Open Access
Noncontiguous operon atlas for the Staphylococcus aureus genome
(Oxford University Press, 2024) Iturbe Sanz, Pablo; San Martín Bernal, Álvaro; Hamamoto, Hiroshi; Marcet Houben, Marina; Galbaldón, Toni; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Ciencias de la Salud; Osasun Zientziak; Universidad Pública de Navarra / Nafarroako Unibertsitate Publikoa
Bacteria synchronize the expression of genes with related functions by organizing genes into operons so that they are cotranscribed together in a single polycistronic messenger RNA. However, some cellular processes may benefit if the simultaneous production of the operon proteins coincides with the inhibition of the expression of an antagonist gene. To coordinate such situations, bacteria have evolved noncontiguous operons (NcOs), a subtype of operons that contain one or more genes that are transcribed in the opposite direction to the other operon genes. This structure results in overlapping transcripts whose expression is mutually repressed. The presence of NcOs cannot be predicted computationally and their identification requires a detailed knowledge of the bacterial transcriptome. In this study, we used direct RNA sequencing methodology to determine the NcOs map in the Staphylococcus aureus genome. We detected the presence of 18 NcOs in the genome of S. aureus and four in the genome of the lysogenic prophage 80α. The identified NcOs comprise genes involved in energy metabolism, metal acquisition and transport, toxin–antitoxin systems, and control of the phage life cycle. Using the menaquinone operon as a proof of concept, we show that disarrangement of the NcO architecture results in a reduction of bacterial fitness due to an increase in menaquinone levels and a decrease in the rate of oxygen consumption. Our study demonstrates the significance of NcO structures in bacterial physiology and emphasizes the importance of combining operon maps with transcriptomic data to uncover previously unnoticed functional relationships between neighbouring genes.
Open Access
Biofilm properties in relation to treatment outcome in patients with first-time periprosthetic hip or knee joint infection
(Elsevier, 2021) Malchau, Karin Svensson; Tillander, Jonatan; Zaborowska, Magdalena; Hoffman, Maria; Lasa Uzcudun, Íñigo; Thomsen, Peter; Malchau, Henrik; Rolfson, Ola; Trobos, Margarita; Ciencias de la Salud; Osasun Zientziak
Background: periprosthetic joint infections (PJI) are challenging complications following arthroplasty. Staphylococci are a frequent cause of PJI and known biofilm producers. Biofilm formation decreases antimicrobial susceptibility, thereby challenging favourable treatment outcomes. The aims of this study were to characterize the biofilm abilities and antimicrobial susceptibilities of staphylococci causing first-time PJI and correlate them to clinical outcome (infection resolution and recurrence). Methods: reoperations for PJI of the hip or knee between 1st January 2012 to 30th June 2015 performed at the Sahlgrenska University Hospital were identified in a local database. Medical records were reviewed and clinical parameters recorded for patients whose intraoperative bacterial isolates had been stored at the clinical laboratory. Staphylococcal strains isolated from reoperations due to first-time PJI were characterised by their ability to form biofilms using the microtiter plate test. Antimicrobial susceptibility of the strains was determined by minimum inhibitory concentration (MIC) when grown planktonically, and by minimum biofilm eradication concentration (MBEC) when grown as biofilms. MBEC determination was conducted using the Calgary biofilm device (CBD) and a custom-made antimicrobial susceptibility plate containing eight clinically relevant antimicrobial agents. Results: the study group included 49 patients (70 bacterial strains) from first-time PJI, whereof 24 (49%) patients had recurrent infection. Strong biofilm production was significantly associated with recurrent infection. Patients infected with strong biofilm producers had a five-fold increased risk for recurrent infection. Strains grown as biofilms were over 8000 times more resistant to antimicrobial agents compared to planktonic cultures. Biofilms were more susceptible to rifampicin compared to other antimicrobials in the assay. Increased biofilm susceptibility (MBEC > MIC) was observed for the majority of the bacterial strains and antimicrobial agents. Conclusions: Strong biofilm production was significantly associated with increased antimicrobial resistance and PJI recurrence. This underscores the importance of determining biofilm production and susceptibility as part of routine diagnostics in PJI. Strong staphylococcal biofilm production may have implications on therapeutic choices and suggest more extensive surgery. Furthermore, despite the increased biofilm resistance to rifampicin, results from this study support its use in staphylococcal PJI. The Translational Potential of this Article: Like for many biomaterial-associated infections, staphylococci are a common cause of PJI. Their ability to adhere to surfaces and produce biofilms on medical devices is proposed to play a role. However, clinical studies where biofilm properties are directly linked to patient outcome are scarce. This study demonstrates that the majority of staphylococci isolated from first-time PJI were biofilm producers with increased antimicrobial resistance. Patients suffering an infection caused by a staphylococcal strain with strong biofilm production ability had a five-fold greater risk of recurrent infection. This novel finding suggests the importance of evaluating biofilm production as a diagnostic procedure for the guidance of treatment decisions in PJI.
