Open Access
Effect of transcriptional activators SoxS, RobA, and RamA on expression of multidrug efflux pump AcrAB-TolC in enterobacter cloacae
(American Society for Microbiology, 2012) Pérez, Astrid; Poza, Margarita; Aranda, Jesús; Latasa Osta, Cristina; Medrano, Francisco Javier; Tomás, María del Mar; Romero, Antonio; Lasa Uzcudun, Íñigo; Bou, Germán; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Control of membrane permeability is a key step in regulating the intracellular concentration of antibiotics. Efflux pumps confer innate resistance to a wide range of toxic compounds such as antibiotics, dyes, detergents, and disinfectants in members of the Enterobacteriaceae. The AcrAB-TolC efflux pump is involved in multidrug resistance in Enterobacter cloacae. However, the underlying mechanism that regulates the system in this microorganism remains unknown. In Escherichia coli, the transcription of acrAB is upregulated under global stress conditions by proteins such as MarA, SoxS, and Rob. In the present study, two clinical isolates of E. cloacae, EcDC64 (a multidrug-resistant strain overexpressing the AcrAB-TolC efflux pump) and Jc194 (a strain with a basal AcrAB-TolC expression level), were used to determine whether similar global stress responses operate in E. cloacae and also to establish the molecular mechanisms underlying this response. A decrease in susceptibility to erythromycin, tetracycline, telithromycin, ciprofloxacin, and chloramphenicol was observed in clinical isolate Jc194 and, to a lesser extent in EcDC64, in the presence of salicylate, decanoate, tetracycline, and paraquat. Increased expression of the acrAB promoter in the presence of the above-described conditions was observed by flow cytometry and reverse transcription-PCR, by using a reporter fusion protein (green fluorescent protein). The expression level of the AcrAB promoter decreased in E. cloacae EcDC64 derivates deficient in SoxS, RobA, and RamA. Accordingly, the expression level of the AcrAB promoter was higher in E. cloacae Jc194 strains overproducing SoxS, RobA, and RamA. Overall, the data showed that SoxS, RobA, and RamA regulators were associated with the upregulation of acrAB, thus conferring antimicrobial resistance as well as a stress response in E. cloacae. In summary, the regulatory proteins SoxS, RobA, and RamA were cloned and sequenced for the first time in this species. The involvement of these proteins in conferring antimicrobial resistance through upregulation of acrAB was demonstrated in E. cloacae.
Open Access
Salmonella biofilm development depends on the phosphorylation status of RcsB
(American Society for Microbiology, 2012) Latasa Osta, Cristina; García Martínez, Begoña; Echeverz Sarasúa, Maite; Toledo Arana, Alejandro; Valle Turrillas, Jaione; Campoy Sánchez, Susana; García del Portillo, Francisco; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIM13329.RI1
The Rcs phosphorelay pathway is a complex signaling pathway involved in the regulation of many cell surface structures in enteric bacteria. In response to environmental stimuli, the sensor histidine kinase (RcsC) autophosphorylates and then transfers the phosphate through intermediary steps to the response regulator (RcsB), which, once phosphorylated, regulates gene expression. Here, we show that Salmonella biofilm development depends on the phosphorylation status of RcsB. Thus, unphosphorylated RcsB, hitherto assumed to be inactive, is essential to activate the expression of the biofilm matrix compounds. The prevention of RcsB phosphorylation either by the disruption of the phosphorelay at the RcsC or RcsD level or by the production of a nonphosphorylatable RcsB allele induces biofilm development. On the contrary, the phosphorylation of RcsB by the constitutive activation of the Rcs pathway inhibits biofilm development, an effect that can be counteracted by the introduction of a nonphosphorylatable RcsB allele. The inhibition of biofilm development by phosphorylated RcsB is due to the repression of CsgD expression, through a mechanism dependent on the accumulation of the small noncoding RNA RprA. Our results indicate that unphosphorylated RcsB plays an active role for integrating environmental signals and, more broadly, that RcsB phosphorylation acts as a key switch between planktonic and sessile life-styles in Salmonella enterica serovar Typhimurium.
