Biomedical subjects

Lucia Bertuccini

Publications and source records attributed to Lucia Bertuccini.

7 recordsLinked to original sources

PubMed2025

Structural obstruction to full DNA replication in terminally differentiated skeletal muscle cells.

Terminal cell differentiation is often associated with permanent withdrawal from proliferation, termed the postmitotic state. Though widespread among vertebrates and determinant for their biology, the molecular underpinnings of this state are poorly understood. Postmitotic skeletal muscle myotubes can be induced to reenter the cell cycle; however, they generally die as a result of their inability to complete DNA replication. Here, we explore the causes of such incompetence. Genomic hybridization of newly synthesized DNA shows that the replicative failure does not concern specific genomic regions, but can stochastically affect any of them. Myoblast and myotube nuclei are incubated in replicative Xenopus egg extract, which provides a full DNA replication machinery. While myoblast nuclei attain complete DNA replication, those from myotubes, even in these conditions, duplicate less than half of their genomes, strongly indicating that the structure of myotube chromatin obstructs DNA replication. Furthermore, disassembling and disorganizing chromatin with a strong salt treatment does not modify the replicative differences between the two types of nuclei, suggesting that they are rooted in the core structure of chromatin.

DNA Replication↗

PubMed2005

Glycosaminoglycans mediate invasion and survival of Enterococcus faecalis into macrophages.

Enterococcus faecalis is responsible for a large variety of nosocomial infections. The intestinal barrier is thought to be one of the preferential portals of entry of enterococci, and the ability of E. faecalis to survive within peritoneal macrophages may contribute to spreading to distant sites. We examined the ability of a polysaccharide-expressing (biofilm-positive) E. faecalis strain and an isogenic biofilm-negative mutant to enter and survive within professional and nonprofessional phagocytes. Biofilm-positive bacteria survived longer in all cell systems than did biofilm-negative bacteria, through a process of receptor-mediated endocytosis that is dependent on functional reorganization of microtubules and polymerization of microfilament and on activation of protein kinases but not ATPases or protein phosphatases. We suggest that glycosaminoglycans--specifically heparin, heparan sulfate, and chondroitin sulfate A--are the host receptors for enterococci on professional and, possibly, nonprofessional phagocytes, allowing entry of enterococci into cell compartments where killing mechanisms are inhibited.

Animals↗

PubMed2004

Internalization of non-toxigenic Corynebacterium diphtheriae by cultured human respiratory epithelial cells.

Although infection by Corynebacterium diphtheriae is a model of extracellular mucosal pathogenesis, and diphtheria is one of the most worried diseases, this microorganism can be associated also with invasive infections such as endocarditis, septic arthritis, and osteomyelitis. Invasive infections are usually caused by non-toxigenic C. diphtheriae strains. Over the last years severe pharyngitis/tonsillitis associated with the isolation of non-toxigenic C. diphtheriae have been described. Penicillin treatment failure of these infections could only partially be explained by penicillin tolerance of the causing strain. Thus, we examined the in vitro ability of non-toxigenic C. diphtheriae throat clinical isolates to adhere to, and enter human respiratory epithelial cells. Trasmission and scanning electron microscopy demonstrated intracellular C. diphtheriae in laryngeal (HEp-2 cells) and pharyngeal (Detroit D562 cells) tissue culture. Live intracellular bacteria were detectable up to 48 h post-infection. Using a variety of compound that act on eukariotic cell structures, the internalization of C. diphtheriae seems to occur via a zipper-like mechanism. It is likely that internalization of C. diphtheriae can be involved in throat colonization contributing to bacterial eradication failure and asymptomatic carriage.

Bacterial Adhesion↗

PubMed2004

A Sphingomonas bacterium interacting with epithelial cells.

Bacteria of the genus Sphingomonas are environmental organisms that have recently been implicated in a variety of community-acquired and nosocomial infections. During studies on bacteria-cell interactions, we incurred a microorganism contaminating our HeLa cell culture, possibly from water utilized for reagent preparation; this bacterium appeared to tightly adhere to cell monolayers and to survive, with only limited growth rate, which did not seem to alter cells as far as shape, growth rate or survival were concerned. The contaminating organism was isolated and partially characterized by morphological, genetic, and biochemical assays. Mechanisms of cell interaction and entry into epithelial cells were investigated by electron microscopy, immunofluorescence, and biochemical inhibitors. Morphological and biochemical features indicated that the microorganism belonged to the genus Sphingomonas. Electron microscopy showed that contact between the Sphingomonas bacterium and epithelial cells leads to a dramatic alteration of the cell surface, with formation of numerous microvillar extensions plus membrane ruffling. Confocal microscopy and the use of inhibitors showed that actin microfilaments were involved during attachment and entry into HeLa cells. Macropinosome formation and an inhibitory effect by amiloride indicate that internalization occurs in part via a macropinocytosis mechanism. Moreover, cholesterol distribution at the site of bacterial binding suggests that Sphingomonas bacteria could use the lipid rafts as initial binding sites.