Open Access
Bacterial biofilm functionalization through Bap amyloid engineering
(Springer Nature, 2022) Matilla Cuenca, Leticia; Taglialegna, Agustina; Gil Puig, Carmen; Toledo Arana, Alejandro; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; Ciencias de la Salud; Osasun Zientziak
Biofilm engineering has emerged as a controllable way to fabricate living structures with programmable functionalities. The amyloidogenic proteins comprising the biofilms can be engineered to create self-assembling extracellular functionalized surfaces. In this regard, facultative amyloids, which play a dual role in biofilm formation by acting as adhesins in their native conformation and as matrix scaffolds when they polymerize into amyloid-like fibrillar structures, are interesting candidates. Here, we report the use of the facultative amyloid-like Bap protein of Staphylococcus aureus as a tool to decorate the extracellular biofilm matrix or the bacterial cell surface with a battery of functional domains or proteins. We demonstrate that the localization of the functional tags can be change by simply modulating the pH of the medium. Using Bap features, we build a tool for trapping and covalent immobilizing molecules at bacterial cell surface or at the biofilm matrix based on the SpyTag/SpyCatcher system. Finally, we show that the cell wall of several Gram-positive bacteria could be functionalized through the external addition of the recombinant engineered Bap-amyloid domain. Overall, this work shows a simple and modulable system for biofilm functionalization based on the facultative protein Bap. © 2022, The Author(s).
Open Access
Antibiofilm activity of flavonoids on staphylococcal biofilms through targeting BAP amyloids
(Nature Research, 2020) Matilla Cuenca, Leticia; Gil Puig, Carmen; Cuesta Ferre, Sergio; Rapún Araiz, Beatriz; Mira, Alex; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; Ziemité, Miglé; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua, PI011 KILL-BACT
The opportunistic pathogen Staphylococcus aureus is responsible for causing infections related to indwelling medical devices, where this pathogen is able to attach and form biofilms. The intrinsic properties given by the self-produced extracellular biofilm matrix confer high resistance to antibiotics, triggering infections difficult to treat. Therefore, novel antibiofilm strategies targeting matrix components are urgently needed. The biofilm associated protein, Bap, expressed by staphylococcal species adopts functional amyloid-like structures as scaffolds of the biofilm matrix. In this work we have focused on identifying agents targeting Bap-related amyloid-like aggregates as a strategy to combat S. aureus biofilm-related infections. We identified that the flavonoids, quercetin, myricetin and scutellarein specifically inhibited Bap-mediated biofilm formation of S. aureus and other staphylococcal species. By using in vitro aggregation assays and the cell-based methodology for generation of amyloid aggregates based on the Curli-Dependent Amyloid Generator system (C-DAG), we demonstrated that these polyphenols prevented the assembly of Bap-related amyloid-like structures. Finally, using an in vivo catheter infection model, we showed that quercetin and myricetin significantly reduced catheter colonization by S. aureus. These results support the use of polyphenols as anti-amyloids molecules that can be used to treat biofilm-related infections.
Open Access
Structural mechanism for modulation of functional amyloid and biofilm formation by Staphylococcal Bap protein switch
(EMBO Press, 2021) Ma, Junfeng; Cheng, Xiang; Xu, Zhonghe; Zhang, Yikan; Valle Turrillas, Jaione; Fan, Xianyang; Lasa Uzcudun, Íñigo; Ciencias de la Salud; Osasun Zientziak
The Staphylococcal Bap proteins sense environmental signals (such as pH, [Ca2+]) to build amyloid scaffold biofilm matrices via unknown mechanisms. We here report the crystal structure of the aggregation-prone region of Staphylococcus aureus Bap which adopts a dumbbell-shaped fold. The middle module (MM) connecting the N-terminal and C-terminal lobes consists of a tandem of novel double-Ca2+-binding motifs involved in cooperative interaction networks, which undergoes Ca2+-dependent order–disorder conformational switches. The N-terminal lobe is sufficient to mediate amyloid aggregation through liquid–liquid phase separation and maturation, and subsequent biofilm formation under acidic conditions. Such processes are promoted by disordered MM at low [Ca2+] but inhibited by ordered MM stabilized by Ca2+ binding, with inhibition efficiency depending on structural integrity of the interaction networks. These studies illustrate a novel protein switch in pathogenic bacteria and provide insights into the mechanistic understanding of Bap proteins in modulation of functional amyloid and biofilm formation, which could be implemented in the anti-biofilm drug design.
