Silvia A. González

Viability and β-galactosidase activity of dairy propionibacteria subjected to digestion by artificial gastric and intestinal fluids.

An important criterion to consider in the selection of strains for dietary adjuncts is the ability of the microorganisms to survive the severe conditions of acidity and bile concentrations usually found in the gastrointestinal tract. In the present work, we report the effects of digestions by artificial gastric and intestinal fluids on beta-galactosidase activity and survival of four strains of dairy propionibacteria previously selected by their bile tolerance and beta-galactosidase activity. The strains were exposed to artificial gastric juice at pH values between 2 and 7 and then subjected to artificial intestinal digestion. Both viability and beta-galactosidase activity were seriously affected at pH 2. Skim milk and Emmental cheese juice exerted a protective effect on the parameters tested. The trypsin present in the intestinal fluid inactivated the enzyme beta-galactosidase in strains of Propionibacterium freudenreichii but not in Propionibacterium acidipropionici. Moreover, the presence of bile salts enhanced the beta-galactosidase activity of these strains by permeabilization of the cells during the first hour of exposure. The intestinal transit rate confirmed the permanence of the bacteria in the intestine for long enough to be permeabilized. These results suggest that P. acidipropionici would be a good source of beta-galactosidase activity in the intestine. We also propose a practical and effective in vitro method as a tool of screening and selection of potential probiotic bacteria.

Rotavirus NS26 is modified by addition of single O-linked residues of N-acetylglucosamine.

We studied the post-translational modification of NS26, the protein product of rotavirus gene 11 segment. Based on the presence of a putative N-glycosylation site and the high content of serine and threonine residues in gene 11 amino acid sequence we investigated whether NS26 is modified by carbohydrate addition. Specific antibodies raised against the gene 11 product expressed in Escherichia coli recognized in infected cells two polypeptides with apparent molecular weight of 26,000 (26-kDa polypeptide) and 28,000 (28-kDa polypeptide). Pulse-chase experiments demonstrated that the 26-kDa product was processed to the 28-kDa polypeptide. Both polypeptides were metabolically labeled with [3H]glucosamine, indicating the presence of a carbohydrate moiety on the protein. NS26 was found to be resistant to endo-beta-N-acetylglucosaminidase H and endo-beta-N-acetylglucosaminidase F/peptide:N-glycosidase F treatment, but sensitive to removal by alkali-induced beta-elimination, suggesting that the saccharide chain was attached to the protein via an O-glycosidic linkage. Chromatographic analysis of total acid hydrolysates of [3H]glucosamine-labeled NS26-bound carbohydrate indicated the presence of N-acetylglucosamine. In addition, mild alkaline treatment of NS26 in the presence of NaB3H4 identified the O-linked carbohydrate moiety as N-acetylglucosamine. Taken together, these data demonstrate that NS26 is processed to a 28-kDa polypeptide by addition of O-linked monosaccharide residues of N-acetylglucosamine.

Lactobacilli isolated from chicken intestines: potential use as probiotics.

Lactobacillus strains were tested for their in vitro probiotic properties. Cell surface hydrophobicity was found to be very high for Lactobacillus fermentum subsp. cellobiosus and Salmonella Gallinarum; high values could indicate a greater ability to adhere to epithelial cells. Studies on Lactobacillus animalis indicated relative cell surface hydrophobicities smaller than those of L. fermentum subsp. cellobiosus and L. fermentum. L. animalis and Enterococcus faecalis were able to coaggregate with L. fermentum subsp. cellobiosus and L. fermentum, respectively, but not with Salmonella Gallinarum. After mixed-culture studies for determining suitable growth behavior, the pair of strains L. animalis plus L. fermentum subsp. cellobiosus was selected for an attempted challenge against Salmonella Gallinarum. Double and triple mixed-culture studies indicated that selected lactobacillus strains were able to retain their beneficial characteristics in the presence of Salmonella Gallinarum such as presence of lectins, production of antimicrobial compounds, and ability to grow and compete. The selected microorganisms can be considered as potential ingredients for a chicken probiotic feed formulation intended to control salmonellosis and also improve poultry sanitation.

