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
Bap, a biofilm matrix protein of Staphylococcus aureus prevents cellular internalization through binding to GP96 host receptor
(Public Library of Science, 2012) Valle Turrillas, Jaione; Latasa Osta, Cristina; Gil Puig, Carmen; Toledo Arana, Alejandro; Solano Goñi, Cristina; Penadés, José R.; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The biofilm matrix, composed of exopolysaccharides, proteins, nucleic acids and lipids, plays a well-known role as a defence structure, protecting bacteria from the host immune system and antimicrobial therapy. However, little is known about its responsibility in the interaction of biofilm cells with host tissues. Staphylococcus aureus, a leading cause of biofilmassociated chronic infections, is able to develop a biofilm built on a proteinaceous Bap-mediated matrix. Here, we used the Bap protein as a model to investigate the role that components of the biofilm matrix play in the interaction of S. aureus with host cells. The results show that Bap promotes the adhesion but prevents the entry of S. aureus into epithelial cells. A broad analysis of potential interaction partners for Bap using ligand overlayer immunoblotting, immunoprecipitation with purified Bap and pull down with intact bacteria, identified a direct binding between Bap and Gp96/GRP94/Hsp90 protein. The interaction of Bap with Gp96 provokes a significant reduction in the capacity of S. aureus to invade epithelial cells by interfering with the fibronectin binding protein invasion pathway. Consistent with these results, Bap deficient bacteria displayed an enhanced capacity to invade mammary gland epithelial cells in a lactating mice mastitis model. Our observations begin to elucidate the mechanisms by which components of the biofilm matrix can facilitate the colonization of host tissues and the establishment of persistent infections.
Open Access
Relative contributions of lipooligosaccharide inner and outer core modifications to nontypeable Haemophilus influenzae pathogenesis
(American Society for Microbiology, 2013) Morey Sancho, Pau; Viadas Martínez, Cristina; Euba, Begoña; Hood, Derek W.; Barberán, Montserrat; Gil Puig, Carmen; Grilló Dolset, María Jesús; Bengoechea Alonso, José Antonio; Garmendia García, Juncal; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Nontypeable Haemophilus influenzae (NTHi) is a frequent commensal of the human nasopharynx that causes opportunistic infection in immunocompromised individuals. Existing evidence associates lipooligosaccharide (LOS) with disease, but the specific and relative contributions of NTHi LOS modifications to virulence properties of the bacterium have not been comprehensively addressed. Using NTHi strain 375, an isolate for which the detailed LOS structure has been determined, we compared systematically a set of isogenic mutant strains expressing sequentially truncated LOS. The relative contributions of 2-keto-3-deoxyoctulosonic acid, the triheptose inner core, oligosaccharide extensions on heptoses I and III, phosphorylcholine, digalactose, and sialic acid to NTHi resistance to antimicrobial peptides (AMP), self-aggregation, biofilm formation, cultured human respiratory epithelial infection, and murine pulmonary infection were assessed. We show that opsX, lgtF, lpsA, lic1, and lic2A contribute to bacterial resistance to AMP; lic1 is related to NTHi self-aggregation; lgtF, lic1, and siaB are involved in biofilm growth; opsX and lgtF participate in epithelial infection; and opsX, lgtF, and lpsA contribute to lung infection. Depending on the phenotype, the involvement of these LOS modifications occurs at different extents, independently or having an additive effect in combination. We discuss the relative contribution of LOS epitopes to NTHi virulence and frame a range of pathogenic traits in the context of infection.
