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 biofilmproducing 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 and Salmonella 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 350 S. 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 model bap was involved in pathogenesis, causing a persistent infection.

The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalis isolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on the E. faecalis isolate, insertional mutagenesis of esp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboring esp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

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.

SummaryWe report the complete sequence of Staphylococcal pathogenicity island bovine 2 (SaPIbov2), encoding the biofilm-associated protein Bap. SaPIbov2 contains 24 open reading frames, including sip , which encodes a functional staphylococcal integrase protein. SaPIbov2 is bordered by 18 bp direct repeats. The integration site into the chromosome lies at the 3 ¢ ¢ ¢ ¢ end of a gene encoding GMP synthase. SaPIbov2 has extensive similarity to previously described pathogenicity islands of Staphylococcus aureus . The principal difference is that toxin genes present in the other pathogenicity islands are exchanged for a transposon-like element that carries the bap gene and genes encoding an ABC transporter and a transposase. Also, SaPIbov2 can be excised to form a circular element and can integrate site-specifically and RecA-independently at a chromosomal att site in a Sip-dependent manner. This was demonstrated both in S. aureus and with plasmid substrates ectopically in Escherichia coli . Thus, SaPIbov2 encodes a functional recombinase of the integrase family that promotes element excision and insertion/integration. In addition, we demonstrated that the presence of SaPIbov2 facilitated the persistence of S. aureus in an intramammary gland infection model. Finally, different bovine isolates of S. aureus were found to carry islands related to SaPIbov2, suggesting the existence of a family of related pathogenicity islands.

Sterol regulatory element binding proteins (SREBPs) regulate the expression of a number of enzymes, which catalyze the synthesis of fatty acids, cholesterol, triglycerides, and phospholipids. SREBP1c is the most relevant isoform in the adult liver, and its expression is controlled by the nutritional state. Transcriptional regulation studies into the SREBP1c gene, performed in the last few years, have improved our knowledge of the variability of signals that converge on its promoter region. Insulin, cholesterol derivatives, T3 and other endogenous molecules have been demonstrated to regulate the SREBP1c expression, particularly in rodents. The present study aimed to perform a detailed analysis of the human SREBP1c gene promoter structure in liver cells by focusing on responses to diverse metabolic signals. Serial deletion and mutation assays reveal that both SREBP (SRE) and LXR (LXRE) response elements are involved in SREBP1c transcription regulation mediated by insulin and cholesterol derivatives. We discovered that peroxisome proliferation-activated receptor alpha (PPAR␣) agonists enhance the activity of the SREBP1c promoter; a DR1 element, at ؊453 in the human promoter was involved in this activation. Moreover, PPAR␣ agonists act in cooperation with LXR or insulin to induce lipogenesis. Collectively, our results identify PPAR␣ as a novel regulatory factor in SREBP1c regulation which plays a relevant role in the interplay between lipids and insulin metabolic regulation.The prevalence of overweight and obesity is increasing worldwide at an alarming rate. An excess amount of body fat not only leads to reduced quality of life and immense healthcare-associated costs but also increases risk of death. Indeed, obesity has been related to a number of cardiovascular and metabolic disorders such as hypertension, type 2 diabetes, hyperinsulinemia, dyslipidemia, and atherosclerosis, all of them defining features of the metabolic syndrome. Beyond obesity and a number of independent factors, the other etiological factor of metabolic syndrome is insulin resistance, commonly considered to be of greater priority in pathogenesis (1, 2).The discovery of sterol regulatory element binding proteins (SREBPs) 4 was critical for our understanding of hepatic cholesterol homeostasis. SREBP1c, one of three SREBPs members of the basic helix-loop-helix family of transcription factors, is essential for the genomic actions of insulin on both carbohydrate and lipid metabolism (3) and plays a central role in the molecular biochemistry of metabolic syndrome. The SREBP1c expression is controlled by nutritional status. Fasting lowers SREBP1c mRNA and protein levels, whereas they are strongly induced in a fed state, followed by a compatible pattern of nutritional changes in lipogenic genes (4). Accordingly, changes in the activity of this transcription factor may be the key to linking insulin resistance with other obesity-associated metabolic disorders.Liver X receptors (LXRs) belong to the nuclear hormone receptor superfamily. The LXR subfamily co...