Biomedical subjects

Paula J Fernandes

Publications and source records attributed to Paula J Fernandes.

5 recordsLinked to original sources

PubMed2006

Interaction and localization studies of enteropathogenic Escherichia coli type IV bundle-forming pilus outer membrane components.

Typical enteropathogenic Escherichia coli strains express an established virulence factor belonging to the type IV pili family, called the bundle-forming pilus (BFP). BFP are present on the cell surface as bundled filamentous appendages, and are assembled and retracted by proteins encoded by the bfp operon. These proteins assemble to form a molecular machine. The BFP machine may be conceptually divided into three components: the cytoplasmic membrane (CM) subassembly, which is composed of CM proteins and cytoplasmic nucleotide-binding proteins; the outer membrane (OM) subassembly and the pilus itself. The authors have previously characterized the CM subassembly and the pilus. In this study, a more complete characterization of the OM subassembly was carried out using a combination of biochemical, biophysical and genetic approaches. It is reported that targeting of BfpG to the OM was influenced by the secretin BfpB. BfpG and BfpU interacted with the amino terminus of BfpB. BfpU had a complex cellular distribution pattern and, along with BfpB and BfpG, was part of the OM subassembly.

Amino Acid Sequence↗

PubMed2005

Structure of the bundle-forming pilus from enteropathogenic Escherichia coli.

Bundle-forming pili (BFP) are essential for the full virulence of enteropathogenic Escherichia coli (EPEC) because they are required for localized adherence to epithelial cells and auto-aggregation. We report the high resolution structure of bundlin, the monomer of BFP, solved by NMR. The structure reveals a new variation in the topology of type IVb pilins with significant differences in the composition and relative orientation of elements of secondary structure. In addition, the structural parameters of native BFP filaments were determined by electron microscopy after negative staining. The solution structure of bundlin was assembled according to these helical parameters to provide a plausible atomic resolution model for the BFP filament. We show that EPEC and Vibriocholerae type IVb pili display distinct differences in their monomer subunits consistent with data showing that bundlin and TcpA cannot complement each other, but assemble into filaments with similar helical organization.

Amino Acid Sequence↗

PubMed2005

Citrobacter rodentium lifA/efa1 is essential for colonic colonization and crypt cell hyperplasia in vivo.

Previously, we have identified a large gene (lifA, for lymphocyte inhibitory factor A) in enteropathogenic Escherichia coli (EPEC) encoding a protein termed lymphostatin that suppresses cytokine expression in vitro. This protein also functions as an adhesion factor for enterohemorrhagic E. coli (EHEC) and Shiga toxin-producing E. coli and is alternatively known as efa1 (EHEC factor for adherence 1). The lifA/efa1 gene is also present in Citrobacter rodentium, an enteric pathogen that causes a disease termed transmissible murine colonic hyperplasia (TMCH), which induces colitis and massive crypt cell proliferation, in mice. To determine if lifA/efa1 is required for C. rodentium-induced colonic pathology in vivo, three in-frame mutations were generated, disrupting the glycosyltransferase (GlM12) and protease (PrMC31) motifs and a domain in between that does not encode any known activity (EID3). In contrast to infection with wild-type C. rodentium, that with any of the lifA/efa1 mutant strains did not induce weight loss or TMCH. Enteric infection with motif mutants GlM12 and PrM31 resulted in significantly reduced colonization counts during the entire 20-day course of infection. In contrast, EID3 was indistinguishable from the wild type during the initial colonic colonization, but cleared rapidly after day 8 of the infection. The colonic epithelium of all infected mice displayed increased epithelial regeneration. However, significantly increased regeneration was observed by day 20 only in mice infected with the wild-type in comparison to those infected with lifA/efa1 mutant EID3. In summary, lifA/efa1 is a critical gene outside the locus for enterocyte effacement that regulates bacterial colonization, crypt cell proliferation, and epithelial cell regeneration.

Animals↗

PubMed2003

Adhesion of enteropathogenic Escherichia coli to host cells.

Enteropathogenic Escherichia coli (EPEC) adhere to the intestinal mucosa and to tissue culture cells in a distinctive fashion, destroying microvilli, altering the cytoskeleton and attaching intimately to the host cell membrane in a manner termed the attaching and effacing effect. Typical EPEC strains also form three-dimensional microcolonies in a pattern termed localized adherence. Attaching and effacing, and in particular intimate attachment requires an outer membrane adhesin called intimin, which binds to the translocated intimin receptor, Tir. Tir is produced by the bacteria and delivered to the host cell via a type III secretion system. In addition to this well-established adhesin-receptor pair, numerous other adhesin interactions between EPEC and host cells have been described including those between intimin and cellular receptors and those involving a bundle-forming pilus and flagella and unknown receptors. Much additional work is needed before a full understanding of EPEC adhesion to host cells comes to light.

Adhesins, Bacterial↗

PubMed2001

Construction of Rhodococcus random mutagenesis libraries using Tn5 transposition complexes.

The ability to generate tagged mutants of Rhodococcus spp. will facilitate a deeper understanding of this medically and commercially important genus. The absence of efficient transposon systems in these organisms has here been overcome by the use of Tn5-based DNA-protein transposition complexes which can transpose at high efficiency. To achieve this, electroporation efficiencies and antibiotic selection were optimized. A Rhodococcus rhodochrous CW25 Tn5 insertion library of 1500 mutants was created. Southern blotting of 23 representative mutants demonstrated random insertion. A number of auxotrophic mutants were isolated and the disrupted regions involved were identified by inverse PCR and subsequent sequencing. Transposition of Tn5 was confirmed by the presence of 9 bp direct repeats of Rhodococcus DNA flanking the transposon insertion site. To further test this system, a Tn5 insertion library was constructed in a wild-type soil isolate of Rhodococcus spp. This is the first viable transposon knockout system reported for Rhodococcus.

Base Sequence↗