Beneficial effects of hepatic cyclooxygenase-2 expression against cholestatic injury after common bile duct ligation in mice.

BACKGROUND AND AIMS: Cyclooxygenase-2 (COX-2) is involved in different liver diseases, but little is known about the significance of COX-2 in cholestatic injury. This study was designed to elucidate the role of COX-2 expression in hepatocytes during the pathogenesis of obstructive cholestasis. METHODS: We used genetically modified mice constitutively expressing human COX-2 in hepatocytes. Transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were either subjected to a mid-abdominal laparotomy or common bile duct ligation (BDL) for 2 or 5 days. Then, we explored the mechanisms underlying the role of COX-2 and its derived prostaglandins in liver function, and the synthesis and excretion of bile acids (BA) in response to cholestatic liver injury. RESULTS: After BDL, hCOX-2-Tg mice showed lower grades of hepatic necrosis and inflammation than Wt mice, in part by a reduced hepatic neutrophil recruitment associated with lower mRNA levels of pro-inflammatory cytokines. Furthermore, hCOX-2-Tg mice displayed a differential metabolic pattern of BA synthesis that led to an improved clearance after BDL-induced accumulation. In addition, an enhanced response to the BDL-induced oxidative stress and hepatic apoptosis was observed. In vitro experiments using hepatic cells that stably express hCOX-2 confirmed the cytoprotective role of prostaglandin E2 against BA toxicity. CONCLUSIONS: Taken together, our data indicate that constitutive expression of COX-2 in hepatocytes ameliorates cholestatic liver injury in mice by reducing inflammation and cell damage and by modulating BA metabolism, pointing to a role for COX-2 as a defensive response against cholestasis-derived BA accumulation and injury.

PBX1 acts as terminal selector for olfactory bulb dopaminergic neurons.

Neuronal specification is a protracted process that begins with the commitment of progenitor cells and culminates with the generation of mature neurons. Many transcription factors are continuously expressed during this process but it is presently unclear how these factors modify their targets as cells transition through different stages of specification. In olfactory bulb adult neurogenesis, the transcription factor PBX1 controls neurogenesis in progenitor cells and the survival of migrating neuroblasts. Here, we show that, at later differentiation stages, PBX1 also acts as a terminal selector for the dopaminergic neuron fate. PBX1 is also required for the morphological maturation of dopaminergic neurons and to repress alternative interneuron fates, findings that expand the known repertoire of terminal-selector actions. Finally, we reveal that the temporal diversification of PBX1 functions in neuronal specification is achieved, at least in part, through the dynamic regulation of alternative splicing. In Caenorhabditis elegans, PBX/CEH-20 also acts as a dopaminergic neuron terminal selector, which suggests an ancient role for PBX factors in the regulation of terminal differentiation of dopaminergic neurons.

Quantitative Proteomics Analysis Reveals That Cyclooxygenase-2 Modulates Mitochondrial Respiratory Chain Complex IV in Cardiomyocytes.

The biochemical mechanisms of cell injury and myocardial cell death after myocardial infarction remain unresolved. Cyclooxygenase 2 (COX-2), a key enzyme in prostanoid synthesis, is expressed in human ischemic myocardium and dilated cardiomyopathy, but it is absent in healthy hearts. To assess the role of COX-2 in cardiovascular physiopathology, we developed transgenic mice that constitutively express functional human COX-2 in cardiomyocytes under the control of the α-myosin heavy chain promoter. These animals had no apparent phenotype but were protected against ischemia-reperfusion injury in isolated hearts, with enhanced functional recovery and diminished cellular necrosis. To further explore the phenotype of this animal model, we carried out a differential proteome analysis of wild-type vs. transgenic cardiomyocytes. The results revealed a tissue-specific proteomic profile dominated by mitochondrial proteins. In particular, an increased expression of respiratory chain complex IV proteins was observed. This correlated with increased catalytic activity, enhanced respiratory capacity, and increased ATP levels in the heart of COX-2 transgenic mice. These data suggest a new link between COX-2 and mitochondria, which might contribute to the protective cardiac effects of COX-2 against ischemia-reperfusion injury.

Protective Role of Hepatocyte Cyclooxygenase-2 Expression Against Liver Ischemia-Reperfusion Injury in Mice.