Open Access
Evaluation of a Salmonella strain lacking the secondary messenger c-di-GMP and RpoS as a live oral vaccine
(Public Library of Science, 2016) Latasa Osta, Cristina; Echeverz Sarasúa, Maite; García Ona, Enrique; Burgui Erice, Saioa; Casares, Noelia; Hervás Stubbs, Sandra; Lasarte, Juan José; Lasa Uzcudun, Íñigo; Solano Goñi, Cristina; García Martínez, Begoña; Gil Puig, Carmen; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIM 13329.RI1
Salmonellosis is one of the most important bacterial zoonotic diseases transmitted through the consumption of contaminated food, with chicken and pig related products being key reservoirs of infection. Although numerous studies on animal vaccination have been performed in order to reduce Salmonella prevalence, there is still a need for an ideal vaccine. Here, with the aim of constructing a novel live attenuated Salmonella vaccine candidate, we firstly analyzed the impact of the absence of cyclic-di-GMP (c-di-GMP) in Salmonella virulence. Cdi-GMP is an intracellular second messenger that controls a wide range of bacterial processes, including biofilm formation and synthesis of virulence factors, and also modulates the host innate immune response. Our results showed that a Salmonella multiple mutant in the twelve genes encoding diguanylate cyclase proteins that, as a consequence, cannot synthesize c-di-GMP, presents a moderate attenuation in a systemic murine infection model. An additional mutation of the rpoS gene resulted in a synergic attenuating effect that led to a highly attenuated strain, referred to as ΔXIII, immunogenic enough to protect mice against a lethal oral challenge of a S. Typhimurium virulent strain. ΔXIII immunogenicity relied on activation of both antibody and cell mediated immune responses characterized by the production of opsonizing antibodies and the induction of significant levels of IFN-γ, TNF- α, IL-2, IL-17 and IL-10. ΔXIII was unable to form a biofilm and did not survive under desiccation conditions, indicating that it could be easily eliminated from the environment. Moreover, ΔXIII shows DIVA features that allow differentiation of infected and vaccinated animals. Altogether, these results show ΔXIII as a safe and effective live DIVA vaccine
Open Access
A systematic evaluation of the two-component systems network reveals that ArlRS is a key regulator of catheter colonization by Staphylococcus aureus
(Frontiers Media, 2018) Burgui Erice, Saioa; Gil Puig, Carmen; Solano Goñi, Cristina; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; Ciencias de la Salud; Osasun Zientziak
Two-component systems (TCS) are modular signal transduction pathways that allow cells to adapt to prevailing environmental conditions by modifying cellular physiology. Staphylococcus aureus has 16 TCSs to adapt to the diverse microenvironments encountered during its life cycle, including host tissues and implanted medical devices. S. aureus is particularly prone to cause infections associated to medical devices, whose surfaces coated by serum proteins constitute a particular environment. Identification of the TCSs involved in the adaptation of S. aureus to colonize and survive on the surface of implanted devices remains largely unexplored. Here, using an in vivo catheter infection model and a collection of mutants in each non-essential TCS of S. aureus, we investigated the requirement of each TCS for colonizing the implanted catheter. Among the 15 mutants in non-essential TCSs, the arl mutant exhibited the strongest deficiency in the capacity to colonize implanted catheters. Moreover, the arl mutant was the only one presenting a major deficit in PNAG production, the main exopolysaccharide of the S. aureus biofilm matrix whose synthesis is mediated by the icaADBC locus. Regulation of PNAG synthesis by ArlRS occurred through repression of IcaR, a transcriptional repressor of icaADBC operon expression. Deficiency in catheter colonization was restored when the arl mutant was complemented with the icaADBC operon. MgrA, a global transcriptional regulator downstream ArlRS that accounts for a large part of the arlRS regulon, was unable to restore PNAG expression and catheter colonization deficiency of the arlRS mutant. These findings indicate that ArlRS is the key TCS to biofilm formation on the surface of implanted catheters and that activation of PNAG exopolysaccharide production is, among the many traits controlled by the ArlRS system, a major contributor to catheter colonization.
Open Access
Characterization of nontypable haemophilus influenzae isolates recovered from adult patients with underlying chronic lung disease reveals genotypic and phenotypic traits associated with persistent infection
(Public Library of Science, 2014) Garmendia García, Juncal; Viadas Martínez, Cristina; Calatayud, Laura; Mell, Joshua Chang; Martí Lliteras, Pau; Euba, Begoña; Llobet, Enrique; Gil Puig, Carmen; Bengoechea Alonso, José Antonio; Redfield, Rosemary J.; Liñares, Josefina; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua, 359/2012
Nontypable Haemophilus influenzae (NTHi) has emerged as an important opportunistic pathogen causing infection in adults suffering obstructive lung diseases. Existing evidence associates chronic infection by NTHi to the progression of the chronic respiratory disease, but specific features of NTHi associated with persistence have not been comprehensively addressed. To provide clues about adaptive strategies adopted by NTHi during persistent infection, we compared sequential persistent isolates with newly acquired isolates in sputa from six patients with chronic obstructive lung disease. Pulse field gel electrophoresis (PFGE) identified three patients with consecutive persistent strains and three with new strains. Phenotypic characterisation included infection of respiratory epithelial cells, bacterial self-aggregation, biofilm formation and resistance to antimicrobial peptides (AMP). Persistent isolates differed from new strains in showing low epithelial adhesion and inability to form biofilms when grown under continuous-flow culture conditions in microfermenters. Self-aggregation clustered the strains by patient, not by persistence. Increasing resistance to AMPs was observed for each series of persistent isolates; this was not associated with lipooligosaccharide decoration with phosphorylcholine or with lipid A acylation. Variation was further analyzed for the series of three persistent isolates recovered from patient 1. These isolates displayed comparable growth rate, natural transformation frequency and murine pulmonary infection. Genome sequencing of these three isolates revealed sequential acquisition of single-nucleotide variants in the AMP permease sapC, the heme acquisition systems hgpB, hgpC, hup and hxuC, the 3-deoxy-D-manno-octulosonic acid kinase kdkA, the long-chain fatty acid transporter ompP1, and the phosphoribosylamine glycine ligase purD. Collectively, we frame a range of pathogenic traits and a repertoire of genetic variants in the context of persistent infection by NTHi.
