The Multitasking Surface Protein of Staphylococcus epidermidis: Accumulation-Associated Protein (Aap)

The stratum corneum is the outermost layer of the epidermis and is thus directly exposed to the environment. It consists mainly of corneocytes, which are keratinocytes in the last stage of differentiation, having neither nuclei nor organelles.

The staphylococcal biofilm protein Aap forms a tetrameric species as a necessary intermediate before amyloidogenesis

The accumulation-associated protein (Aap) from Staphylococcus epidermidis is a biofilm-related protein that was found to be a critical factor for infection using a rat catheter model. The B-repeat superdomain of Aap, composed of 5–17 B-repeats, each containing a Zn2+-binding G5 and a spacer subdomain, is responsible for Zn2+-dependent assembly leading to accumulation of bacteria during biofilm formation. We previously demonstrated that a minimal B-repeat construct (Brpt1.5) forms an antiparallel dimer in the presence of 2–3 Zn2+ ions. More recently, we have reported the presence of functional amyloid-like fibrils composed of Aap within S. epidermidis biofilms and demonstrated that a biologically relevant construct containing five and a half B-repeats (Brpt5.5) forms amyloid-like fibrils similar to those observed in the biofilm. In this study, we analyze the initial assembly events of the Brpt5.5 construct. Analytical ultracentrifugation was utilized to determine hydrodynamic parameters of reversibly associating species and to perform linked equilibrium studies. Linkage studies indicated a mechanism of Zn2+-induced dimerization similar to smaller constructs; however, Brpt5.5 dimers could then undergo further Zn2+-induced assembly into a previously uncharacterized tetramer. This led us to search for potential Zn2+-binding sites outside of the dimer interface. We developed a Brpt5.5 mutant that was unable to form the tetramer and was concordantly incapable of amyloidogenesis. CD and dynamic light scattering indicate that a conformational transition in the tetramer species is a critical step preceding amyloidogenesis. This mechanistic model for B-repeat assembly and amyloidogenesis provides new avenues for potential therapeutic targeting of staphylococcal biofilms.

The biofilm adhesion protein Aap from Staphylococcus epidermidis forms zinc-dependent amyloid fibers

The skin-colonizing commensal bacterium Staphylococcus epidermidis is a leading cause of hospital-acquired and device-related infections. Its pathogenicity in humans is largely due to its propensity to form biofilms, surface-adherent bacterial accumulations that are remarkably resistant to chemical and physical stresses. Accumulation-associated protein (Aap) from S. epidermidis has been shown to be necessary and sufficient for mature biofilm formation and catheter infection. Aap contains up to 17 tandem B-repeat domains, capable of zinc-dependent assembly into twisted, rope-like intercellular filaments in the biofilm. Using microscopic and biophysical techniques, we show here that Aap B-repeat constructs assemble further into zinc-dependent functional amyloid fibers. We observed such amyloid fibers by confocal microscopy during both early and late stages of S. epidermidis biofilm formation, and we confirmed that extracellular fibrils from these biofilms contain Aap. Unlike what has been observed for amyloidogenic biofilm proteins from other bacteria, which typically use chaperones or initiator proteins to initiate amyloid assembly, our findings indicate that Aap from S. epidermidis requires Zn2+ as a catalyst that drives amyloid fiber formation, similar to many mammalian amyloid-forming proteins that require metals for assembly. This work provides detailed insights into S. epidermidis biofilm formation and architecture that improve our understanding of persistent staphylococcal infections.

Accumulation-Associated Protein Enhances Staphylococcus epidermidis Biofilm Formation under Dynamic Conditions and Is Required for Infection in a Rat Catheter Model

