Structural mechanism for modulation of functional amyloid and biofilm formation by Staphylococcal Bap protein switch.

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.

A multifaceted small RNA modulates gene expression upon glucose limitation in Staphylococcus aureus.

Pathogenic bacteria must rapidly adapt to ever-changing environmental signals resulting in metabolism remodeling. The carbon catabolite repression, mediated by the catabolite control protein A (CcpA), is used to express genes involved in utilization and metabolism of the preferred carbon source. Here, we have identified RsaI as a CcpA-repressed small non-coding RNA that is inhibited by high glucose concentrations. When glucose is consumed, RsaI represses translation initiation of mRNAs encoding a permease of glucose uptake and the FN3K enzyme that protects proteins against damage caused by high glucose concentrations. RsaI also binds to the 3' untranslated region of icaR mRNA encoding the transcriptional repressor of exopolysaccharide production and to sRNAs induced by the uptake of glucose-6 phosphate or nitric oxide. Furthermore, RsaI expression is accompanied by a decreased transcription of genes involved in carbon catabolism pathway and an activation of genes involved in energy production, fermentation, and nitric oxide detoxification. This multifaceted RNA can be considered as a metabolic signature when glucose becomes scarce and growth is arrested.

AdrA as a Potential Immunomodulatory Candidate for STING-Mediated Antiviral Therapy That Required Both Type I IFN and TNF-α Production.

Several dinucleotide cyclases, including cyclic GMP-AMP synthase, and their involvement in STING-mediated immunity have been extensively studied. In this study, we tested five bacterial diguanylate cyclases from the Gram-negative bacterium Salmonella Enteritidis, identifying AdrA as the most potent inducer of a STING-mediated IFN response. AdrA wild-type (wt) or its inactive version AdrA mutant (mut) were delivered by an adenovirus (Ad) vector. Dendritic cells obtained from wt mice and infected in vitro with Ad vector containing AdrA wt, but not mut, had increased activation markers and produced large amounts of several immunostimulatory cytokines. For dendritic cells derived from STING-deficient mice, no activation was detected. The potential antiviral activity of AdrA was addressed in hepatitis B virus (HBV)-transgenic and adenovirus-associated virus (AAV)-HBV mouse models. Viremia in serum of Ad AdrA wt-treated mice was reduced significantly compared with that in Ad AdrA mut-injected mice. The viral load in the liver at sacrifice was in line with this finding. To further elucidate the molecular mechanism(s) by which AdrA confers its antiviral function, the response in mice deficient in STING or its downstream effector molecules was analyzed. wt and IFN-αR (IFNAR)-/- animals were additionally treated with anti-TNF-α (Enbrel). Interestingly, albeit less pronounced than in wt mice, in IFNAR-/- and Enbrel-treated wt mice, a reduction of serum viremia was achieved-an observation that was lost in anti-TNF-α-treated IFNAR-/- animals. No effect of AdrA wt was seen in STING-deficient animals. Thus, although STING is indispensable for the antiviral activity of AdrA, type I IFN and TNF-α are both required and act synergistically.

Regulation of Heterogenous LexA Expression in Staphylococcus aureus by an Antisense RNA Originating from Transcriptional Read-Through upon Natural Mispairings in the sbrB Intrinsic Terminator.

Bacterial genomes are pervasively transcribed, generating a wide variety of antisense RNAs (asRNAs). Many of them originate from transcriptional read-through events (TREs) during the transcription termination process. Previous transcriptome analyses revealed that the lexA gene from Staphylococcus aureus, which encodes the main SOS response regulator, is affected by the presence of an asRNA. Here, we show that the lexA antisense RNA (lexA-asRNA) is generated by a TRE on the intrinsic terminator (TTsbrB) of the sbrB gene, which is located downstream of lexA, in the opposite strand. Transcriptional read-through occurs by a natural mutation that destabilizes the TTsbrB structure and modifies the efficiency of the intrinsic terminator. Restoring the mispairing mutation in the hairpin of TTsbrB prevented lexA-asRNA transcription. The level of lexA-asRNA directly correlated with cellular stress since the expressions of sbrB and lexA-asRNA depend on the stress transcription factor SigB. Comparative analyses revealed strain-specific nucleotide polymorphisms within TTsbrB, suggesting that this TT could be prone to accumulating natural mutations. A genome-wide analysis of TREs suggested that mispairings in TT hairpins might provide wider transcriptional connections with downstream genes and, ultimately, transcriptomic variability among S. aureus strains.

