Evaluation of chitosan salt properties in the production of AgNPs materials with antibacterial activity.

In this study, water-soluble chitosan salts (chitosan amine sulfopropyl salts) were prepared from chitosan samples with different molecular weights and deacetylation degrees. These soluble-in-water polymer salts allowed us to produce, in an eco-friendly and facile method, silver nanoparticles (AgNPs) with better control on size and polydispersity, even at large silver concentrations than their corresponding chitosan sample. Chitosan salt-based materials (films and scaffolds) were analyzed in terms of antibacterial properties against Staphylococcus aureus ATCC23915 or Pseudomonas aeruginosa ATCC 27853. 3D scaffolds enhanced the effect of the chitosan-AgNPs combination compared to the equivalent films.

Absence of CD38 delays arrival of neutrophils to the liver and innate immune response development during hepatic amoebiasis by Entamoeba histolytica.

To define the role of CD38 in the migration of neutrophils to the liver and consequently in the induction of an innate immune response during murine hepatic amoebiasis by Entamoeba histolytica, we examined amoebic liver abscess development (ALA), presence of amoebae and neutrophils, and expression levels of cytokines and other inflammation mediators mRNA, in infected wild-type and CD38 Knockout (CD38KO) C57BL/6J mice. Results showed that CD38KO mice undergo a delay in ALA development in comparison with the wild-type strain. The presence of amoebae lasted longer in CD38(-/-), and although neutrophils arrived to the liver in both strains, there was a clear difference in the time between the two strains; whereas in the wild-type strain, neutrophils arrived at early times (6-12 h), in the CD38KO strain, neutrophils arrived later (48-72 h). Cytokines profile during the innate immune response development (TNF-α, IL-1ß, IL-6) was, for WT mice concomitant with, and preceded, for CD38KO mice, the time in which neutrophils were present in the liver lesion. In conclusion, CD38 is important for neutrophils migration during hepatic amoebiasis, and in turn, these cells play an important role in the innate immune response.

Intimate adherence by enteropathogenic Escherichia coli modulates TLR5 localization and proinflammatory host response in intestinal epithelial cells.

Enteropathogenic Escherichia coli (EPEC) causes diarrhoeal disease by altering enterocyte physiology and producing mucosal inflammation. Many details concerning the host response against EPEC remain unknown. We evaluated the role of EPEC virulence factors on the inflammatory response through an analysis of bacterial recognition, cell signalling, and cytokine production using an in vitro epithelial cell infection model. Interestingly, we found that EPEC infection recruits Toll-like receptor 5 (TLR5) to the cell surface. We confirmed that type 3 secretion system (T3SS) and flagellin (FliC) are necessary for efficient extracellular regulated kinases 1 and 2 (ERK1/2) activation and found that intimin could down-regulate this pathway. Besides flagellin, intimin was required to keep nuclear factor kappa B (NF-κB) activated during infection. EPEC infection activated tumour necrosis factor alpha (TNF-α) production and induced interleukin (IL)-1ß and IL-8 release. Virulence factors such as intimin, T3SS, EspA and fliC were required for IL-1ß secretion, whereas intimin and T3SS participated in IL-8 release. Flagellin was essential for late secretion of TNF-α and IL-8 and intimin stimulated cytokine secretion. Initial adherence limited TNF-α release, whereas late attachment sustained TNF-α secretion. We conclude that intimin modulates TLR5 activation and intimate adherence alters the proinflammatory response.

Cell death of chondrocytes is a combination between apoptosis and autophagy during the pathogenesis of Osteoarthritis within an experimental model.