Biofilm formation is the primary virulence factor ofStaphylococcus epidermidis.S. epidermidisbiofilms preferentially form on abiotic surfaces and may contain multiple matrix components, including proteins such as accumulation-associated protein (Aap). Following proteolytic cleavage of the A domain, which has been shown to enhance binding to host cells, B domain homotypic interactions support cell accumulation and biofilm formation. To further define the contribution of Aap to biofilm formation and infection, we constructed anaapallelic replacement mutant and anicaADBC aapdouble mutant. When subjected to fluid shear, strains deficient in Aap production produced significantly less biofilm than Aap-positive strains. To examine thein vivorelevance of our findings, we modified our previously described rat jugular catheter model and validated the importance of immunosuppression and the presence of a foreign body to the establishment of infection. The use of our allelic replacement mutants in the model revealed a significant decrease in bacterial recovery from the catheter and the blood in the absence of Aap, regardless of the production of polysaccharide intercellular adhesin (PIA), a well-characterized, robust matrix molecule. Complementation of theaapmutant with full-length Aap (containing the A domain), but not the B domain alone, increased initial attachment to microtiter plates, as did intransexpression of the A domain in adhesion-deficientStaphylococcus carnosus. These results demonstrate Aap contributes toS. epidermidisinfection, which may in part be due to A domain-mediated attachment to abiotic surfaces.

A Single B-Repeat of Staphylococcus epidermidis Accumulation-Associated Protein Induces Protective Immune Responses in an Experimental Biomaterial-Associated Infection Mouse Model

ABSTRACTNosocomial infections are the fourth leading cause of morbidity and mortality in the United States, resulting in 2 million infections and ∼100,000 deaths each year. More than 60% of these infections are associated with some type of biomedical device.Staphylococcus epidermidisis a commensal bacterium of the human skin and is the most common nosocomial pathogen infecting implanted medical devices, especially those in the cardiovasculature.S. epidermidisantibiotic resistance and biofilm formation on inert surfaces make these infections hard to treat. Accumulation-associated protein (Aap), a cell wall-anchored protein ofS. epidermidis, is considered one of the most important proteins involved in the formation ofS. epidermidisbiofilm. A small recombinant protein vaccine comprising a single B-repeat domain (Brpt1.0) ofS. epidermidisRP62A Aap was developed, and the vaccine's efficacy was evaluatedin vitrowith a biofilm inhibition assay andin vivoin a murine model of biomaterial-associated infection. A high IgG antibody response againstS. epidermidisRP62A was detected in the sera of the mice after two subcutaneous immunizations with Brpt1.0 coadministered with Freund's adjuvant. Sera from Brpt1.0-immunized mice inhibitedin vitroS. epidermidisRP62A biofilm formation in a dose-dependent pattern. After receiving two immunizations, each mouse was surgically implanted with a porous scaffold disk containing 5 × 106CFU ofS. epidermidisRP62A. Weight changes, inflammatory markers, and histological assay results after challenge withS. epidermidisindicated that the mice immunized with Brpt1.0 exhibited significantly higher resistance toS. epidermidisRP62A implant infection than the control mice. Day 8 postchallenge, there was a significantly lower number of bacteria in scaffold sections and surrounding tissues and a lower residual inflammatory response to the infected scaffold disks for the Brpt1.0-immunized mice than for of the ovalbumin (Ova)-immunized mice.

Phenotypic and Genetic Analysis of Biofilm Formation by Staphylococcus epidermidis

Objective. The most important virulence factor of Staphylococcus epidermidis is their capability to form a biofilm on the surfaces of implanted medical devices. The accumulative phase of biofilm formation is linked to the production of intercellular adhesin encoded by the icaADBC operon and accumulation-associated protein by the aap gene. The aim of the study was to investigate biofilm formation phenotypically and genetically in clinical strains of S. epidermidis in comparison with commensal strains. Material and Methods. The study was carried out in 4 hospitals in Riga, Latvia. In total, 105 clinical strains of Staphylococcus epidermidis isolated from patients’ blood (n=67) and intravenous catheters (n=38) in a case of laboratory-confirmed bacteremia were studied. Moreover, 60 Staphylococcus epidermidis commensal strains isolated from nose epithelium of healthy people were included as a control group. Appearance of the icaA and aap genes was tested by polymerase chain reaction. The microtiter plate method was used. Results. Biofilm formation was detected in 50 (47%) of Staphylococcus epidermidis isolates in the clinical group and 15 (25%) of isolates in the control group (P=0.0049). Among 50 biofilmforming clinical isolates, 46 (92%) were positive for the icaA and/or aap genes. The icaA and aap genes were not found only in 4 strains. Conclusions. The clinical isolates of Staphylococcus epidermidis were more likely to form biofilms than the commensal strains. The carriage of the icaA or aap gene alone, or their absence, is not applicable as a molecular marker for the discrimination invasive Staphylococcus epidermidis strains from contaminants.