Open Access
Increased biofilm formation by nontypeable Haemophilus influenzae isolates from patients with invasive disease or otitis media versus strains recovered from cases of respiratory infections
(American Society for Microbiology, 2014) Gil Puig, Carmen; Domenech, Arnau; Garmendia García, Juncal; Langereis, Jeroen D.; Mayer, Pascal; Calatayud, Laura; Liñares, Josefina; Ardanuy, Carmen; Martí, Sara; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Biofilm formation by nontypeable (NT) Haemophilus influenzae remains a controversial topic. Nevertheless, biofilm-like structures have been observed in the middle-ear mucosa of experimental chinchilla models of otitis media (OM). To date, there have been no studies of biofilm formation in large collections of clinical isolates. This study aimed to investigate the initial adhesion to a solid surface and biofilm formation by NT H. influenzae by comparing isolates from healthy carriers, those with noninvasive respiratory disease, and those with invasive respiratory disease. We used 352 isolates from patients with nonbacteremic community-acquired pneumonia (NB-CAP), chronic obstructive pulmonary disease (COPD), OM, and invasive disease and a group of healthy colonized children. We then determined the speed of initial adhesion to a solid surface by the BioFilm ring test and quantified biofilm formation by crystal violet staining. Isolates from different clinical sources displayed high levels of biofilm formation on a static solid support after growth for 24 h. We observed clear differences in initial attachment and biofilm formation depending on the pathology associated with NT H. influenzae isolation, with significantly increased biofilm formation for NT H. influenzae isolates collected from patients with invasive disease and OM compared with NT H. influenzae isolates from patients with NB-CAP or COPD and healthy colonized subjects. In all cases, biofilm structures were detached by proteinase K treatment, suggesting an important role for proteins in the initial adhesion and static biofilm formation measured by crystal violet staining.
Open Access
Biofilm matrix exoproteins induce a protective immune response against Staphylococcus aureus biofilm infection
(American Society for Microbiology, 2014) Gil Puig, Carmen; Solano Goñi, Cristina; Burgui Erice, Saioa; Latasa Osta, Cristina; García Martínez, Begoña; Toledo Arana, Alejandro; Lasa Uzcudun, Íñigo; Valle Turrillas, Jaione; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua: IIQ14066.RI1
The Staphylococcus aureus biofilm mode of growth is associated with several chronic infections that are very difficult to treat due to the recalcitrant nature of biofilms to clearance by antimicrobials. Accordingly, there is an increasing interest in preventing the formation of S. aureus biofilms and developing efficient antibiofilm vaccines. Given the fact that during a biofilm-associated infection, the first primary interface between the host and the bacteria is the self-produced extracellular matrix, in this study we analyzed the potential of extracellular proteins found in the biofilm matrix to induce a protective immune response against S. aureus infections. By using proteomic approaches, we characterized the exoproteomes of exopolysaccharide-based and proteinbased biofilm matrices produced by two clinical S. aureus strains. Remarkably, results showed that independently of the nature of the biofilm matrix, a common core of secreted proteins is contained in both types of exoproteomes. Intradermal administration of an exoproteome extract of an exopolysaccharide-dependent biofilm induced a humoral immune response and elicited the production of interleukin 10 (IL-10) and IL-17 in mice. Antibodies against such an extract promoted opsonophagocytosis and killing of S. aureus. Immunization with the biofilm matrix exoproteome significantly reduced the number of bacterial cells inside a biofilm and on the surrounding tissue, using an in vivo model of mesh-associated biofilm infection. Furthermore, immunized mice also showed limited organ colonization by bacteria released from the matrix at the dispersive stage of the biofilm cycle. Altogether, these data illustrate the potential of biofilm matrix exoproteins as a promising candidate multivalent vaccine against S. aureus biofilm-associated infections.