Advances in bacterial transcriptome understanding: From overlapping transcription to the excludon concept.

In the last decade, the implementation of high-throughput methods for RNA profiling has uncovered that a large part of the bacterial genome is transcribed well beyond the boundaries of known genes. Therefore, the transcriptional space of a gene very often invades the space of a neighbouring gene, creating large regions of overlapping transcription. The biological significance of these findings was initially regarded with scepticism. However, mounting evidence suggests that overlapping transcription between neighbouring genes conforms to regulatory purposes and provides new strategies for coordinating bacterial gene expression. In this MicroReview, considering the discoveries made in a pioneering transcriptome analysis performed on Listeria monocytogenes as a starting point, we discuss the progress in understanding the biological meaning of overlapping transcription that has given rise to the excludon concept. We also discuss new conditional transcriptional termination events that create antisense RNAs depending on the metabolite concentrations and new genomic arrangements, known as noncontiguous operons, which contain an interspersed gene that is transcribed in the opposite direction to the rest of the operon.

Regulation of gene expression by non-phosphorylated response regulators.

Two-component systems (TCSs) are a prominent sensory system in bacteria. A prototypical TCS comprises a membrane-bound sensor histidine kinase (HK) responsible for sensing the signal and a cytoplasmic response regulator (RR) that controls target gene expression. Signal binding activates a phosphotransfer cascade from the HK to the RR. As a result, the phosphorylated RR undergoes a conformational change that leads to activation of the response. Growing experimental evidence indicates that unphosphorylated RRs may also have regulatory functions, and thus, the classical view that the RR is only active when it is phosphorylated needs to be revisited. In this review, we highlight the most recent findings showing that RRs in the non-phosphorylated state control critical bacterial processes that range from secretion of factors to the host, antibiotic resistance, iron transport, stress response, and cell-wall metabolism to biofilm development.

A DIVA vaccine strain lacking RpoS and the secondary messenger c-di-GMP for protection against salmonellosis in pigs.

Salmonellosis is the second most common food-borne zoonosis in the European Union, with pigs being a major reservoir of this pathogen. Salmonella control in pig production requires multiple measures amongst which vaccination may be used to reduce subclinical carriage and shedding of prevalent serovars, such as Salmonella enterica serovar Typhimurium. Live attenuated vaccine strains offer advantages in terms of enhancing cell mediated immunity and allowing inoculation by the oral route. However, main failures of these vaccines are the limited cross-protection achieved against heterologous serovars and interference with serological monitoring for infection. We have recently shown that an attenuated S. Enteritidis strain (ΔXIII) is protective against S. Typhimurium in a murine infection model. ΔXIII strain harbours 13 chromosomal deletions that make it unable to produce the sigma factor RpoS and synthesize cyclic-di-GMP (c-di-GMP). In this study, our objectives were to test the protective effects of ΔXIII strain in swine and to investigate if the use of ΔXIII permits the discrimination of vaccinated from infected pigs. Results show that oral vaccination of pre-weaned piglets with ΔXIII cross-protected against a challenge with S. Typhimurium by reducing faecal shedding and ileocaecal lymph nodes colonization, both at the time of weaning and slaughter. Vaccinated pigs showed neither faecal shedding nor tissue persistence of the vaccine strain at weaning, ensuring the absence of ΔXIII strain by the time of slaughter. Moreover, lack of the SEN4316 protein in ΔXIII strain allowed the development of a serological test that enabled the differentiation of infected from vaccinated animals (DIVA).

The regulon of the RNA chaperone CspA and its auto-regulation in Staphylococcus aureus.