Actins↗

PubMed2004

Survey for virulence determinants among Enterococcus faecalis isolated from different sources.

A collection of Enterococcus faecalis strains from clinical isolates, healthy individuals and the environment was screened for the presence of virulence factor genes, such as those for collagen-binding protein (ace), endocarditis antigen (efaA), haemolysin activator (cylA), gelatinase (gelE), aggregation substances (asa1 and asa373), a surface protein (esp) and two novel putative surface antigens (EF0591 and EF3314). Apart from some genes that were present in all strains (ace, efaA and EF3314), the gelE gene was the most common factor, although its presence did not correlate with its expression. The genes that encode Esp and CylA were never detected in endocarditis isolates, whereas an association was noted between the esp gene and isolates from urinary tract infection (UTI) and bacteraemia. An aggregation substance gene was always present in commensal strains. As for gelatinase, the presence of the cylA and asa genes did not correlate completely with their phenotypic expression. Generally, isolates from endocarditis, biliary stents and the environment were equipped with fewer virulence factors than isolates from other sources. UTI strains possessed the highest number of factors.

Adhesins, Bacterial↗

PubMed2004

Receptor-mediated endocytosis of biofilm-forming Enterococcus faecalis by rat peritoneal macrophages.

BACKGROUND & OBJECTIVES: Enterococci are important nosocomial pathogens that are increasingly difficult to treat due to intrinsic and acquired resistance to antibiotics. Studies were taken up to identify virulence factors and to characterise pathogenic mechanisms of such infections to evaluate potential targets for treatments alternative to antibiotic therapy. This study was carried out to evaluate the contribution of extracellular polysaccharide expressed by Enterococcus faecalis to resistance to phagocytosis and survival within rat peritoneal macrophages. METHODS: Six E. faecalis clinical isolates were tested for their ability to survive within rat peritoneal macrophages. Cytochalasin D, colchicine and monodansylcadaverine were used to investigate the route of enterococcal entry inside macrophages. RESULTS: Four of the isolates were able to produce extracellular polysaccharide and form biofilm after growth in glucose-supplemented medium, while no production could be detected in glucose deficient medium. Two isolates were polysaccharide-negative in both conditions. Isolates expressing extracellular polysaccharide were able to survive for more than 24 h compared to polysaccharide-negative bacterial cells of the same strain grown in glucose-deficient medium, which were readily cleared. Cytochalasin D virtually abolished the number of viable intracellular bacteria, after growth in either trypticase soy broth (TSB) or TSB supplemented with glucose; colchicine and monodansylcadaverine strongly affected survival of polysaccharide-positive bacteria, significantly more than that of polysaccharide-negative ones. INTERPRETATION & CONCLUSION: Biofilm-forming E. faecalis survived within rat peritoneal macrophages significantly better than polysaccharide-negative isolates. Perturbators of cytoskeleton and of surface receptors turnover, indicated receptors-mediated endocytosis as the most likely route for enterococcal entry into macrophages.

Animals↗

PubMed2002

Invasion of HeLa cells by Enterococcus faecalis clinical isolates.

We examined the in vitro ability of Enterococcus faecalis clinical isolates to adhere to and to invade HeLa cells, suggested to be a valuable model system to study bacteria-directed endocytosis. Using a variety of compounds that act on eukaryotic cell structures, both microtubules and microfilaments were found to be involved in enterococcal entry into cells. Two distinct modes of interaction were observed: in one, a close proximity of bacteria with the cell membrane was observed, possibly leading to direct engulfment of the bacterial cell. In the other mode, cellular pseudopodal formation seemed to be stimulated by vicinity of bacterial cells; in some cases, such associations involved formation of clathrin-coated-like vesicles before internalizing enterococci. The above-mentioned experimental data together with the use of monodansylcadaverine, amiloride and NH4Cl, all involved in cytosol acidification and inhibition of receptor-mediated endocytosis (RME), led us to conclude that E. faecalis is internalized within HeLa cells by more than one invasion pathway. One, sensitive to amiloride, is most likely a macropinocytic, actin-dependent uptake mechanism, which determines the production of large smooth-membrane vacuoles engulfing enterococci. The other is RME, in which entry is dependent on both microfilament and microtubule structural integrity.

Acids↗