Liver ischemia and reperfusion injury (IRI) remains a serious clinical problem affecting liver transplantation outcomes. IRI causes up to 10% of early organ failure and predisposes to chronic rejection. Cyclooxygenase-2 (COX-2) is involved in different liver diseases, but the significance of COX-2 in IRI is a matter of controversy. This study was designed to elucidate the role of COX-2 induction in hepatocytes against liver IRI. In the present work, hepatocyte-specific COX-2 transgenic mice (hCOX-2-Tg) and their wild-type (Wt) littermates were subjected to IRI. hCOX-2-Tg mice exhibited lower grades of necrosis and inflammation than Wt mice, in part by reduced hepatic recruitment and infiltration of neutrophils, with a concomitant decrease in serum levels of proinflammatory cytokines. Moreover, hCOX-2-Tg mice showed a significant attenuation of the IRI-induced increase in oxidative stress and hepatic apoptosis, an increase in autophagic flux, and a decrease in endoplasmic reticulum stress compared to Wt mice. Interestingly, ischemic preconditioning of Wt mice resembles the beneficial effects observed in hCOX-2-Tg mice against IRI due to a preconditioning-derived increase in endogenous COX-2, which is mainly localized in hepatocytes. Furthermore, measurement of prostaglandin E2 (PGE2 ) levels in plasma from patients who underwent liver transplantation revealed a significantly positive correlation of PGE2 levels and graft function and an inverse correlation with the time of ischemia. Conclusion: These data support the view of a protective effect of hepatic COX-2 induction and the consequent rise of derived prostaglandins against IRI.

Stable SREBP-1a knockdown decreases the cell proliferation rate in human preadipocyte cells without inducing senescence.

Sterol regulatory element binding proteins (SREBP), encoded by the Srebf1 and Srebf2 genes, are important regulators of genes involved in cholesterol and fatty acid metabolism. Whereas SREBP-2 controls the cholesterol synthesis, SREBP-1 proteins (-1a and -1c) function as the central hubs in lipid metabolism. Despite the key function of these transcription factors to promote adipocyte differentiation, the roles of SREBP-1 proteins during the preadipocyte state remain unknown. Here, we evaluate the role of SREBP-1 in preadipocyte proliferation using RNA interference technology. Knockdown of the SREBP-1a gene decreased the proliferation rate in human SGBS preadipocyte cell strain without inducing senescence. Furthermore, our data identified retinoblastoma binding protein 8 and cyclin-dependent kinase inhibitor 3 genes as new potential SREBP-1 targets, in addition to cyclin-dependent kinase inhibitor 1A which had already been described as a gene regulated by SREBP-1a. These data suggested a new role of SREBP-1 in adipogenesis via regulation of preadipocyte proliferation.

Identification of a novel Pfkfb1 mRNA variant in rat fetal liver.

The bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) catalyzes the synthesis and degradation of fructose-2,6-bisphosphate, a key metabolite in the glucose homeostasis. Four genes, Pfkfb1-4, have been characterized in mammals that code for several isoforms generated by alternative splicing through the control of several promoters and 5' non-coding exons. Here, we characterize in fetal rat liver new mRNA variants which are transcribed from a new Pfkfb1 gene promoter. The long variant codes to a new isoform (FL-PFK-2) that would be of relevant function to modulate the transition of fetal to adult liver metabolism.

Progression of liver oncogenesis in the double transgenic mice c-myc/TGF α is not enhanced by cyclooxygenase-2 expression.

Cyclooxygenase-2 (COX-2) has been associated with cell growth regulation, tissue remodeling and carcinogenesis. Overexpression of COX-2 in hepatocytes constitutes an ideal condition to evaluate the role of prostaglandins (PGs) in liver pathogenesis. The effect of COX-2-dependent PGs in genetic hepatocarcinogenesis has been investigated in triple c-myc/transforming growth factor α (TGF-α) transgenic mice that express human COX-2 in hepatocytes on a B6CBAxCD1xB6DBA2 background. Analysis of the contribution of COX-2-dependent PGs to the development of hepatocarcinogenesis, evaluated in this model, suggested a minor role of COX-2-dependent prostaglandins to liver oncogenesis as indicated by liver histopathology, morphometric analysis and specific markers of tumor progression. This allows concluding that COX-2 is insufficient for modifying the hepatocarcinogenesis course mediated by c-myc/TGF-α.

Advances in Understanding the Role of NRF2 in Liver Pathophysiology and Its Relationship with Hepatic-Specific Cyclooxygenase-2 Expression.

