Methods Enzymol

Methods Enzymol

Methods Enzymol. in a number of outbreaks in the developed world (13, 37, 49, 77). EAEC-mediated diarrhea is definitely characterized by the formation of a solid mucus gel within the intestinal mucosa and by mucosal damage. EAEC diarrhea is definitely mainly secretory in nature; stools contain mucus and often blood but generally no polymorphonuclear leukocytes (17, 50). Although enterotoxins have been explained in EAEC (23, 74), the full picture of EAEC pathogenesis has not yet been explained. is closely related genetically to represents a subspecies of (40, 64, 69). However, elicits a distinctive and complex disease, bacillary dysentery, caused by invasion of the colonic epithelial cells and characterized by an intense inflammatory response (57). Notably, however, many instances of shigellosis are manifested as watery Theophylline-7-acetic acid diarrhea, which may be mediated by one or more enterotoxins. Study on pathogenicity offers focused mainly within the plasmid-encoded genes necessary for penetration and intercellular dissemination (48, 72). Similarly, factors associated with EAEC-mediated diarrhea have been localized to a 65-MDa plasmid, which is required for manifestation of aggregative adherence fimbriae (18) and several putative toxins (23, 73). However, evidence is present for chromosomal virulence factors in both (54, 65) and EAEC (19). In this article we statement the cloning, nucleotide sequence analysis, and expression of the gene encoding a 116-kDa secreted protein explained MGC18216 by Eslava et al. (24), which is located within the chromosome of both EAEC and strains. We have termed this gene and the gene product Pic (for protein involved in intestinal colonization). This protein is an extracellular serine protease which displays in vitro mucinolytic activity, serum resistance, and hemagglutination. The protease is definitely synthesized as a large precursor, which is definitely processed during secretion from the autotransporter secretion mechanism. MATERIALS AND METHODS Bacterial strains and plasmids. The bacterial strains and plasmids used in this study are outlined in Table ?Table1.1. 042, a known diarrheal pathogen, was isolated from a child with diarrhea during the course of an epidemiological study in Peru (50). HB101 was utilized for genetic manipulations. Strains were passed regularly on Luria-Bertani broth (L-broth) or agar with the following antibiotic health supplements where appropriate: ampicillin (100 g/ml), kanamycin (50 g/ml), nalidixic acid (50 g/ml), and tetracycline (10 g/ml). All strains were stored at ?70C in Trypticase soy broth with 15% glycerol. TABLE 1 Bacterial strains and plasmids used in this?study ((((for 10 min at 4C. Envelopes were prepared by a modification of the method layed out by Caffrey and Owen (10). Briefly, envelopes isolated following French pressure lysis of bacterial cells were sedimented by centrifugation (48,000 for 60 min at 4C) and washed twice in 30 ml of 10 mM Tris-HCl (pH 7.2) and once in 3 ml of the same buffer. The standard conditions for sedimentation of envelope fractions were 48,000 for 60 min at 4C. The envelopes were finally resuspended in 1 ml of the same buffer and aliquoted for storage at ?70C for further manipulations. To prepare Theophylline-7-acetic acid tradition supernatant fractions, strains were grown over night at 37C in 100 ml of L-broth. After centrifugation at 12,000 for 10 min, supernatants were concentrated and size fractionated with Ultrafilters (Millipore) having a 100-kDa cutoff. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (43) was performed with 12.5% acrylamide separating gels and 4.5% Theophylline-7-acetic acid acrylamide stacking gels. Samples were routinely heated for 5 min at 100C in Laemmli (43) sample buffer before becoming loaded. Proteins were recognized by staining with Coomassie amazing blue R250. Western immunoblotting was performed essentially as explained by Caffrey et al. (9). Dried skim milk (5%) was used as a obstructing reagent. Alkaline phosphatase-conjugated goat anti-rabbit immunoglobulin G (IgG) was used as the localizing reagent, and reacting antigens were visualized with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium as explained elsewhere (3). To determine N-terminal amino acid sequences, the proteins were separated by SDS-PAGE as explained by Laemmli (43) and transferred to Immobilon polyvinylidene difluoride membranes (Millipore) prior to amino-terminal sequencing. Amino-terminal sequencing was performed by automated Edman degradation in the Protein and Nucleic Acid Facility, Stanford University or college, Palo Alto, Calif. Preparation of antisera. Rabbit antiserum specific for Pic was raised by subcutaneous injection of Pic preparations in Freund’s adjuvant as explained previously (33). Preparations of immunoglobulins were adsorbed successively by incubation at 4C for 24 h with an protein-agarose suspension (Sigma). PCR methods. Amplifications were performed with 500 ng of purified chromosomal DNA as themes and 0.2 mM each primer inside a 100-l reaction mixture containing 2 U.