CcpA Is Important for Growth and Virulence of Enterococcus faecium

Abstract

The collagen adhesin Acm was the first virulence determinant reported to be important for the pathogenesis of Enterococcus faecium in a rat infective endocarditis model. We had previously reported that there was a slight growth delay associated with acm allelic replacement (cat) mutant strain TX6051 used in that study. Recently, we generated a nonpolar markerless acm deletion mutant and did not observe a delay in growth. We therefore performed comparative genome sequence analysis of wild-type strain TX82 and TX6051 and found a single mutation, a nonsense mutation in the ccpA gene of TX6051. After correcting this mutation, the growth defect of TX6051 was abolished, implicating a role for CcpA in the growth of E. faecium. To confirm this, we created a ccpA deletion mutant of TX82, which also exhibited a slight delay in growth. Furthermore, the ccpA deletion mutant was attenuated (P ‫؍‬ 0.0024) in a mixed-inoculum (TX82 plus TX82 ⌬ccpA) rat endocarditis model and also in an in vitro competitive growth assay; a ccpA-complemented strain showed neither reduced growth nor reduced virulence. We also found attenuation in the endocarditis model with the new acm deletion mutant although not as great as that previously observed with TX6051 carrying the ccpA mutation. Taken together, our data confirm the role of Acm in the pathogenesis of endocarditis. We also show that CcpA affects the growth of E. faecium, that an intact ccpA gene is important for full virulence, and that a ccpA mutation was partly responsible for the highly attenuated phenotype of TX6051. Enterococcus faecium has become one of the most problematic causes of nosocomial infections in the United States over the last 2 decades and is found as a cause of various infections, including endocarditis, bacteremia in neutropenic patients, urinary tract infections, and transplant-and device-associated infections (1). A recent survey indicated that slightly more than one-third of the hospital-associated infections caused by enterococci can be attributed to E. faecium when the organisms were identified to the species level (2). Infections with multidrug-resistant E. faecium strains are a major clinical threat due to limited treatment options. The emergence of resistance to ampicillin and the subsequent acquisition of resistance to vancomycin have been associated with the progression of this harmless commensal organism into an important nosocomial pathogen in the United State (3-5). In addition, E. faecium isolates recovered from hospital-associated infections (HA clade or subclade A1) (4, 6) are also characterized by the presence of many putative or confirmed virulence-associated traits, including the expression of collagen adherence mediated by Acm (adhesin of collagen from E. faecium) (7), Esp (enterococcal surface protein) (8), a plasmid encoding a hyaluronidase-like protein (Hyl fm , now annotated family 84 glycosyltransferase) (9), MSCRAMMs (microbial surface component recognizing adhesive matrix molecules) (10, 11), EmpABC (forming E. faecium pili [previously kno...

The Fibronectin-Binding Protein Fnm Contributes to Adherence to Extracellular Matrix Components and Virulence of Enterococcus faecium

“…The reintroduction of fnm into its original chromosomal location resulted in a fully restored wild-type phenotype in 40% of the infected animals (data not shown); the genetic basis for the lack of restoration of Fnm function in all animals is under investigation in our laboratory. Interestingly, we have observed a similar "bimodal" pattern, with one or two animals showing the reverse of the results for the others, with several other enterococcal mutants tested previously (35,47,58).…”

Vancomycin-Resistant Enterococci

“…The role of Ace and Acm in supporting infection was shown in a rat model of IE, as deletion mutants in both species were found to be attenuated as compared to the wild type. 30,31 In addition, antibodies against Ace provided protection against developing IE in this same model. 31 Further, in community-associated isolates of E faecium, the acm allele is often a pseudogene, which does not express a functional product, resulting in a phenotype that is unable to bind collagen.…”

Stress Physiology of Lactic Acid Bacteria

“…An S. pneumoniae mutant lacking Rel, the major (p)ppGpp synthetase activating the stringent response, was severely attenuated and displayed a significantly altered course of disease progression in a murine pneumonia model (126). Deletion of ccpA in E. faecium also resulted in attenuated virulence in a rat endocarditis model (550). The levels of (p)ppGpp in E. faecalis correlated with stress responses, antibiotic resistance, and virulence (123,124,130).…”