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.

Lack of the PGA exopolysaccharide in Salmonella as an adaptive trait for survival in the host.

Many bacteria build biofilm matrices using a conserved exopolysaccharide named PGA or PNAG (poly-ß-1,6-N-acetyl-D-glucosamine). Interestingly, while E. coli and other members of the family Enterobacteriaceae encode the pgaABCD operon responsible for PGA synthesis, Salmonella lacks it. The evolutionary force driving this difference remains to be determined. Here, we report that Salmonella lost the pgaABCD operon after the divergence of Salmonella and Citrobacter clades, and previous to the diversification of the currently sequenced Salmonella strains. Reconstitution of the PGA machinery endows Salmonella with the capacity to produce PGA in a cyclic dimeric GMP (c-di-GMP) dependent manner. Outside the host, the PGA polysaccharide does not seem to provide any significant benefit to Salmonella: resistance against chlorine treatment, ultraviolet light irradiation, heavy metal stress and phage infection remained the same as in a strain producing cellulose, the main biofilm exopolysaccharide naturally produced by Salmonella. In contrast, PGA production proved to be deleterious to Salmonella survival inside the host, since it increased susceptibility to bile salts and oxidative stress, and hindered the capacity of S. Enteritidis to survive inside macrophages and to colonize extraintestinal organs, including the gallbladder. Altogether, our observations indicate that PGA is an antivirulence factor whose loss may have been a necessary event during Salmonella speciation to permit survival inside the host.

σB Inhibits Poly-N-Acetylglucosamine Exopolysaccharide Synthesis and Biofilm Formation in Staphylococcus aureus.

Staphylococcus aureus clinical strains are able to produce at least two distinct types of biofilm matrixes: biofilm matrixes made of the polysaccharide intercellular adhesin (PIA) or poly-N-acetylglucosamine (PNAG), whose synthesis is mediated by the icaADBC locus, and biofilm matrixes built of proteins (polysaccharide independent). σB is a conserved alternative sigma factor that regulates the expression of more than 100 genes in response to changes in environmental conditions. While numerous studies agree that σB is required for polysaccharide-independent biofilms, controversy persists over the role of σB in the regulation of PIA/PNAG-dependent biofilm development. Here, we show that genetically unrelated S. aureus σB-deficient strains produced stronger biofilms under both static and flow conditions and accumulated higher levels of PIA/PNAG exopolysaccharide than their corresponding wild-type strains. The increased accumulation of PIA/PNAG in the σB mutants correlated with a greater accumulation of the IcaC protein showed that it was not due to adjustments in icaADBC operon transcription and/or icaADBC mRNA stability. Overall, our results reveal that in the presence of active σB, the turnover of Ica proteins is accelerated, reducing the synthesis of PIA/PNAG exopolysaccharide and consequently the PIA/PNAG-dependent biofilm formation capacity.IMPORTANCE Due to its multifaceted lifestyle, Staphylococcus aureus needs a complex regulatory network to connect environmental signals with cellular physiology. One particular transcription factor, named σB (SigB), is involved in the general stress response and the expression of virulence factors. For many years, great confusion has existed about the role of σB in the regulation of the biofilm lifestyle in S. aureus Our study demonstrated that σB is not necessary for exopolysaccharide-dependent biofilms and, even more, that S. aureus produces stronger biofilms in the absence of σB The increased accumulation of exopolysaccharide correlates with higher stability of the proteins responsible for its synthesis. The present findings reveal an additional regulatory layer to control biofilm exopolysaccharide synthesis under stress conditions.

A pyrene-inhibitor fluorescent probe with large Stokes shift for the staining of Aß1-42, α-synuclein, and amylin amyloid fibrils as well as amyloid-containing Staphylococcus aureus biofilms.

