Use of in-biofilm expression technology to identify genes involved in Pseudomonas aeruginosa biofilm development

Mature Pseudomonas aeruginosa biofilms form complex three-dimensional architecture and are tolerant of antibiotics and other antimicrobial compounds. In this work, an in vivo expression technology system, originally designed to study virulence-associated genes in complex mammalian environments, was used to identify genes up-regulated in P. aeruginosa grown to a mature (5-day) biofilm. Five unique cloned promoters unable to promote in vitro growth in the absence of purines after recovery from the biofilm environment were identified. The open reading frames downstream of the cloned promoter regions were identified, and knockout mutants were generated. Insertional mutation of PA5065, a homologue of Escherichia coli ubiB, was lethal, while inactivation of PA0240 (a porin homologue), PA3710 (a putative alcohol dehydrogenase), and PA3782 (a homologue of the Streptomyces griseus developmental regulator adpA) had no effect on planktonic growth but caused defects in biofilm formation in static and flowing systems. In competition experiments, mutants demonstrated reduced fitness compared with the parent strain, comprising less than 0.0001% of total biofilm cells after 5 days. Therefore, using in-biofilm expression technology, we have identified novel genes that do not affect planktonic growth but are important for biofilm formation, development, and fitness.     

Abnormal phospholipid composition impairs HDL biogenesis and maturation in mice lacking Abca1

Recent studies have demonstrated that the ATP-binding cassette transporter A1 (ABCA1) facilitates the efflux of phospholipids and cholesterol to apoprotein acceptors, leading to the synthesis of HDL. The purpose of this study was to determine the changes in the lipoprotein fractions in Abca1-deficient mice and study the mechanisms responsible for the low levels of HDL when ABCA1 is absent. Plasma phospholipid concentration was decreased by more than 75%, mostly due to a reduction of phosphatidylcholine (PC) in HDL. Abca1(-/-) HDL represents less than 2% of wild-type levels and is smaller and enriched in phospholipids (11.2-fold more than HDL from controls). Compared to wild-type littermates, Abca1(-/-) HDL had a 4-fold increase in PC, whereas lysophosphatidylcholine (LPC) (125-fold), sphingomyelin (SPH) (49-fold), and phosphatidylethanolamine (PE) (18-fold) showed even higher increases. As a consequence, the ratios of LPC/PC, SPH/PC, PE/PC, and phosphatidylinositol + phosphatidylserine (PI+PS)/PC were all much higher in HDL from Abca1(-/-), compared to wild-type HDL. Plasma phospholipid transfer protein (PLTP) and lecithin cholesterol acyltransferase (LCAT) activities were decreased by more than 80%, suggesting that the maturation of HDL is affected. To test this hypothesis, plasma from Abca1(-/-) mice was incubated with CHO cells that are known to express high levels of ABCA1 with the intent of restoring the flux of phospholipid and cholesterol onto apoAI. Compared to native plasma, no change in maturation of HDL was observed. In contrast, a 220% increase in the formation of mature HDL was observed when ABCA1 function and LCAT activities were restored. Taken together, these observations suggest that ABCA1 is necessary for the adequate lipidation of apoAI, which enables the interaction with LCAT and subsequent maturation.     

Three rhamnosyltransferases responsible for assembly of the A-band D-rhamnan polysaccharide in Pseudomonas aeruginosa: a fourth transferase, WbpL, is required for the initiation of both A-band and B-band lipopolysaccharide synthesis

The Pseudomonas aeruginosa A-band lipopolysaccharide (LPS) molecule has an O-polysaccharide region composed of trisaccharide repeat units of α1 → 2, α1 → 3, α1 → 3 linked D -rhamnose (Rha). The A-band polysaccharide is assembled by the α-D -rhamnosyltransferases, WbpX, WbpY and WbpZ. WbpZ probably transfers the first Rha residue onto the A-band accepting molecule, while WbpY and WbpX subsequently transfer two α1 → 3 linked Rha residues and one α1 → 2 linked Rha respectively. The last two transferases are predicted to be processive, alternating in their activities to complete the A-band polymer. The genes coding for these transferases were identified at the 3′ end of the A-band biosynthetic cluster. Two additional genes, psecoA and uvrD, border the 3′ end of the cluster and are predicted to encode a co-enzyme A transferase and a DNA helicase II enzyme respectively. Chromosomal wbpX, wbpY and wbpZ mutants were generated, and Western immunoblot analysis demonstrates that these mutants are unable to synthesize A-band LPS, while B-band synthesis is unaffected. WbpL, a transferase encoded within the B-band biosynthetic cluster, was previously proposed to initiate B-band biosynthesis through the addition of Fuc2NAc (2-acetamido-2,6-dideoxy-D -galactose) to undecaprenol phosphate (Und-P). In this study, chromosomal wbpL mutants were generated that did not express A band or B band, indicating that WbpL initiates the synthesis of both LPS molecules. Cross-complementation experiments using WbpL and its homologue, Escherichia coli WecA, demonstrates that WbpL is bifunctional, initiating B-band synthesis with a Fuc2NAc residue and A-band synthesis with either a GlcNAc (N-acetylglucosamine) or GalNAc (N-acetylgalactosamine) residue. These data indicate that A-band polysaccharide assembly requires four glycosyltransferases, one of which is necessary for initiating both A-band and B-band LPS synthesis.     

