Infections involving Staphylococcus aureus are often more severe and difficult to treat when the organism assumes a biofilm mode of growth. The polysaccharide poly-N-acetylglucosamine (PNAG), also known as polysaccharide intercellular adhesin, is synthesized by the products of the intercellular adhesin (ica) locus and plays a key role in biofilm formation. Numerous conditions and exogenous factors influence ica transcription and PNAG synthesis, but the regulatory factors and pathways through which these environmental stimuli act have been only partially characterized. We developed a DNA affinity chromatography system to purify potential regulatory proteins that bind to the ica promoter region. Using this technique, we isolated four proteins, including the staphylococcal gene regulator SarA, a MarR family transcriptional regulator of the teicoplaninassociated locus TcaR, DNA-binding protein II, and topoisomerase IV, that bound to the ica promoter. Site-directed deletion mutagenesis of tcaR indicated that TcaR was a negative regulator of ica transcription, but deletion of tcaR alone did not induce any changes in PNAG production or in adherence to polystyrene. We also investigated the role of IcaR, encoded within the ica locus but divergently transcribed from the biosynthetic genes. As has been shown previously in Staphylococcus epidermidis, we found that IcaR was also a negative regulator of ica transcription in S. aureus. We also demonstrate that mutation of icaR augmented PNAG production and adherence to polystyrene. Transcription of the ica locus, PNAG production, and adherence to polystyrene were further increased in a tcaR icaR double mutant. In summary, TcaR appeared to be a weak negative regulator of transcription of the ica locus, whereas IcaR was a strong negative regulator, and in their absence PNAG production and biofilm formation were enhanced.

Poly-N-acetyl-glucosamine (PNAG) is a staphylococcal surface polysaccharide influencing biofilm formation that is also under investigation for its vaccine potential. Antibodies that bind to PNAG with either low (<15%) or high (>90%) levels of acetate are superior at opsonic and protective activity compared with antibodies that bind to PNAG with only high levels (>70%) of acetate. PNAG is synthesized by four proteins encoded within the intercellular adhesin (ica) locus icaADBC. In Staphylococcus epidermidis, icaB encodes a deacetylase needed for the surface retention of PNAG and optimal biofilm formation. In this study, we confirmed that icaB plays a similar role in Staphylococcus aureus and found that an icaB mutant of S. aureus expressed significantly less surface-associated PNAG, was highly susceptible to antibody-independent opsonic killing that could not be enhanced with antibody raised against deacetylated PNAG (dPNAG), and had reduced survival capacity in a murine model of bacteremia. In contrast, an icaB-overexpressing strain produced primarily surface-associated PNAG, was more susceptible to opsonophagocytosis with antibody to dPNAG, and had increased survival in a murine bacteremia model. The highly acetylated secreted PNAG was more effective at blocking opsonic killing mediated by a human monoclonal antibody (mAb) to native PNAG than it was at blocking killing mediated by a human mAb to dPNAG, which by itself was a more effective opsonin. Retention of dPNAG on the surface of S. aureus is key to increased survival during bacteremia and also provides a molecular mechanism explaining the superior opsonic and protective activity of antibody to dPNAG.Staphylococcus aureus and coagulase-negative staphylococci are the most frequent causes of nosocomial bloodstream infections (43). A critical virulence determinant in such infections is the production of a high-molecular-weight polymer of ␤-1-6-linked N-acetyl-glucosamine (PNAG) that is involved in adherence to polymeric substrates, bacterial intercellular adhesion, biofilm formation, and protection against antibodyindependent opsonic killing (3,4,15,26,40). Proteins encoded by the icaADBC genes of the intercellular adhesin (ica) locus synthesize PNAG (6, 10-12, 30, 31). IcaA is a trans-membrane glucosyltransferase and can synthesize short PNAG polymers in vitro using UDP-N-acetyl-glucosamine as a substrate (10). IcaD increases the biosynthetic efficiency of IcaA (10). IcaC is also a transmembrane protein and appears to be involved in linking short polymers to make longer oligomers of PNAG (10). In its mature form, PNAG, also referred to by some researchers as polysaccharide intercellular adhesin (PIA) (12,44), is a mixture of polymers such that about 10 to 20% of the amino groups are not acetylated, which could give some of the polymers within the polysaccharide complex a net positive charge (16,27). It is not known whether PNAG consists of a minority of highly deacetylated molecules mixed in with a majority of highly acetylated molecules or if there is a rang...

Objective: To determine brain magnetic resonance imaging (MRI) measures of cerebrospinal fluid (CSF) and whole brain volume of full-term and premature infants following surgical treatment for thoracic non-cardiac congenital anomalies requiring critical care. Methods: Full-term ( n = 13) and pre-term ( n = 13) patients with long-gap esophageal atresia, and full-term naïve controls ( n = 19) < 1 year corrected age, underwent non-sedated brain MRI following completion of thoracic non-cardiac surgery and critical care treatment. Qualitative MRI findings were reviewed and reported by a pediatric neuroradiologist and neurologist. Several linear brain metrics were measured using structural T1-weighted images, while T2-weighted images were required for segmentation of total CSF and whole brain tissue using the M orphologically A daptive N eonatal T issue S egmentation ( MANTiS ) tool. Group differences in absolute (mm, cm 3 ) and normalized (%) data were analyzed using a univariate general linear model with age at scan as a covariate. Mean normalized values were assessed using one-way ANOVA. Results: Qualitative brain findings suggest brain atrophy in both full-term and pre-term patients. Both linear and volumetric MRI analyses confirmed significantly greater total CSF and extra-axial space, and decreased whole brain size in both full-term and pre-term patients compared to naïve controls. Although linear analysis suggests greater ventricular volumes in all patients, volumetric analysis showed that normalized ventricular volumes were higher only in premature patients compared to controls. Discussion: Linear brain metrics paralleled volumetric MRI analysis of total CSF and extra-axial space, but not ventricular size. Full-term infants appear to demonstrate similar brain vulnerability in the context of life-saving thoracic non-cardiac surgery requiring critical care as premature infants.