Signature tagged mutagenesis has recently revealed that the Ssp serine protease (V8 protease) contributes to in vivo growth and survival of Staphylococcus aureus in different infection models, and our previous work indicated that Ssp could play a role in controlling microbial adhesion. In this study, we describe an operon structure within the ssp locus of S. aureus RN6390. The ssp gene encoding V8 protease is designated as sspA, and is followed by sspB, which encodes a 40.6-kDa cysteine protease, and sspC, which encodes a 12.9-kDa protein of unknown function. S. aureus SP6391 is an isogenic derivative of RN6390, in which specific loss of SspA function was achieved through a nonpolar allelic replacement mutation. In addition to losing SspA, the culture supernatant of SP6391 showed a loss of 22-to 23-kDa proteins and the appearance of a 40-kDa protein corresponding to SspB. Although the 40-kDa SspB protein could degrade denatured collagen, our data establish that this is a precursor form which is normally processed by SspA to form a mature cysteine protease. Culture supernatant of SP6391 also showed a new 42-kDa glucosaminidase and enhanced glucosaminidase activity in the 29 to 32 kDa range. Although nonpolar inactivation of sspA exerted a pleiotropic effect, S. aureus SP6391 exhibited enhanced virulence in a tissue abscess infection model relative to RN6390. Therefore, we conclude that SspA is required for maturation of SspB and plays a role in controlling autolytic activity but does not by itself exert a significant contribution to the development of tissue abscess infections.The serine protease of Staphylococcus aureus strain V8 (Ssp, also known as V8 protease) was one of the first secreted enzymes of S. aureus to be purified and characterized in detail (16). It is a member of the glutamyl endopeptidase family of enzymes (24) and has been widely used in this capacity as a specific tool for determining protein structure. However, its contributions to the growth and survival of S. aureus have not been elucidated. S. aureus is a major cause of infectious morbidity and mortality in both the community and hospital settings (30), and although capable of expressing several different toxins, it is not generally equated with other pathogens that cause illness primarily through the elaboration of specific toxins (17). Rather, the hallmarks of S. aureus disease are its rapid multiplication and induction of inflammation at the site of infection and its ability to disseminate and initiate metastatic infection (50, 51). This is facilitated by an accessory gene regulator locus, agr, which at high cell density is responsible for inducing the expression of secreted toxins and exoenzymes, while simultaneously promoting the reduced expression of cell surface adhesins and colonization factors (18,38,41,48). Therefore, agr-null mutants demonstrate enhanced expression of colonization factors and a pleiotropic defect in expression of secreted virulence factors.Due to the inability to express secreted virulence factors, agr-null strai...

Passive protection of neonatal piglets against fatal enteric colibacillosis was achieved with powder preparations of specific antibodies against K88, K99, and 987P fimbrial adhesins of enterotoxigenic Escherichia coil. The antibody powders were obtained by spray drying the water-soluble protein fraction of egg yolks from immunized hens after the lipid components were precipitated with an aqueous dispersion of acrylic resins (Eudragit L3OD-55; Rohm pharma). The anti-K88,-K99, and-987P antibody preparations reacted specifically against the corresponding fimbrial antigens in an enzyme-linked immunosorbent assay. The orally administered antibodies protected in a dose-dependent fashion against infection with each of the three homologous strains of E. coli in passive immunization trials with a colostrum-deprived piglet model of enterotoxigenic E. coli diarrhea. Scanning electron microscopy revealed adherence of enterotoxigenic E. coli in intestinal epithelial surfaces of control piglets, whereas in treated piglets treated with high-titer antibodies, a resistance to bacterial adhesion was observed. An enzyme immunoassay with avidin-biotin complex demonstrated specific local antibody activity in target areas of the small intestines. In vitro, E. coli K88+, K99+, and 987P+ strains adhered equally to porcine duodenal and ileal epithelial cells but failed to do so in the presence of homologous anti-fimbrial antibodies. Absorption of egg yolk antibodies with fimbrial immunosorbent removed the anti-fimbrial antibody fraction and reduced significantly the protective nature of the antibody preparation in a passive immunization experiment, suggesting that anti-fimbrial antibodies were the active components. MATERUILS AND METHODS Animals. A total of 76 colostrum-deprived, newborn Large White pigs were utilized in protection trials with antibody 998 Vol. 60, No. 3 on September 4, 2020 by guest http://iai.asm.org/ Downloaded from PROTECTIVE EFFECT OF CHICKEN EGG YOLK ANTIBODIES 999 powder preparations and absorbed antibody solutions. Fivemonth-old White Leghorn chickens (strain Hyline W36) were utilized for immunization, and New Zealand White rabbits (3 kg) and Japanese Black cattle (4 years old) were used for antiserum production. Bacteria and cultivation conditions. ETEC strains 19304

The L-type Ca current (I(Ca,L)), essential for normal cardiac function, also regulates dynamic action potential (AP) properties that promote ventricular fibrillation. Blocking I(Ca,L) can prevent ventricular fibrillation, but only at levels suppressing contractility. We speculated that, instead of blocking I(Ca,L), modifying its shape by altering kinetic features could produce equivalent anti-fibrillatory effects without depressing contractility. To test this concept experimentally, we overexpressed a mutant Ca-insensitive calmodulin (CaM(1234)) in rabbit ventricular myocytes to inhibit Ca-dependent I(Ca,L) inactivation, combined with the ATP-sensitive K current agonist pinacidil or I(Ca,L) blocker verapamil to maintain AP duration (APD) near control levels. Cell shortening was enhanced in pinacidil-treated myocytes, but depressed in verapamil-treated myocytes. Both combinations flattened APD restitution slope and prevented APD alternans, similar to I(Ca,L) blockade. To predict the arrhythmogenic consequences, we simulated the cellular effects using a new AP model, which reproduced flattening of APD restitution slope and prevention of APD/Ca(i) transient alternans but maintained a normal Ca(i) transient. In simulated two-dimensional cardiac tissue, these changes prevented the arrhythmogenic spatially discordant APD/Ca(i) transient alternans and spiral wave breakup. These findings provide a proof-of-concept test that I(Ca,L) can be targeted to increase dynamic wave stability without depressing contractility, which may have promise as an antifibrillatory strategy.