Grouping of multiple-lentigines/LEOPARD and Noonan syndromes on the PTPN11 gene

Multiple-lentigines (ML)/LEOPARD (multiple lentigines, electrocardiographic-conduction abnormalities, ocular hypertelorism, pulmonary stenosis, abnormal genitalia, retardation of growth, and sensorineural deafness) syndrome is an autosomal dominant condition--characterized by lentigines and café au lait spots, facial anomalies, cardiac defects--that shares several clinical features with Noonan syndrome (NS). We screened nine patients with ML/LEOPARD syndrome (including a mother-daughter pair) and two children with NS who had multiple café au lait spots, for mutations in the NS gene, PTPN11, and found, in 10 of 11 patients, one of two new missense mutations, in exon 7 or exon 12. Both mutations affect the PTPN11 phosphotyrosine phosphatase domain, which is involved in <30% of the NS PTPN11 mutations. The study demonstrates that ML/LEOPARD syndrome and NS are allelic disorders. The detected mutations suggest that distinct molecular and pathogenetic mechanisms cause the peculiar cutaneous manifestations of the ML/LEOPARD-syndrome subtype of NS.     

Different signal transduction by epidermal growth factor may be responsible for the difference in modulation of amino acid transport between fetal and adult hepatocytes

[1-¹⁴C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP₃) and calcium, is concerned, substantial differences were found: EGF increased IP₃ production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP₃ and calcium transients, suggesting that this transduction pathway is not activated during fetal life.     

Enterocin 226NWC, a bacteriocin produced by Enterococcus faecalis 226, active against Listeria monocytogenes

F. VILLANI, G. SALZANO, E. SORRENTINO, O. PEPE, P. MARINO AND S. COPPOLA. 1993. Enterococcus faecalis 226, isolated from natural whey cultures utilized as starters in the manufacture of mozzarella cheese from water-buffalo milk, produces a bacteriocin designated enterocin 226NWC. The bacteriocin was isolated from culture supernatant fluids of the producer strain and was active against strains of the same species and Listeria monocytogenes, but not against useful lactic acid bacteria. Enterocin 226NWC is a protein with an apparent molecular weight of about 5800; it is relatively heat-stable and has a bactericidal mode of action. Listeria monocytogenes, growing in the presence of the enterocin 226NWC producer strain in broth and in reconstituted skim milk, was inhibited.     

Prevention of systemic lupus erythematosus in MRL/lpr mice by administration of an immunoglobulin-binding peptide

Systemic lupus erythematosus (SLE) is a multisystem chronic inflammatory disease of unknown etiology that affects many organs, including the kidney. The presence of multiple autoantibodies and other immunological abnormalities point to basic defects in immunoregulatory controls that normally maintain self-tolerance. The deposition on kidney tissue of autoantibodies as immune complexes (ICs) through the interaction with Fc-receptor gamma-chains is thought to trigger an inflammatory response typical of SLE, leading to glomerulonephritis. Using combinatorial chemistry approaches, we have identified a peptide able to bind to immunoglobulins and to interfere with Fcgamma-receptor recognition. Administration of this peptide to MRL/lpr mice, an animal model used to study SLE, resulted in a remarkable enhancement of the survival rate (80%) compared to placebo-treated animals (10%). Consistent with this was a significant reduction of proteinuria, a clinical sign of SLE. Kidney histological examination of treated animals confirmed the preservation of tissue integrity and a remarkable reduction in IC deposition. These results support the role of Fcgamma receptors in SLE pathogenesis and open new avenues for the development of drugs to treat autoimmune disorders.     

Human alpha-fetoprotein primary structure: a mass spectrometric study.

The amino acid sequence of human alpha-fetoprotein, a 67-kDa protein present in mammalian embryonic serum, was verified by fast atom bombardment mass spectrometric (FAB/MS) analyses of three different enzymatic digests of the protein. Human alpha-fetoprotein obtained from a large-scale cell culture was digested with trypsin and V-8 protease either separately on two different samples or combined on the same one. The V-8 protease digest of the protein was partially fractionated by HPLC; the other samples were directly analyzed by FAB/MS without previous purification steps. About 90% of the alpha-fetoprotein amino acid sequence was verified by mass spectrometric analysis; this also confirmed that the cell-derived protein is identical with the hepatoma-derived protein. FAB analysis revealed that the N terminus of the mature protein is arginine rather than threonine, with the threonine occupying the second position. Therefore, the processing site of the alpha-fetoprotein signal peptide during maturation of the protein occurs at the N-terminal side of the arginine residue formerly indicated as residue-1. Thus mature alpha-fetoprotein contains 591 amino acids rather than 590.     

Surgery for epilepsy. Selective partial microsurgical callosotomy for intractable multiform seizures: criteria for clinical selection and results

A series of 35 patients have been submitted to microsurgical callosotomy since February 1978, their age ranging from 6 to 42 years. The surgical procedure consisted either of a frontal parasagittal trephination or a larger frontoparietal rectangular craniotomy to allow the investigation of the surgical areas of the hemispheres with electrocorticography under local anesthesia. This allowed us to better follow the electrographic abnormalities and plan the extent of the callosal section suited to each case, in particular rostral and genual section, section of the trunk only or subtotal section sparing the splenium. In 2 cases, cortical resection was associated with the split. The callosal fibers which were divided by careful section under the resected portion provided a critical postoperative control of the extent of the lesion. Results and selection criteria will also be analyzed.