While originally characterized as a collection of related syndromes, cystic fibrosis (CF) is now recognized as a single disease whose diverse symptoms stem from the wide tissue distribution of the gene product that is defective in CF, the ion channel and regulator, cystic fibrosis transmembrane conductance regulator (CFTR). Defective CFTR protein impacts the function of the pancreas and alters the consistency of mucosal secretions. The latter of these effects probably plays an important role in the defective resistance of CF patients to many pathogens. As the modalities of CF research have changed over the decades from empirical histological studies to include biophysical measurements of CFTR function, the clinical management of this disease has similarly evolved to effectively address the ever-changing spectrum of CF-related infectious diseases. These factors have led to the successful management of many CF-related infections with the notable exception of chronic lung infection with the gram-negative bacterium Pseudomonas aeruginosa. The virulence of P. aeruginosa stems from multiple bacterial attributes, including antibiotic resistance, the ability to utilize quorum-sensing signals to form biofilms, the destructive potential of a multitude of its microbial toxins, and the ability to acquire a mucoid phenotype, which renders this microbe resistant to both the innate and acquired immunologic defenses of the host

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The long-term antimicrobial efficacy of silver dressings against bacterial biofilms was investigated in a 7-day treatment in vitro model where the protein-rich medium was refreshed daily in order to mimic the conditions found in a wound bed. The use of plate-to-plate transfer assays demonstrated measurable differences in the effectivenesses of several silver dressings on the viability of biofilm bacteria and their susceptibility to antibiotics. Whereas after the first day of treatment, all dressings used resulted in a significant reduction in the number of viable cells in the biofilms and disruption of the biofilm colonies, during prolonged treatment, the efficacy of dressings with hydrophilic base materials diminished with daily transfers, and bacterial populations recovered. For dressings with hydrophobic base materials, the level of efficacy correlated with the silver species loaded. Biofilm bacteria, which survived the initial silver treatment, were susceptible to tobramycin, ciprofloxacin, and trimethoprim-sulfamethoxazole, in contrast to untreated biofilms, which were highly tolerant to the same antibiotics. This acquired susceptibility was unaffected by the longevity of pretreatment with the silver dressings but depended on the dressing used. The antimicrobial efficacy of the dressings correlated with the type of the dressing base material and silver species loaded.

The cystic fibrosis transmembrane conductance regulator (CFTR) protein is an epithelial receptor mediating the translocation of Salmonella enterica serovar Typhi to the gastric submucosa. Since the level of cell surface CFTR is directly related to the efficiency of serovar Typhi translocation, the goal of this study was to measure CFTR expression by the intestinal epithelium during infection. CFTR protein initially present in the epithelial cell cytoplasm was rapidly trafficked to the plasma membrane following exposure to live serovar Typhi or bacterial extracts. CFTR-dependent bacterial uptake by epithelial cells increased (>100-fold) following CFTR redistribution. The bacterial factor which triggers CFTR redistribution is heat and protease sensitive. These data suggest that serovar Typhi induces intestinal epithelial cells to increase membrane CFTR levels, leading to enhanced bacterial ingestion and submucosal translocation. This could be a key, early step in the infectious process leading to typhoid fever.Salmonella enterica serovar Typhi causes typhoid or enteric fever and initiates infection by entering the enterocytes and specialized M cells of the small intestine, after which the bacterium is translocated to the intestinal submucosa and ingested by host macrophages. The mechanism by which serovar Typhi enters enterocytes is complex and involves intercellular adhesins (5) and intracellular signaling components (18) which modify the host cell cytoskeletal (6) and vacuolar (7) organization. This process is initiated when adhesins and signaling components are delivered into the host cell by the bacterium via the bacterial type III secretion apparatus (4). Since assembly of the type III secretion apparatus is stimulated by bacterium-epithelial cell contact (9), early interactions between serovar Typhi and the epithelial cell play an important role in the initiation of infection.