Oxidative stress and inflammation play an important role in the pathophysiological changes of liver diseases. Nuclear factor erythroid 2-related factor 2 (NRF2) is a transcription factor that positively regulates the basal and inducible expression of a large battery of cytoprotective genes, thus playing a key role in protecting against oxidative damage. Cyclooxygenase-2 (COX-2) is a key enzyme in prostaglandin biosynthesis. Its expression has always been associated with the induction of inflammation, but we have shown that, in addition to possessing other benefits, the constitutive expression of COX-2 in hepatocytes is beneficial in reducing inflammation and oxidative stress in multiple liver diseases. In this review, we summarized the role of NRF2 as a main agent in the resolution of oxidative stress, the crucial role of NRF2 signaling pathways during the development of chronic liver diseases, and, finally we related its action to that of COX-2, where it appears to operate as its partner in providing a hepatoprotective effect.

Human SREBP1c expression in liver is directly regulated by peroxisome proliferator-activated receptor alpha (PPARalpha).

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 (PPARα) agonists enhance the activity of the SREBP1c promoter; a DR1 element, at -453 in the human promoter was involved in this activation. Moreover, PPARα agonists act in cooperation with LXR or insulin to induce lipogenesis. Collectively, our results identify PPARα as a novel regulatory factor in SREBP1c regulation which plays a relevant role in the interplay between lipids and insulin metabolic regulation.

Transgenic mice expressing cyclooxygenase-2 in hepatocytes reveal a minor contribution of this enzyme to chemical hepatocarcinogenesis.

Cyclooxygenase-2 (COX-2) has been associated with cell growth regulation, tissue remodeling, and carcinogenesis. Ectopic expression of COX-2 in hepatocytes constitutes a nonphysiological condition ideal for evaluating the role of prostaglandins (PGs) in liver pathogenesis. The effect of COX-2-dependent PGs in chronic liver disease, hepatitis, fibrosis, and chemical hepatocarcinogenesis, has been investigated in transgenic (Tg) mice that express human COX-2 in hepatocytes and in Tg hepatic human cell lines. We have used three different complementary approaches: i) diethylnitrosamine (DEN)-induced chemical hepatocarcinogenesis in COX-2 Tg mice, ii) DEN/phenobarbital treatment of human COX-2 Tg hepatocyte-like cells, and iii) COX-2 Tg hepatocyte-like cells implants in nude mice. The data suggest that PGs produced by COX-2 in hepatocytes promoted mild hepatitis in 60-week-old mice, as assessed by histological examination, but failed to contribute to the development of liver fibrogenesis after methionine- and choline-deficient diet treatment. Moreover, liver injury, collagen content, and hepatic stellate cell activation were equally severe in wild-type and COX-2 Tg mice. The contribution of COX-2-dependent PGs to the development of DEN-induced hepatocarcinogenesis was evaluated in Tg mice, Tg hepatocyte-like cells, and nude mice and the analysis revealed that COX-2 expression favors the development of preneoplastic foci without affecting malignant transformation. Endogenous COX-2 expression in wild-type mice is a late event in the development of hepatocellular carcinoma.

COX-2 Expression in Hepatocytes Improves Mitochondrial Function after Hepatic Ischemia-Reperfusion Injury.

Cyclooxygenase 2 (COX-2) is a key enzyme in prostanoid biosynthesis. The constitutive hepatocyte expression of COX-2 has a protective role in hepatic ischemia-reperfusion (I/R) injury (IRI), decreasing necrosis, reducing reactive oxygen species (ROS) levels, and increasing autophagy and antioxidant and anti-inflammatory response. The physiopathology of IRI directly impacts mitochondrial activity, causing ATP depletion and being the main source of ROS. Using genetically modified mice expressing human COX-2 (h-COX-2 Tg) specifically in hepatocytes, and performing I/R surgery on the liver, we demonstrate that COX-2 expression has a beneficial effect at the mitochondrial level. Mitochondria derived from h-COX-2 Tg mice livers have an increased respiratory rate associated with complex I electron-feeding pathways compared to Wild-type (Wt) littermates, without affecting complex I expression or assembly. Furthermore, Wt-derived mitochondria show a loss of mitochondrial membrane potential (ΔΨm) that correlates to increased proteolysis of fusion-related OPA1 through OMA1 protease activity. All these effects are not observed in h-COX-2 Tg mitochondria, which behave similarly to the Sham condition. These results suggest that COX-2 attenuates IRI at a mitochondrial level, preserving the proteolytic processing of OPA1, in addition to the maintenance of mitochondrial respiration.

Characterization of the human insulin-induced gene 2 (INSIG2) promoter: the role of Ets-binding motifs.