Amyloid fibrils formed by a variety of peptides are biological markers of different human diseases, such as Alzheimer's disease, Parkinson's disease, and type II diabetes, and are structural constituents of bacterial biofilms. Novel fluorescent probes offering improved sensitivity or specificity toward 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 Aß1-42 peptide. All the derivatives tested retain the interaction with the amyloid architecture and allow its staining. The most soluble derivative, N-acetyl-2-(2-methyl-4-oxo-5,6,7,8-tetrahydro-4H-benzo[4,5]thieno[2,3-d][1,3]oxazin-7-yl)-N-(pyren-1-ylmethyl)acetamide (compound 1D), stains similarly well amyloid fibrils formed by Aß1-42, α-synuclein, or amylin, provides a sensitivity only slightly lower than that of thioflavin T, displays a large Stokes shift, allows efficient excitation in the UV spectral region, and 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, Staphylococcus aureus biofilms containing amyloid-forming phenol-soluble modulins from those lacking them. Graphical abstract ᅟ.

Staphylococcal Bap Proteins Build Amyloid Scaffold Biofilm Matrices in Response to Environmental Signals.

Biofilms are communities of bacteria that grow encased in an extracellular matrix that often contains proteins. The spatial organization and the molecular interactions between matrix scaffold proteins remain in most cases largely unknown. Here, we report that Bap protein of Staphylococcus aureus self-assembles into functional amyloid aggregates to build the biofilm matrix in response to environmental conditions. Specifically, Bap is processed and fragments containing at least the N-terminus of the protein become aggregation-prone and self-assemble into amyloid-like structures under acidic pHs and low concentrations of calcium. The molten globule-like state of Bap fragments is stabilized upon binding of the cation, hindering its self-assembly into amyloid fibers. These findings define a dual function for Bap, first as a sensor and then as a scaffold protein to promote biofilm development under specific environmental conditions. Since the pH-driven multicellular behavior mediated by Bap occurs in coagulase-negative staphylococci and many other bacteria exploit Bap-like proteins to build a biofilm matrix, the mechanism of amyloid-like aggregation described here may be widespread among pathogenic bacteria.

Amyloid Structures as Biofilm Matrix Scaffolds.

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.

Moonlighting bacteriophage proteins derepress staphylococcal pathogenicity islands.

Staphylococcal superantigen-carrying pathogenicity islands (SaPIs) are discrete, chromosomally integrated units of approximately 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 transfer. 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 genes. 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 packaged, this strategy assures that SaPIs will be transferred once induced. Several different SaPIs are induced by helper phage 80alpha 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.

Base pairing interaction between 5'- and 3'-UTRs controls icaR mRNA translation in Staphylococcus aureus.

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.

Unravelling bacteriophage ϕ11 requirements for packaging and transfer of mobile genetic elements in Staphylococcus aureus.

Bacteriophages play a major role in spreading mobile genetic elements (MGEs)-encoded genes among bacterial populations. In spite of this, the molecular requirements for building phage transducing particles have not been completely deciphered. Here, we systematically inactivated each ORF from the packaging and lysis modules of the staphylococcal phage ϕ11, used as a model for the Siphoviridae phages infecting Gram-positive bacteria, and determined their functional role in transferring different MGEs including plasmids, staphylococcal pathogenicity islands (SaPIs) and the phage itself. In a previous report, we identified seven of these ORFs as being required for the production of functional phage or SaPI particles. In this report, we have completed the mutational analysis and have identified and characterized 15 additional phage-encoded proteins required for the production of mature phage, SaPI, or transducing particles. Apart from these, we have not yet ascertained any specific function for the six remaining ϕ11 genes, though they are highly conserved among the staphylococcal bacteriophages. To the best of our knowledge, this study represents the first systematic deletion analysis of all the ORFs comprising the morphogenetic and lysis modules of a phage, clearly defining the molecular requirements involved in phage-mediated MGEs transfer.

Auranofin efficacy against MDR Streptococcus pneumoniae and Staphylococcus aureus infections.

