Open Access
Expression of the biofilm-associated protein interferes with host protein receptors of Staphylococcus aureus and alters the infective process
(American Society for Microbiology, 2002) Cucarella, Carme; Tormo Más, María Ángeles; Knecht, Erwin; Amorena Zabalza, Beatriz; Lasa Uzcudun, Íñigo; Foster, Timothy J.; Penadés, José R.; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The adherence of Staphylococcus aureus to soluble proteins and extracellular-matrix components of the host is one of the key steps in the pathogenesis of staphylococcal infections. S. aureus presents a family of adhesins called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that specifically recognize host matrix components. We examined the influence of biofilm-associated protein (Bap) expression on S. aureus adherence to host proteins, epithelial cell cultures, and mammary gland sections and on colonization of the mammary gland in an in vivo infection model. Bap-positive strain V329 showed lower adherence to immobilized fibrinogen and fibronectin than isogenic Bap-deficient strain m556. Bacterial adherence to histological sections of mammary gland and bacterial internalization into 293 cells were significantly lower in the Bap-positive strains. In addition, the Bap-negative strain showed significantly higher colonization in vivo of sheep mammary glands than the Bap-positive strain. Taken together, these results strongly suggest that the expression of the Bap protein interferes with functional properties of the MSCRAMM proteins, preventing initial bacterial attachment to host tissues and cellular internalization.
Open Access
Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands
(Nature Research, 2010) Tormo Más, María Ángeles; Mir, Ignacio; Shrestha, Archana; Tallent, Sandra M.; Campoy Sánchez, Susana; Lasa Uzcudun, Íñigo; Barbé, Jordi; Novick, Richard P.; Christie, Gail E.; Penadés, José R.; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Staphylococcal superantigen-carrying pathogenicity islands (SaPIs) are discrete, chromosomally integrated units of ∼15 kilobases that are induced by helper phages to excise and replicate. SaPI DNA is then efficiently encapsidated in phage-like infectious particles, leading to extremely high frequencies of intra- as well as intergeneric transfer1,2,3. In the absence of helper phage lytic growth, the island is maintained in a quiescent prophage-like state by a global repressor, Stl, which controls expression of most of the SaPI genes4. Here we show that SaPI derepression is effected by a specific, non-essential phage protein that binds to Stl, disrupting the Stl–DNA complex and thereby initiating the excision-replication-packaging cycle of the island. Because SaPIs require phage proteins to be packaged5,6, this strategy assures that SaPIs will be transferred once induced. Several different SaPIs are induced by helper phage 80α and, in each case, the SaPI commandeers a different non-essential phage protein for its derepression. The highly specific interactions between different SaPI repressors and helper-phage-encoded antirepressors represent a remarkable evolutionary adaptation involved in pathogenicity island mobilization.
Open Access
Protein A-mediated multicellular behavior in Staphylococcus aureus
(American Society for Microbiology, 2008) Merino Barberá, Nekane; Toledo Arana, Alejandro; Vergara Irigaray, Marta; Valle Turrillas, Jaione; Solano Goñi, Cristina; Calvo, Enrique; Lopez, Juan Antonio; Foster, Timothy J.; Penadés, José R.; Lasa Uzcudun, Íñigo; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
The capacity of Staphylococcus aureus to form biofilms on host tissues and implanted medical devices is one of the major virulence traits underlying persistent and chronic infections. The matrix in which S. aureus cells are encased in a biofilm often consists of the polysaccharide intercellular adhesin (PIA) or poly-N-acetyl glucosamine (PNAG). However, surface proteins capable of promoting biofilm development in the absence of PIA/PNAG exopolysaccharide have been described. Here, we used two-dimensional nano-liquid chromatography and mass spectrometry to investigate the composition of a proteinaceous biofilm matrix and identified protein A (spa) as an essential component of the biofilm; protein A induced bacterial aggregation in liquid medium and biofilm formation under standing and flow conditions. Exogenous addition of synthetic protein A or supernatants containing secreted protein A to growth media induced biofilm development, indicating that protein A can promote biofilm development without being covalently anchored to the cell wall. Protein A-mediated biofilm formation was completely inhibited in a dose-dependent manner by addition of serum, purified immunoglobulin G, or anti-protein A-specific antibodies. A murine model of subcutaneous catheter infection unveiled a significant role for protein A in the development of biofilm-associated infections, as the amount of protein A-deficient bacteria recovered from the catheter was significantly lower than that of wild-type bacteria when both strains were used to coinfect the implanted medical device. Our results suggest a novel role for protein A complementary to its known capacity to interact with multiple immunologically important eukaryotic receptors.
Open Access
Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus
(American Society for Microbiology, 2004) Cucarella, Carme; Tormo Más, María Ángeles; Ubeda, Carles; Trotonda, María Pilar; Monzón, Marta; Peris, Cristòfol; Amorena Zabalza, Beatriz; Lasa Uzcudun, Íñigo; Penadés, José R.; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Staphylococcus aureus is a common cause of intramammary infections, which frequently become chronic, associated with the ability of the bacteria to produce biofilm. Here, we report a relationship between the ability to produce chronic bovine mastitis and biofilm formation. We have classified bovine mastitis S. aureus isolates into three groups based on the presence of particular genetic elements required for biofilm formation: group 1 (ica+ bap+), group 2 (ica+, bap negative), and group 3 (ica negative, bap negative). Overall, animals naturally infected with group 1 and 2 isolates had a lower milk somatic cell count than those infected with isolates of group 3. In addition, Bap-positive isolates were significantly more able to colonize and persist in the bovine mammary gland in vivo and were less susceptible to antibiotic treatments when forming biofilms in vitro. Analysis of the structural bap gene revealed the existence of alternate forms of expression of the Bap protein in S. aureus isolates obtained under field conditions throughout the animal's life. The presence of anti-Bap antibodies in serum samples taken from animals with confirmed S. aureus infections indicated the production of Bap during infection. Furthermore, disruption of the ica operon in a bap-positive strain had no effect on in vitro biofilm formation, a finding which strongly suggested that Bap could compensate for the deficiency of the PIA/PNAG product (a biofilm matrix polysaccharide). Altogether, these results demonstrate that, in the bovine intramammary gland, the presence of Bap may facilitate a biofilm formation connected with the persistence of S. aureus.