Open Access
Evaluation of surface microtopography engineered by direct laser interference for bacterial anti-biofouling
(2015) Valle Turrillas, Jaione; Burgui Erice, Saioa; Langheinrich, Denise; Gil Puig, Carmen; Solano Goñi, Cristina; Toledo Arana, Alejandro; Helbig, Ralf; Lasagni, Andrés; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIQ14066.RI1
Biofilm formation by bacterial pathogens on the surface of medical and industrial settings is a 25 serious health problem. Modification of the biomaterial surface topography is a promising 26 strategy to prevent bacterial attachment and biofilm development. However, fabrication of 27 functional biomaterials at large scale with periodic network-topology is still problematic. In this 28 study, we use direct laser interference (DLIP), an easily scalable process, to modify polystyrene 29 surface (PS) topography at sub-micrometer scale. The resulting structure surfaces were 30 interrogated for their capacity to prevent adhesion and biofilm formation of the major human 31 pathogen Staphylococcus aureus. The results revealed that three-dimensional micrometer 32 periodic structures on PS have a profound impact on bacterial adhesion capacity. Thus, line- 33 and pillar-like topographical patterns enhanced S. aureus adhesion, whereas complex lamella 34 microtopography reduced S. aureus adhesion both in static and continuous flow culture 35 conditions. Interestingly, lamella-like textured surfaces retained the capacity to inhibit S. aureus 36 adhesion both when the surface is coated with human serum proteins in vitro and when the 37 material is implanted subcutaneously in a foreign-body associated infection model. Our results 38 establish that the DLIP technology can be used to functionalize polymeric surfaces for the 39 inhibition of bacterial adhesion to surfaces.
Open Access
An effort to make sense of antisense transcription in bacteria
(Taylor & Francis, 2012) Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; Gingeras, Thomas R.; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Analysis of bacterial transcriptomes have shown the existence of a genome-wide process of overlapping transcription due to the presence of antisense RNAs, as well as mRNAs that overlapped in their entire length or in some portion of the 5′- and 3′-UTR regions. The biological advantages of such overlapping transcription are unclear but may play important regulatory roles at the level of transcription, RNA stability and translation. In a recent report, the human pathogen Staphylococcus aureus is observed to generate genome-wide overlapping transcription in the same bacterial cells leading to a collection of short RNA fragments generated by the endoribonuclease III, RNase III. This processing appears most prominently in Gram-positive bacteria. The implications of both the use of pervasive overlapping transcription and the processing of these double stranded templates into short RNAs are explored and the consequences discussed.
Open Access
Amyloid structures as biofilm matrix scaffolds
(American Society for Microbiology, 2016) Taglialegna, Agustina; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Recent insights into bacterial biofilm matrix structures have induced a paradigm shift toward the recognition of amyloid fibers as common building block structures that confer stability to the exopolysaccharide matrix. Here we describe the functional amyloid systems related to biofilm matrix formation in both Gram-negative and Gram-positive bacteria and recent knowledge regarding the interaction of amyloids with other biofilm matrix components such as extracellular DNA (eDNA) and the host immune system. In addition, we summarize the efforts to identify compounds that target amyloid fibers for therapeutic purposes and recent developments that take advantage of the amyloid structure to engineer amyloid fibers of bacterial biofilm matrices for biotechnological applications.
Open Access
Polymicrobial infections: do bacteria behave differently depending on their neighbours?
(Taylor & Francis, 2018) Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; Ciencias de la Salud; Osasun Zientziak
Despite the number of examples that correlate interspecies interactions in polymicrobial infections with variations in pathogenicity and antibiotic susceptibility of individual organisms, antibiotic therapies are selected to target the most relevant pathogen, with no consideration of the consequences that the presence of other bacterial species may have in the pathogenicity and response to antimicrobial agents. In this issue of Virulence, Garcia-Perez et al. [ 10 ] applied replica plating of used wound dressings to assess the topography of distinct S. aureus types in chronic wounds of patients with the genetic blistering disease epidermolysis bullosa, which is characterized by the development of chronic wounds upon simple mechanical trauma. This approach led to the identification of two strains of S. aureus coexisting with Bacillus thuringiensis and Klebsiella oxytoca. S. aureus is highly prevalent in chronic wound infections, whereas B. thuringiensis and K. oxytoca are regarded as opportunistic pathogens. These bacterial species did not inhibit each other's growth under laboratory conditions, suggesting that they do not compete through the production of inhibitory compounds. Using a top-down proteomic approach to explore the inherent relationships between these co-existing bacteria, the exoproteomes of the staphylococcal isolates in monoculture and co-culture with B. thuringiensis or K. oxytoca were characterized by Mass Spectrometry.
Open Access
A pyrene-inhibitor fluorescent probe with large stokes shift for the staining of Aβ1–42, α-synuclein, and amylin amyloid fibrils as well as amyloid-containing staphylococcus aureus biofilms
(Springer, 2019) Mahía, Alejandro; Conde-Giménez, María; Salillas, Sandra; Pallarés, Irantzu; Galano-Frutos, Juan J.; Lasa Uzcudun, Íñigo; Ventura, Salvador; Díaz-de-Villegas, María D.; Gálvez, José A.; Sancho, Javier; Ciencias de la Salud; Osasun Zientziak
Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer, Parkinson or Type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity towards that diversity of amyloid fibrils, or providing alternative spectral windows are needed to improve the detection or the identification of amyloid structures. One potential source for such new probes is offered by molecules known to interact with fibrils, such as the inhibitors of amyloid aggregation found in drug discovery projects. Here, we show the feasibility of the approach by designing, synthesizing and testing several pyrene-based fluorescent derivatives of a previously discovered inhibitor of the aggregation of the Aβ1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The more soluble derivative, compound 1D, stains similarly well amyloid fibrils formed by Aβ1-42, α-synuclein or amylin, provides a sensitivity only slightly lower than that of Thioflavin T, displays a large Stokes shift, allows an efficient excitation in the UV spectral region,and it is not cytotoxic. Compound 1D can also stain amyloid fibrils formed by Staphylococcal peptides present in biofilm matrices and can be used to distinguish, by direct staining,S. aureus biofilms containing amyloid forming phenol soluble modulins from those lacking them.
