Library Screen Identifies Enterococcus faecalis CcpA, the Catabolite Control Protein A, as an Effector of Ace, a Collagen Adhesion Protein Linked to Virulence

Abstract

The Enterococcus faecalis cell wall-anchored protein Ace is an important virulence factor involved in cell adhesion and infection. Expression of Ace on the cell surface is affected by many factors, including stage of growth, culture temperature, and environmental components, such as serum, urine, and collagen. However, the mechanisms that regulate or modulate Ace display are not well understood. With interest in identifying genes associated with Ace expression, we utilized a whole-cell enzyme-linked immunosorbent assay (ELISA)-based screening method to identify mutants from a transposon insertion mutant library which exhibited distinct Ace surface expression profiles. We identified a ccpA insertion mutant which showed significantly decreased levels of Ace surface expression at early growth phase versus those of wild-type OG1RF. Confirmation of the observation was achieved through flow cytometry and complementation analysis. Compared to the wild type, the E. faecalis ccpA mutant had an impaired ability to adhere to collagen when grown to early exponential phase, consistent with the lack of Ace expression in the early growth phase. As a key component of carbon catabolite regulation, CcpA has been previously reported to play a critical role in regulating expression of proteins involved in E. faecalis carbohydrate uptake and utilization. Our discovery is the first to associate CcpA with the production of a major E. faecalis virulence factor, providing new insights into the regulation of E. faecalis pathogenesis. Enterococcus faecalis, a commensal organism of the gastrointestinal tract, is also known to be an opportunistic pathogen, a major cause of hospital-acquired infections, and a growing public health concern due to its increasing resistance to multiple antibiotics. A group of surface proteins, the microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), play an important role in the virulence of E. faecalis by encouraging adherence and colonization to host tissue. Among them, Ace (adhesin of collagen from E. faecalis) plays an important role in adherence, presumably by mediating binding to extracellular matrix proteins, including both collagen and laminin (1). Ace has significant sequence homology with and a structural organization similar to that of Cna, a known collagen binding virulence factor of Staphylococcus aureus (2), and homology to Acm, a collagen adhesion protein in Enterococcus faecium (3). The association of Ace expression with virulence has been reported in many publications, including recent reports that an ace deletion mutant was significantly attenuated in both a rat endocarditis model and a murine urinary tract infection model, suggesting that Ace is involved in the pathogenesis of E. faecalis in both infectious endocarditis and urinary tract infections (4-6).The surface expression of Ace has been shown to be regulated by many factors, including growth phase, temperature, and medium components, such as serum and collagen. As previously reported, Ace is maximally displayed...

CcpA Is Important for Growth and Virulence of Enterococcus faecium

“…CcpA has been linked to biofilm formation in many bacteria, including S. aureus, Staphylococcus epidermidis, Bacillus subtilis, and Clostridium perfringens (36)(37)(38)(39). Interestingly, CcpA-dependent regulation has also been demonstrated for the expression of many virulence factors in Gram-positive bacterial pathogens, including S. aureus, Bacillus anthracis, group A streptococci, and E. faecalis (30,(40)(41)(42)(43). Moreover, deletion of ccpA led to decreased virulence of S. pneumoniae in a pneumonia model and reduced colonization of the nasopharynx (41).…”

Regulating the Intersection of Metabolism and Pathogenesis in Gram-positive Bacteria

“…In E. faecalis , CcpA activates expression of a collagen-binding colonization factor (124). In Listeria monocytogenes , effects of carbon availability on virulence gene expression are mediated by HPr independently of CcpA.…”

The Two-Component System GrvRS (EtaRS) Regulates ace Expression in Enterococcus faecalis OG1RF

“…Previous works have identified several environmental factors regulating ace expression; e.g., transcription of ace was increased when E. faecalis was grown at 46°C and grown in the presence of 40% horse serum, urine, and bile salts (5,8,9). In addition, levels of Ace on the cell surface are dependent on the E. faecalis strain and growth phase (10)(11)(12). With E. faecalis OG1RF, Ace is increased in the early exponential phase but reduced in the stationary phase; however, with E. faecalis JH2-2, it is maintained in later growth phases (10)(11)(12).…”

“…In E. faecalis OG1RF, this regulator does not seem to play a role, as deletion of ers did not affect ace expression under the various tested conditions (13). Deletion of ccpA encoding the transcriptional regulator CcpA (catabolite control protein A) from OG1RF resulted in significantly decreased levels of Ace surface expression in the early growth phase and an impaired ability to adhere to collagen in comparison to the wild-type (12). However, transcriptional levels of ace were similar in both OG1RF and the ccpA mutant, indicating that CcpA is not directly involved in regulating ace transcription.…”

“…In addition, levels of Ace on the cell surface are dependent on the E. faecalis strain and growth phase (10)(11)(12). With E. faecalis OG1RF, Ace is increased in the early exponential phase but reduced in the stationary phase; however, with E. faecalis JH2-2, it is maintained in later growth phases (10)(11)(12). The decrease in stationary-phase Ace in strain OG1RF was shown to be dependent on a functional fsr quorum-sensing system controlling the expression of gelatinase (GelE), which cleaves Ace from the OG1RF cell surface in latephase cultures (11).…”