Four human IgE isoforms produced by alternative splicing of the epsilon primary transcript were expressed as chimeric mouse/human anti 5-dimethylamino-1-naphthalenesulfonyl antibodies in the murine myeloma cell line Sp2/0. The four isoforms include the classic secreted form and three novel isoforms with altered carboxyl termini. All of these isoforms lack the transmembrane region encoded by the M1/M1 exon and are therefore predicted to be secreted proteins. When expressed in Sp2/0 cells, three of the IgE isoforms are assembled into complete molecules of two Ig heavy chains and two Ig light chains, whereas the fourth isoform is predominately assembled into half-molecules of one Ig heavy chain and one Ig light chain. All four isoforms are secreted with similar kinetics. In contrast, when the isoform containing the C⑀4 domain joined directly to the M2 exon (IgE grandé ) is expressed in the J558L cell line, it is degraded intracellularly, suggesting a cell line-dependent regulation of secretion. These data show that these novel isoforms of human IgE, predicted to occur from in vivo and in vitro mRNA analysis, can be produced and secreted by mammalian cells. The different forms of IgE may have physiologically relevant but distinct roles in human IgE-mediated immune inflammation. The availability of purified recombinant human IgE isoforms makes it possible to analyze the functional differences among them.Alternative RNA splicing determines the production of secreted versus membrane-bound forms of immunoglobulins (1, 2). This is accomplished in mammals by the alternative usage of either a secreted terminus at the end of the last constant region domain or two downstream exons (M1 and M2) that encode the transmembrane and intracellular amino acids. Splicing to the M exons removes from the transcript the nucleotides that encode the hydrophilic COOH terminus and polyadenylation signal for the smaller, secreted form of the Ig.The one functional genomic locus encoding human epsilon heavy chain contains four Ig domain exons (C⑀1 to C⑀4) and the two membrane exons (M1 and M2). We (3-5) and others (6, 7) have previously shown that RNA prepared from the IgE-producing human cell line AF-10 and from fresh B lymphocytes stimulated to make IgE contain a variety of epsilon mRNAs produced by alternative splicing. In contrast to what is observed with other isotypes, the most common form of mRNA encoding membrane IgE is produced by splicing to a novel splice acceptor 156 base pairs upstream of the normal M1 acceptor site (4, 7). The M1Ј exon produced using this splice acceptor encodes 52 novel amino acids that are largely hydrophilic followed by the amino acids normally encoded by M1.Other alternatively spliced epsilon mRNAs are present that encode a series of potentially secreted proteins. The splicing events that generate these mRNAs utilize several novel exons including M2Ј, M2Љ, and C⑀5 in addition to the classic secreted form (see Fig. 1A). The M2Ј exon is created by splicing directly from C⑀4 to the normal M2 splice acceptor. The ...

SummaryThe invasion of Pseudomonas aeruginosa and Salmonella enterica serovar Typhi into epithelial cells depends on the cystic fibrosis transmembrane conductance regulator (CFTR) protein as an epithelial receptor. In the case of P. aeruginosa, the bacterial ligand for CFTR is the outer core oligosaccharide portion of the lipopolysaccharide (LPS). To determine whether serovar Typhi LPS is also a bacterial ligand mediating internalization, we used both P. aeruginosa and serovar Typhi LPS as a competitive inhibitor of serovar Typhi invasion into the epithelial cell line T84. P. aeruginosa LPS containing a complete core efficiently inhibited serovar Typhi invasion. However, neither killed wild-type Typhi cells nor purified LPS were effective inhibitors. LPS from mutant Typhi strains defective in O side-chain synthesis, but with an apparently normal core, was capable of inhibiting invasion, but LPS obtained from a deeper rough mutant strain with alterations in fast-migrating core oligosaccharide failed to inhibit invasion. Lastly, exposure of wild-type serovar Typhi to T84 cultures before heat killing resulted in a structural alteration in its LPS that allowed the heat-killed cells to inhibit invasion of wild-type serovar Typhi. These data indicate that the serovar Typhi LPS core, like the P. aeruginosa LPS core, is a ligand mediating internalization of bacteria by epithelial cells, and that exposure of this ligand on wild-type Typhi is induced by the bacteria's interaction with host cells.