Open Access
The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus
(Oxford University Press, 2018) Caballero Sánchez, Carlos; Menéndez Gil, Pilar; Catalán Moreno, Arancha; Vergara Irigaray, Marta; García Martínez, Begoña; Segura, Víctor; Irurzun Domínguez, Naiara; Villanueva San Martín, Maite; Ruiz de los Mozos Aliaga, Igor; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
RNA-binding proteins (RBPs) are essential to finetune gene expression. RBPs containing the coldshock 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 RBPimmunoprecipitation- 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 posttranscriptionally 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.
Open Access
RsaI, un ARN régulateur aux multiples facettes, module le métabolisme du pathogène opportuniste Staphylococcus aureus
(EDP Sciences, 2019) Desgranges, Emma; Bronesky, Delphine; Corvaglia, Anna; François, Patrice; Caballero Sánchez, Carlos; Prado, Laura; Toledo Arana, Alejandro; Lasa Uzcudun, Íñigo; Moreau, Karen; Vandenesch, François; Marzi, Stefano; Romby, Pascale; Caldelari, Isabelle; Ciencias de la Salud; Osasun Zientziak
Staphylococcus aureus est une bactérie commensale retrouvée chez environ 30 % des individus sains dont elle colonise la peau et la muqueuse nasale. Cependant, c’est également une bactérie pathogène opportuniste responsable d’infections diverses telles que orgelet, ostéomyélite, endocardite, ou encore septicémie en envahissant un grand nombre de tissus et d’organes. Cette bactérie est capable de s’adapter à des conditions hostiles et variées, telles que carence nutritive et stress osmotique, oxydant, ou thermique, ainsi qu’à la réponse immunitaire de l’hôte, car elle produit une grande diversité de facteurs de virulence. La synthèse de ces facteurs est finement régulée par des protéines et des ARN régulateurs majoritairement non codants, souvent désignés par l’abréviation sARN (dérivée de l’anglais, small RNA). Les facteurs de transcription et les systèmes à deux composants contrôlent l’expression des gènes impliqués non seulement dans le métabolisme, mais aussi dans la réponse au stress et la virulence [1]. Par exemple, la protéine du contrôle catabolique (carbon catabolite control protein A, CcpA) a un rôle essentiel dans le choix de la source carbonée en régulant le métabolisme central de la bactérie ainsi que la virulence [2, 3]. CcpA se fixe à une séquence promotrice spécifique appelée cre (catabolite-responsive element), qui est très conservée chez les bactéries à Gram positif [2]. Quant aux sARN, ils interagissent principalement avec leurs ARN messagers (ARNm) cibles. L’hybridation peut conduire à la stabilisation/ déstabilisation de l’ARNm ou à l’activation/répression de sa traduction [4]. Nous avons montré que la transcription du sARN RsaI (RNA Staphylococcus aureus I) est réprimée par CcpA en présence de glucose [5]. L’induction de la synthèse de RsaI signale que la concentration en glucose diminue dans le milieu extracellulaire et que la croissance des bactéries est ralentie. En interagissant avec ses ARNm cibles ou d’autres sARN, il permet à la population bactérienne de modifier son métabolisme lorsque la source carbonée primaire est consommée.
Open Access
Base pairing interaction between 5′- and 3′-UTRs controls icaR mRNA translation in Staphylococcus aureus
(Public Library of Science, 2013) Ruiz de los Mozos Aliaga, Igor; Vergara Irigaray, Marta; Segura, Víctor; Villanueva San Martín, Maite; Bitarte Manzanal, Nerea; Saramago, Margarida; Domingues, Susana; Arraiano, Cecilia M.; Fechter, Pierre; Romby, Pascale; Valle Turrillas, Jaione; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The presence of regulatory sequences in the 39 untranslated region (39-UTR) of eukaryotic mRNAs controlling RNA stability and translation efficiency is widely recognized. In contrast, the relevance of 39-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 39-UTRs longer than 100-nt and thus, potential regulatory functions. We selected the long 39-UTR of icaR, which codes for the repressor of the main exopolysaccharidic compound of the S. aureus biofilm matrix, to evaluate the role that 39-UTRs may play in controlling mRNA expression. We showed that base pairing between the 39- 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 39-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 39-UTR with the 59-UTR of the same mRNA.