Insulin-induced gene 2 (INSIG2) and its homolog INSIG1 encode closely related endoplasmic reticulum proteins that regulate the proteolytic activation of sterol regulatory element-binding proteins, transcription factors that activate the synthesis of cholesterol and fatty acids in animal cells. Several studies have been carried out to identify INSIG2 genetic variants associated with metabolic diseases. However, few data have been published regarding the regulation of INSIG2 gene expression. Two Insig2 transcripts have been described in rodents through the use of different promoters that produce different noncoding first exons that splice into a common second exon. Herein we report the cloning and characterization of the human INSIG2 promoter and the detection of an INSIG2-specific transcript homologous to the Insig2b mouse variant in human liver. Deletion analyses on 3 kb of 5'-flanking DNA of the human INSIG2 gene revealed the functional importance of a 350-bp region upstream of the transcription start site. Mutated analyses, chromatin immunoprecipitation assays, and RNA interference analyses unveiled the significance of an Ets-consensus motif in the proximal region and the interaction of the Ets family member SAP1a (serum response factor (SRF) accessory protein-1a) with this region of the human INSIG2 promoter. Moreover, our findings suggest that insulin activated the human INSIG2 promoter in a process mediated by phosphorylated SAP1a. Overall, these results map the functional elements in the human INSIG2 promoter sequence and suggest an unexpected regulation of INSIG2 gene expression in human liver.

Hepatic COX-2 expression protects mice from an alcohol-high fat diet-induced metabolic disorder by involving protein acetylation related energy metabolism.

PURPOSE: A diet high in fat and ethanol often results in chronic metabolic disorder, hepatic steatosis, and liver inflammation. Constitutive hepatic cyclooxygenase-2 (COX-2) expression could protect from high fat-induced metabolism disturbance in a murine model. In this study, we explored the influence of hCOX-2 transgenic [TG] to high fat with ethanol-induced metabolic disorder and liver injury using a mouse animal model. METHODS: 12-week-old male hepatic hCOX-2 transgenic (TG) or wild type mice (WT) were fed either a high fat and ethanol liquid diet (HF+Eth) or a regular control diet (RCD) for 5 weeks (four groups: RCD/WT, RCD/TG; HF+Eth/TG, HF+Eth/WT). We assessed metabolic biomarkers, cytokine profiles, histomorphology, and gene expression to study the impact of persistent hepatic COX-2 expression on diet-induced liver injury. RESULTS: In the HF+Eth diet, constitutively hepatic human COX-2 expression protects mice from body weight gain and white adipose tissue accumulation, accompanied by improved IPGTT response, serum triglyceride/cholesterol levels, and lower levels of serum and liver inflammatory cytokines. Histologically, hCOX-2 mice showed decreased hepatic lipid droplets accumulation, decreased hepatocyte ballooning, and improved steatosis scores. Hepatic hCOX-2 overexpression enhanced AKT insulin signaling and increased fatty acid synthesis in both RCD and HF+Eth diet groups. The anti-lipogenic effect of hCOX-2 TG in the HF+Eth diet animals was mediated by increasing lipid disposal through enhanced ß-oxidation via elevations in the expression of PPARα and PPARγ, and increased hepatic autophagy as assessed by the ratio of autophagy markers LC3 II/I in hepatic tissue. Various protein acetylation pathway components, including HAT, HDAC1, SIRT1, and SNAIL1, were modulated in hCOX-2 TG mice in either RCD or HF+Eth diet. CONCLUSIONS: Hepatic human COX-2 expression protected mice from the metabolic disorder and liver injury induced by a high fat and ethanol diet by enhancing hepatic lipid expenditure. Epigenetic reprogramming of diverse metabolic genes might be involved in the anti-lipogenic effect of COX-2.

Hepatic cyclooxygenase-2 expression protects against diet-induced steatosis, obesity, and insulin resistance.

Accumulation evidence links obesity-induced inflammation as an important contributor to the development of insulin resistance, which plays a key role in the pathophysiology of obesity-related diseases such as type 2 diabetes and nonalcoholic fatty liver disease. Cyclooxygenase (COX)-1 and -2 catalyze the first step in prostanoid biosynthesis. Because adult hepatocytes fail to induce COX-2 expression regardless of the proinflammatory stimuli used, we have evaluated whether this lack of expression under mild proinflammatory conditions might constitute a permissive condition for the onset of insulin resistance. Our results show that constitutive expression of human COX-2 (hCOX-2) in hepatocytes protects against adiposity, inflammation, and, hence, insulin resistance induced by a high-fat diet, as demonstrated by decreased hepatic steatosis, adiposity, plasmatic and hepatic triglycerides and free fatty acids, increased adiponectin-to-leptin ratio, and decreased levels of proinflammatory cytokines, together with an enhancement of insulin sensitivity and glucose tolerance. Furthermore, hCOX-2 transgenic mice exhibited increased whole-body energy expenditure due in part by induction of thermogenesis and fatty acid oxidation. The analysis of hepatic insulin signaling revealed an increase in insulin receptor-mediated Akt phosphorylation in hCOX-2 transgenic mice. In conclusion, our results point to COX-2 as a potential therapeutic target against obesity-associated metabolic dysfunction.