Open Access
Characterization of the common genetic variation in the spanish population of Navarre
(MDPI, 2024) Maíllo Ruiz de Infante, Alberto; Huergo, Estefanía; Apellániz Ruiz, María Valvanera; Urrutia Lafuente, Edurne; Miranda, María; Salgado Garrido, Josefa; Pasalodos Sánchez, Sara; Delgado-Mora, Luna; Teijido Hermida, Óscar; Goicoechea, Ibai; Carmona, Rosario; Pérez-Florido, Javier; Aquino, Virginia; López-López, Daniel; Peña-Chilet, María; Beltrán, Sergi; Dopazo, Joaquín; Lasa Uzcudun, Íñigo; Beloqui, Juan José; NAGEN-Scheme; Alonso Sánchez, Ángel Miguel; Gómez-Cabrero, David; Ciencias de la Salud; Osasun Zientziak
Large-scale genomic studies have significantly increased our knowledge of genetic variability across populations. Regional genetic profiling is essential for distinguishing common benign variants from disease-causing ones. To this end, we conducted a comprehensive characterization of exonic variants in the population of Navarre (Spain), utilizing whole genome sequencing data from 358 unrelated individuals of Spanish origin. Our analysis revealed 61,410 biallelic single nucleotide variants (SNV) within the Navarrese cohort, with 35% classified as common (MAF > 1%). By comparing allele frequency data from 1000 Genome Project (excluding the Iberian cohort of Spain, IBS), Genome Aggregation Database, and a Spanish cohort (including IBS individuals and data from Medical Genome Project), we identified 1069 SNVs common in Navarre but rare (MAF ≤ 1%) in all other populations. We further corroborated this observation with a second regional cohort of 239 unrelated exomes, which confirmed 676 of the 1069 SNVs as common in Navarre. In conclusion, this study highlights the importance of population-specific characterization of genetic variation to improve allele frequency filtering in sequencing data analysis to identify disease-causing variants.
Open Access
The biofilm-associated surface protein Esp of Enterococcus faecalis forms amyloid-like fibers
(Nature Research, 2020) Taglialegna, Agustina; Matilla Cuenca, Leticia; Dorado Morales, Pedro; Navarro, Susanna; Ventura, Salvador; Garnett, James A.; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; Ciencias de la Salud; Osasun Zientziak
Functional amyloids are considered as common building block structures of the biofilm matrix in different bacteria. In previous work, we have shown that the staphylococcal surface protein Bap, a member of the Biofilm-Associated Proteins (BAP) family, is processed and the fragments containing the N-terminal region become aggregation-prone and self-assemble into amyloid-like structures. Here, we report that Esp, a Bap-orthologous protein produced by Enterococcus faecalis, displays a similar amyloidogenic behavior. We demonstrate that at acidic pH the N-terminal region of Esp forms aggregates with an amyloid-like conformation, as evidenced by biophysical analysis and the binding of protein aggregates to amyloid-indicative dyes. Expression of a chimeric protein, with its Esp N-terminal domain anchored to the cell wall through the R domain of clumping factor A, showed that the Esp N-terminal region is sufficient to confer multicellular behavior through the formation of an extracellular amyloid-like material. These results suggest that the mechanism of amyloid-like aggregation to build the biofilm matrix might be widespread among BAP-like proteins. This amyloid-based mechanism may not only have strong relevance for bacteria lifestyle but could also contribute to the amyloid burden to which the human physiology is potentially exposed.
Open Access
Biofilm morphology and antibiotic susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) on poly-D,L-lactide-co-poly(ethylene glycol) (PDLLA-PEG) coated titanium
(Elsevier, 2024-10-05) Turner, Adam Benedict; Zermeño-Pérez, David; Mysior, Margaritha M.; Giraldo-Osorno, Paula Milena; García Martínez, Begoña; O'Gorman, Elizabeth; Oubihi, Shafik; Simpson, Jeremy C.; Lasa Uzcudun, Íñigo; Ó'Cróinín, Tadhg; Trobos, Margarita; Ciencias de la Salud; Osasun Zientziak
Biodegradable polymeric coatings are being explored as a preventive strategy for orthopaedic device-related infection. In this study, titanium surfaces (Ti) were coated with poly-D,L-lactide (PDLLA, (P)), polyethylene-glycol poly-D,L-lactide (PEGylated-PDLLA, (PP20)), or multi-layered PEGylated-PDLLA (M), with or without 1 % silver sulfadiazine. The aim was to evaluate their cytocompatibility, resistance to Staphylococcus aureus biofilm formation, and their potential to enhance the susceptibility of any biofilm formed to antibiotics. Using automated high-content screening confocal microscopy, biofilm formation of a clinical methicillin-resistant Staphylococcus aureus (MRSA) isolate expressing GFP was quantified, along with isogenic mutants that were unable to form polysaccharidic or proteinaceous biofilm matrices. The results showed that PEGylated-PDLLA coatings exhibited significant antibiofilm properties, with M showing the highest effect. This inhibitory effect was stronger in S. aureus biofilms with a matrix composed of proteins compared to those with an exopolysaccharide (PIA) biofilm matrix. Our data suggest that the antibiofilm effect may have been due to (i) inhibition of the initial attachment through microbial surface components recognising adhesive matrix molecules (MSCRAMMs), since PEG reduces protein surface adsorption via surface hydration layer and steric repulsion; and (ii) mechanical disaggregation and dispersal of microcolonies due to the bioresorbable/degradable nature of the polymers, which undergo hydration and hydrolysis over time. The disruption of biofilm morphology by the PDLLA-PEG co-polymers increased S. aureus susceptibility to antibiotics like rifampicin and fusidic acid. Adding 1 % AgSD provided additional early bactericidal effects on both biofilm and planktonic S. aureus. Additionally, the coatings were cytocompatible with immune cells, indicating their potential to enhance bacterial clearance and reduce bacterial colonisation of titanium-based orthopaedic biomaterials.