The death of chondrocytes and the loss of extracellular matrix are the central features in cartilage degeneration during Osteoarthritis (OA) pathogenesis. The mechanism by which chondrocytes are removed in OA cartilage are still not totally defined, although previous reports support the presence of apoptotic as well as non apoptotic signals. In addition, in 2004 Roach and co-workers suggested the term "Chondroptosis" to design the type of cell death present in articular cartilage, which include the presence of some apoptotic and autophagic processes. To identify the mechanisms, as well as the chronology by which chondrocytes are eliminated during OA pathogenesis, we decided to evaluate apoptosis (by active caspase 3 and TUNEL signal) and autophagy (by LC3II molecule and cytoplasmic vacuolization) using Immunohistochemistry and Western blot techniques in an animal OA model. During OA pathogenesis, chondrocytes exhibit modifications in their death process in each zone of the cartilage. At early stages of OA, the death of chondrocytes starts with apoptosis in the superficial and part of the middle zones of the cartilage, probably as a consequence of a constant mechanical damage in the joint. As the degenerative process progresses, high incidence of active caspase 3 as well as LC3II expression are observed in the same cell, which indicate a combination of both death processes. In contrast, in the deep zone, due the abnormal subchondral bone ossification during the OA pathogenesis, apoptosis is the only mechanism observed.

Strategies for the identification of virulence determinants in human pathogenic fungi.

The incidence of fungal infections is increasing in different countries. The current available therapy of these infections does not satisfy all requirements in terms of specificity and therapeutic index, a fact that has stimulated the scientific community to identify fungal virulence determinants. Several pathogenic fungi are opportunistic and, therefore, identification of virulence genes is difficult, given their close relationship with host cells. In recent years, the development of genetic tools in several pathogenic fungi has enabled the development of genetic strategies for their identification. These include several strategies based on the phenotypic analysis of strains or environmental conditions in which the expression of the putative gene(s) is either altered or deleted; and this is accomplished through the development of in vitro or in vivo systems. In the near future, this research will produce a better picture of fungal pathogenesis and therefore define novel promising targets in antifungal therapy.

Virulence genes in the pathogenic yeast Candida albicans.

In recent years, the incidence of fungal infections has been rising all over the world. Although the amount of research in the field of pathogenic fungi has also increased, there is still a need for the identification of reliable determinants of virulence. In this review, we focus on identified Candida albicans genes whose deletant strains have been tested in experimental virulence assays. We discuss the putative relationship of these genes to virulence and also outline the use of new different systems to examine the precise effect in virulence of different genes.

Plasmid-encoded toxin of enteroaggregative Escherichia coli is internalized by epithelial cells.

We have previously described a 104-kDa protein termed Pet (for plasmid-encoded toxin) secreted by some strains of enteroaggregative Escherichia coli (EAEC). Through an unknown mechanism, this toxin (i) raises transepithelial short-circuit current (Isc) and decreases the electrical resistance of rat jejunum mounted in the Ussing chamber, (ii) causes cytoskeletal alterations in HEp-2 cells and HT29/C1 cells, and (iii) is required for histopathologic effects of EAEC on human intestinal mucosa. Pet is a member of the autotransporter class of secreted proteins and together with Tsh, EspP, EspC, ShMu, and SepA proteins comprises the SPATE subfamily. Here, we show that Pet is internalized by HEp-2 cells and that internalization appears to be required for the induction of cytopathic effects. Evidence supporting Pet internalization includes the facts that (i) the effects of Pet on epithelial cells were inhibited by brefeldin A, which interferes with various steps of intracellular vesicular transport; (ii) immunoblots using anti-Pet antibodies detected Pet in the cytoplasmic fraction of intoxicated HEp-2 cells; (iii) Pet was detected inside HEp-2 cells by confocal microscopy; and (iv) a mutant in the passenger domain cleavage site, which prevents Pet release from the bacterial outer membrane, did not produce cytopathic effects on epithelial cells, whereas the release of mutant Pet from the outer membrane with trypsin yielded active toxin. We have also shown that the Pet serine protease motif is required to produce cytopathic effects but not for Pet secretion. Our results suggest an intracellular mode of action for the Pet protease and are consistent with we our recent report suggesting an intracellular mode of action for Pet.

espC pathogenicity island of enteropathogenic Escherichia coli encodes an enterotoxin.