Characterization of the Lactic Acid Bacteria in Ewe's Milk and Cheese from Northwest Argentina

Indigenous lactic acid bacteria in ewes milk and artisanal cheese were studied in four samples of fresh raw milk and four 1-month-old cheeses from the provinces of northwest Argentina. Mean growth counts on M17, MRS, and MSE agar media did not show significant differences (P < 0.05) in raw milk and cheeses. Isolates of lactic acid bacteria from milk were identified as Enterococcus (48%), lactococci (14%), leuconostocs (8%), and lactobacilli (30%). All lactococci were identified as Lactococcus lactis (subsp. lactis and subsp. cremoris). Lactobacilli were identified as Lactobacillus plantarum (92%) and Lactobacillus acidophilus (8%). Enterococci (59%) and lactobacilli (41%) were isolated from cheeses. L. plantarum (93%), L. acidophilus (5%), and Lactobacillus casei (2%) were most frequently isolated. L. lactis subsp. lactis biovar diacetylactis strains were considered as fast acid producers. L. lactis subsp. cremoris strains were slow acid producers. L. plantarum and L. casei strains identified from the cheeses showed slow acid production. The majority of the lactobacilli and Lactococcus lactis strains utilized citrate and produced diacetyl and acetoin in milk. Enzyme activities (API-ZYM tests) of lactococci were low, but activities of L. plantarum strains were considerably higher. The predominance of L. plantarum in artisanal cheese is probably important in the ripening of these cheeses due to their physiological and biochemical characteristics.

Study of the morphology of the cell walls of some strains of lactic acid bacteria and related species

The objective of the present study was to find an explanation for the biological effect of the bacteria present in a biotherapeutic milk (Lactobacillus casei CRL 431 and Lactobacillus acidophilus CRL 730). The ability of bacterial cell walls to induce an immune response when introduced into an organism is well known. In this paper we specifically analyzed the morphology of these cell walls. Besides the two bacterial strains used in the fermented milk, two other lactic acid bacteria, belonging to another genus and unable to induce an immune system response, as well as a strain of Propionibacterium, of which the immune modulating capacity is known, were used in this work. We found a structural particularly in strains with immunostimulating capacity (L. casei CRL 431 and P. acidopropionici CRL 1198): molecules which protrude from the cell surface. In L. casei CRL 431 these molecules were identified as lectins because they are able to agglutinate yeast cells treated with glutaraldehyde and glycine. The structures protruding from P. acidipropionici CRL 1198 cells were teichoic acids. Teichoic acid and lectin-like structures can participate in adhesion to intestinal cells. The immunostimulation observed can be induced by the adhesion phenomenon.

Structure of rearranged genome segment 11 in two different rotavirus strains generated by a similar mechanism.

The structures of the rearranged genomic segment 11 of two spontaneous swine rotavirus strains were determined. We found that the rearrangements involved the duplication of normal segment 11 in a head-to-tail orientation, and partial deletions in both monomers. The open reading frame for VP11, the protein encoded by normal segment 11, was maintained. We also showed that the two rearranged genes were transcribed into RNA molecules of the same length as their corresponding genomic segments.

A Family of Diverse Kunitz Inhibitors from Echinococcus granulosus Potentially Involved in Host-Parasite Cross-Talk

The cestode Echinococcus granulosus, the agent of hydatidosis/echinococcosis, is remarkably well adapted to its definitive host. However, the molecular mechanisms underlying the successful establishment of larval worms (protoscoleces) in the dog duodenum are unknown. With the aim of identifying molecules participating in the E. granulosus-dog cross-talk, we surveyed the transcriptomes of protoscoleces and protoscoleces treated with pepsin at pH 2. This analysis identified a multigene family of secreted monodomain Kunitz proteins associated mostly with pepsin/H+-treated worms, suggesting that they play a role at the onset of infection. We present the relevant molecular features of eight members of the E. granulosus Kunitz family (EgKU-1 – EgKU-8). Although diverse, the family includes three pairs of close paralogs (EgKU-1/EgKU-4; EgKU-3/EgKU-8; EgKU-6/EgKU-7), which would be the products of recent gene duplications. In addition, we describe the purification of EgKU-1 and EgKU-8 from larval worms, and provide data indicating that some members of the family (notably, EgKU-3 and EgKU-8) are secreted by protoscoleces. Detailed kinetic studies with native EgKU-1 and EgKU-8 highlighted their functional diversity. Like most monodomain Kunitz proteins, EgKU-8 behaved as a slow, tight-binding inhibitor of serine proteases, with global inhibition constants (K I *) versus trypsins in the picomolar range. In sharp contrast, EgKU-1 did not inhibit any of the assayed peptidases. Interestingly, molecular modeling revealed structural elements associated with activity in Kunitz cation-channel blockers. We propose that this family of inhibitors has the potential to act at the E. granulosus-dog interface and interfere with host physiological processes at the initial stages of infection.