BACKGROUND: Auranofin is an FDA-approved, gold-containing compound in clinical use for the oral treatment of rheumatoid arthritis and has been recently granted by the regulatory authorities due to its antiprotozoal properties. METHODS: A reprofiling strategy was performed with a Streptococcus pneumoniae phenotypic screen and a proprietary library of compounds, consisting of both FDA-approved and unapproved bioactive compounds. Two different multiresistant S. pneumoniae strains were employed in a sepsis mouse model of infection. In addition, an MRSA strain was tested using both the thigh model and a mesh-associated biofilm infection in mice. RESULTS: The repurposing approach showed the high potency of auranofin against multiresistant clinical isolates of S. pneumoniae and Staphylococcus aureus in vitro and in vivo. Efficacy in the S. pneumoniae sepsis model was obtained using auranofin by the oral route in the dose ranges used for the treatment of rheumatoid arthritis. Thioglucose replacement by alkyl chains showed that this moiety was not essential for the antibacterial activity and led to the discovery of a new gold derivative (MH05) with remarkable activity in vitro and in vivo. CONCLUSIONS: Auranofin and the new gold derivative MH05 showed encouraging in vivo activity against multiresistant clinical isolates of S. pneumoniae and S. aureus. The clinical management of auranofin, alone or in combination with other antibiotics, deserves further exploration before use in patients presenting therapeutic failure caused by infections with multiresistant Gram-positive pathogens. Decades of clinical use mean that this compound is safe to use and may accelerate its evaluation in humans.

A super-family of transcriptional activators regulates bacteriophage packaging and lysis in Gram-positive bacteria.

The propagation of bacteriophages and other mobile genetic elements requires exploitation of the phage mechanisms involved in virion assembly and DNA packaging. Here, we identified and characterized four different families of phage-encoded proteins that function as activators required for transcription of the late operons (morphogenetic and lysis genes) in a large group of phages infecting Gram-positive bacteria. These regulators constitute a super-family of proteins, here named late transcriptional regulators (Ltr), which share common structural, biochemical and functional characteristics and are unique to this group of phages. They are all small basic proteins, encoded by genes present at the end of the early gene cluster in their respective phage genomes and expressed under cI repressor control. To control expression of the late operon, the Ltr proteins bind to a DNA repeat region situated upstream of the terS gene, activating its transcription. This involves the C-terminal part of the Ltr proteins, which control specificity for the DNA repeat region. Finally, we show that the Ltr proteins are the only phage-encoded proteins required for the activation of the packaging and lysis modules. In summary, we provide evidence that phage packaging and lysis is a conserved mechanism in Siphoviridae infecting a wide variety of Gram-positive bacteria.

Audiological results after total ossicular reconstruction for stapes fixation.

The objective was to assess audiological results after total ossicular reconstruction for stapes fixation. The study is a retrospective evaluation conducted in a tertiary referral centre. The patients were 16 adults with conductive or mixed hearing loss and stapes fixation due to tympanosclerosis or otosclerosis. A total or partial stapedectomy with perichondrium interposition on the oval window and ossicular reconstruction with titanium total prosthesis were done. To assess pre- and post-operative (1 and 4 years) air and bone-conduction thresholds (frequencies 0.5, 1, 2, 3 kHz), pure-tone average air and bone conduction, and air-bone gaps were measured and the number of decibels of closure of the air-bone gap at 1 year and at 4 years were compared. One year after surgery, air conduction thresholds and pure-tone average air conduction were improved for all frequencies, and there were no significant differences in bone conduction thresholds or in pure-tone average bone conduction. There were no differences in air and bone conduction thresholds, pure-tone average air or bone conduction between 1 and 4 years. The air-bone gap was significantly reduced 1 year after surgery and remained so at 4 years. (Preoperative air-bone gap, 34.04 dB; at 1 year, 16.40 dB; at 4 years, 17.3 dB. Decibels of closure of the air-bone gap at 1 year, 17.64 dB; at 4 years, 16.74 dB.) No differences were found between otosclerosis subjects and all other cases combined. Total ossicular reconstruction in stapes fixation due to tympanosclerosis or otosclerosis produces satisfactory short- and long-term auditory results.

Biofilm matrix exoproteins induce a protective immune response against Staphylococcus aureus biofilm infection.

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 protein-based 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.