Previous findings indicate that the cystic fibrosis transmembrane conductance regulator (CFTR) is a ligand for Pseudomonas aeruginosa ingestion into respiratory epithelial cells. In experimental murine keratitis, P. aeruginosa enters corneal epithelial cells. We determined the importance of CFTR-mediated uptake of P. aeruginosa by corneal cells in experimental eye infections. Entry of noncytotoxic (exoU) P. aeruginosa into human and rabbit corneal cell cultures was inhibited with monoclonal antibodies and peptides specific to CFTR amino acids 108 to 117. Immunofluorescence microscopy and flow cytometry demonstrated CFTR in the intact murine corneal epithelium, and electron microscopy showed that CFTR binds to P. aeruginosa following corneal cell ingestion. In experimental murine eye infections, multiple additions of 5 nM CFTR peptide 103-117 to inocula of either cytotoxic (exoU +) or noncytotoxic P. aeruginosa resulted in large reductions in bacteria in the eye and markedly lessened eye pathology. Compared with wild-type C57BL/6 mice, heterozygous ΔF508 Cftr mice infected with P. aeruginosa had an approximately 10-fold reduction in bacterial levels in the eye and consequent reductions in eye pathology. Homozygous ΔF508 Cftr mice were nearly completely resistant to P. aeruginosa corneal infection. CFTR-mediated internalization of P. aeruginosa by buried corneal epithelial cells is critical to the pathogenesis of experimental eye infection, while in the lung, P. aeruginosa uptake by surface epithelial cells enhances P. aeruginosa clearance from this tissue.

). Here we show that (i) trypsinization of DTx does indeed produce nucleolytically active DTA, (ii) reduction of electroeluted, unreduced, cleaved DTx (58 kDa) yields nuclease-active DTA (24 kDa), and (iii) fractionation of DTx and DTA by anion-exchange chromatography leads to coelution of nuclease activity with both forms of the toxin, even though each form elutes at a distinct salt concentration. In addition, we show that Escherichia coli-derived DTA also expresses nuclease activity. These studies confirm our initial assertion that the nuclease activity observed in DTx preparations is intrinsic to the DTA portion of DTx.Diphtheria toxin (DTx) is synthesized as a single polypeptide chain that can be "nicked" by limited proteolysis to generate A and B subunits (DTA and DTB, respectively) that remain connected by a cystine bridge (7,14,16). DTB is involved in receptor binding; following sequestration in endosomes and subsequent acidification of the endosomal compartment, DTA is transported to the cytosol (8,14,22). DTA is involved in the ADP ribosylation of elongation factor 2, which leads to the inhibition of protein synthesis (7,14,16). We and others have noted that extensive toxin-induced translation inhibition is not correlated with cell death in human K562 cells (4,19); these cells are lysis resistant to doses of DTx as high as 7.5 ,ug/ml despite the rapid and complete abrogation of protein synthesis (4). In addition, extensive inhibition of protein synthesis by unrelated treatments does not lead to the prelytic intemucleosomal DNA cleavage that we have observed in DTx-intoxicated cells or to cell lysis (4). These and other findings (5) led us to propose that DTx triggers the programmed cell death pathway (4). Testing this hypothesis resulted in the discovery of a nuclease activity intrinsic to the DTx molecule (3). Detection of this activity is highly reproducible (10), optimal reaction conditions have been established (13), and an ADP ribosyltransferase (ADPrT)-defective form of DTx (called CRM197) exhibits greater nuclease activity than DTx itself (2). Moreover, nuclease activity was found to comigrate with the DTA portions of both DTx and CRM197 during electrophoresis in DNA-containing sodium dodecyl sulfate (SDS) gels (2, 3, 10) and with whole DTx and CRM197 during native gel electrophoresis (2), even though each of these forms of DTx migrates with a distinctive mobility in each gel system. We now report that the nuclease activity exhibited by DTx is intrinsic to DTA. chromatography of DTx and trypsin-generated DTA (21); therefore, they proposed that the nuclease activity of their DTx preparations resides in an uncharacterized contaminating protein. Moreover, they showed that trypsin cleavage of intact DTx results in a severe decrease in total observed nuclease activity and a simultaneous increase in ADPrT activity (21). In contrast, we observed that endoproteinase ArgC-cleaved DTx remains fully active and that the amount of DTx-associated nuclease activity corresponds to the amount of DTA generated ...