Protection against Fas-induced liver apoptosis in transgenic mice expressing cyclooxygenase 2 in hepatocytes.

UNLABELLED: Cyclooxygenase-2 (COX-2) is upregulated in many cancers, and the prostanoids synthesized increase proliferation, improve angiogenesis, and inhibit apoptosis in several tissues. To explore the function of COX-2 in liver, transgenic (Tg) mice were generated containing a fusion gene (LIVhCOX-2) consisting of human COX-2 cDNA under the control of the human ApoE promoter. Six lines were developed; all of them expressed the LIVhCOX-2 transgene selectively in hepatocytes. The Tg mice exhibited a normal phenotype, and the increased levels of PGE2 found were due to the constitutively expressed COX-2. Histological analysis of different tissues and macroscopic examination of the liver showed no differences between wild-type (Wt) and Tg animals. However, Tg animals were resistant to Fas-mediated liver injury, as demonstrated by low levels of plasmatic aminotransferases, a lesser caspase-3 activation, and Bax levels and an increase in Bcl-2, Mcl-1, and xIAP proteins, when compared with the Wt animals. Moreover, the resistance to Fas-mediated apoptosis is suppressed in the presence of COX-2-selective inhibitors, which prevented prostaglandin accumulation in the liver of Tg mice. CONCLUSION: These results demonstrate that expression of COX-2-dependent prostaglandins exerted a protection against liver apoptosis.

Phase-variable expression of the biofilm-associated protein (Bap) in Staphylococcus aureus.

A process of phase variation is described that affects the expression of Bap (biofilm-associated protein) in Staphylococcus aureus. Upon subculture of the Bap-positive S. aureus strain V329 on Congo red agar, spontaneous smooth biofilm-negative colonies appeared at a low frequency (5 x 10(-4)). Northern blot analysis of these variants with a bap-specific gene probe showed that transcription of the bap gene did not occur. However, DNA typing, Southern blot hybridization and DNA sequencing did not show any differences between the parent V329 strain and the biofilm-negative variants. The biofilm-negative phenotype reverted to wild-type at a similar frequency upon subculture of Bap-negative variants in liquid media. Experimental infection of ovine mammary glands with Bap-negative variants showed that phase variation occurred in vivo, because Bap-expressing, biofilm-positive revertants were isolated from infected mammary glands. The absence of Bap correlated with increased adherence to fibrinogen and fibronectin. It is possible that S. aureus can detach from a biofilm by switching to a Bap-negative state.

Sip, an integrase protein with excision, circularization and integration activities, defines a new family of mobile Staphylococcus aureus pathogenicity islands.

We 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.

Expression of the biofilm-associated protein interferes with host protein receptors of Staphylococcus aureus and alters the infective process.

The adherence of Staphylococcus aureus to soluble proteins and extracellular-matrix components of the host is one of the key steps in the pathogenesis of staphylococcal infections. S. aureus presents a family of adhesins called MSCRAMMs (microbial surface components recognizing adhesive matrix molecules) that specifically recognize host matrix components. We examined the influence of biofilm-associated protein (Bap) expression on S. aureus adherence to host proteins, epithelial cell cultures, and mammary gland sections and on colonization of the mammary gland in an in vivo infection model. Bap-positive strain V329 showed lower adherence to immobilized fibrinogen and fibronectin than isogenic Bap-deficient strain m556. Bacterial adherence to histological sections of mammary gland and bacterial internalization into 293 cells were significantly lower in the Bap-positive strains. In addition, the Bap-negative strain showed significantly higher colonization in vivo of sheep mammary glands than the Bap-positive strain. Taken together, these results strongly suggest that the expression of the Bap protein interferes with functional properties of the MSCRAMM proteins, preventing initial bacterial attachment to host tissues and cellular internalization.

Role of biofilm-associated protein bap in the pathogenesis of bovine Staphylococcus aureus.

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.