by the accumulation of discrete antisense transcripts. These results suggest the production of pervasive but hidden antisense transcription used to process sense transcripts by means of creating double-stranded substrates. This process of RNase III-mediated digestion of overlapping transcripts can be observed in several evolutionarily diverse Gram-positive bacteria and is capable of providing a unique genome-wide posttranscriptional mechanism to adjust mRNA levels.
Iñigo Lasa; Alejandro Toledo-Arana; Alexander Dobin; Maite Villanueva; Igor Ruiz De Los Mozos; Marta Vergara-Irigaray; Víctor Segura; Delphine Fagegaltier; José R Penadés; Jaione Valle; Cristina Solano; Thomas R. Gingeras. Genome-wide antisense transcription drives mRNA processing in bacteria. Proceedings of the National Academy of Sciences 2011, 108, 20172 -20177.
AMA StyleIñigo Lasa, Alejandro Toledo-Arana, Alexander Dobin, Maite Villanueva, Igor Ruiz De Los Mozos, Marta Vergara-Irigaray, Víctor Segura, Delphine Fagegaltier, José R Penadés, Jaione Valle, Cristina Solano, Thomas R. Gingeras. Genome-wide antisense transcription drives mRNA processing in bacteria. Proceedings of the National Academy of Sciences. 2011; 108 (50):20172-20177.
Chicago/Turabian StyleIñigo Lasa; Alejandro Toledo-Arana; Alexander Dobin; Maite Villanueva; Igor Ruiz De Los Mozos; Marta Vergara-Irigaray; Víctor Segura; Delphine Fagegaltier; José R Penadés; Jaione Valle; Cristina Solano; Thomas R. Gingeras. 2011. "Genome-wide antisense transcription drives mRNA processing in bacteria." Proceedings of the National Academy of Sciences 108, no. 50: 20172-20177.