Open Access
Beta-lactam antibiotics induce the SOS response and horizontal transfer of virulence factors in Staphylococcus aureus
(American Society for Microbiology, 2006) Maiques, Elisa; Ubeda, Carles; Campoy Sánchez, Susana; Salvador, Noelia; Lasa Uzcudun, Íñigo; Novick, Richard P.; Barbé, Jordi; Penadés, José R.; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Antibiotics that interfere with DNA replication and cell viability activate the SOS response. In Staphylococcus aureus, the antibiotic-induced SOS response promotes replication and high-frequency horizontal transfer of pathogenicity island-encoded virulence factors. Here we report that β-lactams induce a bona fide SOS response in S. aureus, characterized by the activation of the RecA and LexA proteins, the two master regulators of the SOS response. Moreover, we show that β-lactams are capable of triggering staphylococcal prophage induction in S. aureus lysogens. Consequently, and as previously described for SOS induction by commonly used fluoroquinolone antibiotics, β-lactam-mediated phage induction also resulted in replication and high-frequency transfer of the staphylococcal pathogenicity islands, showing that such antibiotics may have the unintended consequence of promoting the spread of bacterial virulence factors.
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
Genome-wide antisense transcription drives mRNA processing in bacteria
(National Academy of Sciences, 2011) Lasa Uzcudun, Íñigo; Toledo Arana, Alejandro; Dobin, Alexander; Villanueva San Martín, Maite; Ruiz de los Mozos Aliaga, Igor; Vergara Irigaray, Marta; Segura, Víctor; Fagegaltier, Delphine; Penadés, José R.; Valle Turrillas, Jaione; Solano Goñi, Cristina; Gingeras, Thomas R.; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
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.
Open Access
B regulates IS256-mediated Staphylococcus aureus biofilm phenotypic variation
(American Society for Microbiology, 2007) Valle Turrillas, Jaione; Vergara Irigaray, Marta; Merino Barberá, Nekane; Penadés, José R.; Lasa Uzcudun, Íñigo; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua
Biofilm formation in Staphylococcus aureus is subject to phase variation, and biofilm-negative derivatives emerge sporadically from a biofilm-positive bacterial population. To date, the only known mechanism for generating biofilm phenotypic variation in staphylococci is the reversible insertion/excision of IS256 in biofilm-essential genes. In this study, we present evidence suggesting that the absence of the σB transcription factor dramatically increases the rate of switching to the biofilm-negative phenotype in the clinical isolate S. aureus 15981, under both steady-state and flow conditions. The phenotypic switching correlates with a dramatic increase in the number of IS256 copies in the chromosomes of biofilm-negative variants, as well as with an augmented IS256 insertion frequency into the icaC and the sarA genes. IS256-mediated biofilm switching is reversible, and biofilm-positive variants could emerge from biofilm-negative σB mutants. Analysis of the chromosomal insertion frequency using a recombinant IS256 element tagged with an erythromycin marker showed an almost three-times-higher transposition frequency in a ΔσB strain. However, regulation of IS256 activity by σB appears to be indirect, since transposase transcription is not affected in the absence of σB and IS256 activity is inhibited to wild-type levels in a ΔσB strain under NaCl stress. Overall, our results identify a new role for σB as a negative regulator of insertion sequence transposition and support the idea that deregulation of IS256 activity abrogates biofilm formation capacity in S. aureus.
Open Access
Bap, a Staphylococcus aureus surface protein involved in biofilm formation
(American Society for Microbiology, 2001) Cucarella, Carme; Solano Goñi, Cristina; Valle Turrillas, Jaione; Amorena Zabalza, Beatriz; Lasa Uzcudun, Íñigo; Penadés, José R.; Nekazaritza Ekoizpena; Producción Agraria; IdAB. Instituto de Agrobiotecnología / Agrobioteknologiako Institutua; Gobierno de Navarra / Nafarroako Gobernua
Identification of new genes involved in biofilm formation is needed to understand the molecular basis of strain variation and the pathogenic mechanisms implicated in chronic staphylococcal infections. A biofilm-producing Staphylococcus aureus isolate was used to generate biofilm-negative transposon (Tn917) insertion mutants. Two mutants were found with a significant decrease in attachment to inert surfaces (early adherence), intercellular adhesion, and biofilm formation. The transposon was inserted at the same locus in both mutants. This locus (bap [for biofilm associated protein]) encodes a novel cell wall associated protein of 2,276 amino acids (Bap), which shows global organizational similarities to surface proteins of gram-negative (Pseudomonas aeruginosa andSalmonella enterica serovar Typhi) and gram-positive (Enteroccocus faecalis) microorganisms. Bap's core region represents 52% of the protein and consists of 13 successive nearly identical repeats, each containing 86 amino acids. bap was present in a small fraction of bovine mastitis isolates (5% of the 350S. aureus isolates tested), but it was absent from the 75 clinical human S. aureus isolates analyzed. All staphylococcal isolates harboring bap were highly adherent and strong biofilm producers. In a mouse infection modelbap was involved in pathogenesis, causing a persistent infection.
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.