Open Access
Bacteriophages avoid autoimmunity from cognate immune systems as an intrinsic part of their life cycles
(Nature Research, 2024) Rostøl, Jakob T.; Quiles Puchalt, Nuria; Iturbe Sanz, Pablo; Lasa Uzcudun, Íñigo; Penadés, José R.; Ciencias de la Salud; Osasun Zientziak
Dormant prophages protect lysogenic cells by expressing diverse immune systems, which must avoid targeting their cognate prophages upon activation. Here we report that multiple Staphylococcus aureus prophages encode Tha (tail-activated, HEPN (higher eukaryotes and prokaryotes nucleotide-binding) domain-containing anti-phage system), a defence system activated by structural tail proteins of incoming phages. We demonstrate the function of two Tha systems, Tha-1 and Tha-2, activated by distinct tail proteins. Interestingly, Tha systems can also block reproduction of the induced tha-positive prophages. To prevent autoimmunity after prophage induction, these systems are inhibited by the product of a small overlapping antisense gene previously believed to encode an excisionase. This genetic organization, conserved in S. aureus prophages, allows Tha systems to protect prophages and their bacterial hosts against phage predation and to be turned of during prophage induction, balancing immunity and autoimmunity. Our results show that the fne regulation of these processes is essential for the correct development of prophages’ life cycle.
Open Access
Evaluation of the use of sonication combined with enzymatic treatment for biofilm removal in the microbiological diagnosis of prosthetic joint infection
(American Society for Microbiology, 2024) Henríquez, Lucía; Martín Contero, María del Carmen; Echeverz Sarasúa, Maite; Lasa Uzcudun, Íñigo; Ezpeleta Baquedano, María Carmen; Portillo, Eugenia; Ciencias de la Salud; Osasun Zientziak; Gobierno de Navarra / Nafarroako Gobernua
Sonicating explanted prosthetic implants to physically remove biofilms is a recognized method for improving the microbiological diagnosis of prosthetic joint infection (PJI); however, chemical and enzymatic treatments have been investigated as alternative biofilm removal methods. We compared the biofilm dislodging efficacy of sonication followed by the addition of enzyme cocktails with different activity spectra in the diagnosis of PJI with that of the sonication of fluid cultures alone. Consecutive patients who underwent prosthesis explantation due to infection at our institution were prospectively enrolled for 1 year. The diagnostic procedure included the collection of five intraoperative tissue cultures, sonication of the removed devices, and conventional culture of the sonication fluid. The resulting sonication fluid was also treated with an enzyme cocktail consisting of homemade dispersin B (0.04 ¿g/mL) and proteinase K (Sigma; 100 ¿g/mL) for 45 minutes at 37°C. The resulting sonication (S) and sonication with subsequent enzymatic treatment (SE) fluids were plated for aerobic and anaerobic culture broth for 7 days (aerobic) or 14 days (anaerobic). Identification was performed by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (Bruker). We included 107 patients from whom a prosthetic implant had been removed, among which PJI was diagnosed in 36 (34%). The sensitivity of S alone was significantly greater than that of SE alone (82% vs 71%; P < 0.05). Four patients with PJI were positive after sonication alone but negative after sonication plus enzymatic treatment. The four microorganisms missed after the addition of the enzyme cocktail were Staphylococcus aureus, two coagulase-negative Staphylococci, and Cutibacterium acnes. In conclusion, sonication alone was more sensitive than sonication followed by enzymatic treatment. The combination of these two methods had no synergistic effect; in contrast, the results suggest that the combination of both dislodgment methods affects the viability of gram-positive microorganisms.