Positive and Negative Modulation of Virus Infectivity and Envelope Glycoprotein Incorporation into Virions by Amino Acid Substitutions at the N Terminus of the Simian Immunodeficiency Virus Matrix Protein

ABSTRACT The matrix (MA) protein of the simian immunodeficiency viruses (SIVs) is encoded by the amino-terminal region of the Gag precursor and is the component of the viral capsid that lines the inner surface of the virus envelope. Previously, we identified domains in the SIV MA that are involved in the transport of Gag to the plasma membrane and in particle assembly. In this study, we characterized the role in the SIV life cycle of highly conserved residues within the SIV MA region spanning the two N-terminal α-helices H1 and H2. Our analyses identified two classes of MA mutants: (i) viruses encoding amino acid substitutions within α-helices H1 or H2 that were defective in envelope (Env) glycoprotein incorporation and exhibited impaired infectivity and (ii) viruses harboring mutations in the β-turn connecting helices H1 and H2 that were more infectious than the wild-type virus and displayed an enhanced ability to incorporate the Env glycoprotein. Remarkably, among the latter group of MA mutants, the R22L/G24L double amino acid substitution increased virus infectivity eightfold relative to the wild-type virus in single-cycle infectivity assays, an effect that correlated with a similar increase in Env incorporation. Furthermore, the R22L/G24L MA mutation partially or fully complemented single-point MA mutations that severely impair or block Env incorporation and virus infectivity. Our finding that the incorporation of the Env glycoprotein into virions can be upregulated by specific mutations within the SIV MA amino terminus strongly supports the notion that the SIV MA domain mediates Gag-Env association during particle formation.

Esterolytic and Lipolytic Activities of Lactic Acid Bacteria Isolated from Ewe's Milk and Cheese

In the present work, we report on the esterase and lipase activities of lactic acid bacteria representing the genera Lactococcus, Leuconostoc, Lactobacillus, and Enterococcus isolated from ewes milk and cheeses. Esterase activity was studied using alpha- and beta-naphthyl derivatives of 2 to 12 carbon atoms and postelectrophoretic detection. The lactic acid bacteria evaluated had intracellular esterase activities, which preferentially degraded the alpha- and beta-naphthyl derivatives of 2 to 6 carbon atoms. By studying postelectrophoretic patterns, it was found that some strains presented more than one esterase. Lactobacillus plantarum O236 showed four enzymes that hydrolyze carboxyl ester linkages with different specificity. Lipase activity was studied in intracellular and extracellular fractions using tributyrin, tricaprylin, triolein, and milk fat as substrates. The intracellular and extracellular fractions of Leuconostoc mesenteroides O257, Lactobacillus plantarum O236, and Lactobacillus acidophilus O177 were able to hydrolyze tributyrin. L. plantarum O186, L. acidophilus O252, Enterococcus faecium O174 and O426, and Enterococcus faecalis Ov409 showed lipase activity associated with the intracellular fraction on tributyrin. Lactococcus lactis O233, L. plantarum O155, and Lactobacillus casei O190 did not hydrolyze triglycerides. Not all strains that showed esterase activity exhibited high activity on triglycerides. Esterase and lipase activities were species- and strain-specific. Wide variations in activity between strains highlight the need for selecting appropriate starters to produce enzyme-modified cheese as well as accelerated ripened cheese.

Mutational analysis of the feline immunodeficiency virus matrix protein

To study the process of feline immunodeficiency virus (FIV) assembly, we examined the suitability of the vaccinia vector system to reproduce FIV particle formation. To this end, we constructed a recombinant vaccinia virus carrying the FIV gag gene. Biochemical and electron microscopy analyses of cells infected with this recombinant virus showed that the FIV Gag polyprotein self-assembled into lentivirus-like particles that were released into the culture medium. As a first step in the identification of molecular determinants in FIV Gag that are involved in virus assembly, we performed a site-directed mutagenesis analysis of the N-terminal matrix (MA) domain of the FIV Gag precursor. To this end, a series of amino acid substitutions and small in-frame deletions were introduced into the FIV MA and the mutated FIV gag gene constructs were expressed by means of the vaccinia system. Characterization of the assembly phenotype of these FIV Gag mutants led to the identification of amino acidic regions within the MA domain that are necessary for efficient transport of the Gag precursor to the plasma membrane and particle assembly. Our results reveal the role that the FIV MA plays in virus morphogenesis and contribute to the understanding of the assembly process in non-primate lentiviruses.