At least five proteins are secreted extracellularly by enteropathogenic Escherichia coli (EPEC), a leading cause of infant diarrhea in developing countries. However only one, EspC, is known to be secreted independently of the type III secretion apparatus encoded by genes located within the 35.6-kb locus of enterocyte effacement pathogenicity island. EspC is a member of the autotransporter family of proteins, and the secreted portion of the molecule is 110 kDa. Here we determine that the espC gene is located within a second EPEC pathogenicity island at 60 min on the chromosome of E. coli. We also show that EspC is an enterotoxin, indicated by rises in short-circuit current and potential difference in rat jejunal tissue mounted in Ussing chambers. In addition, preincubation with antiserum against the homologous Pet enterotoxin of enteroaggregative E. coli eliminated EspC enterotoxin activity. Like the EAF plasmid, the espC pathogenicity island was found only in a subset of EPEC, suggesting that EspC may play a role as an accessory virulence factor in some but not all EPEC strains.

Signal transduction pathways and cell-wall construction in Candida albicans.

Signal transduction pathways are the molecular mechanisms responsible for detecting and transmitting changes in the surrounding environment to the nucleus where appropriate responses are generated. The cell wall is the most external structure of the fungal cell and, in pathogenic fungi, is responsible for specifically interacting with the mammalian host cell in a highly dynamic interplay. Recent work has shown the role that some signal transduction pathways, involving members of the MAP kinase family, have in this process in the nonpathogenic model organism Saccharomyces cerevisiae. However, as yet little is known about these phenomena in pathogenic fungi. The aim of this review is to characterize the existing signal transduction pathways in Candida albicans and their relationship with the cell-wall construction.

Immunodulation of rat serum and mucosal antibody responses to Entamoeba histolytica trophozoites by beta-1,3-glucan and cholera toxin.

Systemic and mucosal and immune responses can be manipulated with immunomodulators. Here we show the modulatory effects of cholera toxin (CT) and beta-1,3-glucan (GLU) on the rat antiamebic serum and fecal antibody responses to one or four intraperitoneal (IP) or intragastric (IG) doses of glutaraldehyde-fixed Entamoeba histolytica trophozoites (GFT). One IP dose of GFT maximized serum IgM and IgG antiamebic antibodies on days 4 and 9, respectively; CT coadministration increased IgM antibodies, whereas IgG titers increased with CT or GLU; coproantibodies were undetectable after GFT alone or coadministered with GLU, whereas CT coadministration maximized fecal IgA antibodies on day 6. One IG dose of GFT alone increased serum IgM and IgG antibodies 2.5 times and no further increases were detected using GLU, whereas CT doubled serum IgG antibodies; GFT did not affect the coproantibody responses, whereas GLU coadministration maximized IgG coproantibody levels on day 6 and CT increased IgG and IgA coproantibody levels on the same day. On the other hand, four IG doses of GFT alone or with GLU induced tolerance, whereas GFT alone via the IP route increased serum antibodies slightly and GLU coadministration increased serum IgG antibody titers 300-fold. CT coadministration by both routes increased IgA coproantibodies, and simultaneous CT+GLU coadministration induced lower responses than either CT or GLU. Different antiamebic immune responses might therefore be attained through the use of different immunization routes and immunomodulators to induce protective immunity against intestinal or extraintestinal amebiasis.

Pet toxin from enteroaggregative Escherichia coli produces cellular damage associated with fodrin disruption.

Pet toxin is a serine protease from enteroaggregative Escherichia coli which has been described as causing enterotoxic and cytotoxic effects. In this paper we show that Pet produces spectrin and fodrin (nonerythroid spectrin) disruption. Using purified erythrocyte membranes treated with Pet toxin, we observed degradation of alpha- and beta-spectrin chains; this effect was dose and time dependent, and a 120-kDa protein fraction was observed as a breakdown product. Spectrin degradation and production of the 120-kDa subproduct were confirmed using specific antibodies against the alpha- and beta-spectrin chains. The same degradation effect was observed in alpha-fodrin from epithelial HEp-2 cells, both in purified cell membranes and in cultured cells which had been held in suspension for 36 h; these effects were confirmed using antifodrin rabbit antibodies. The spectrin and fodrin degradation caused by Pet is related to the Pet serine protease motif. Fluorescence and light microscopy of HEp-2 Pet-treated cells showed morphological alterations, which were associated with irregular distribution of fodrin in situ. Spectrin and fodrin degradation by Pet toxin were inhibited by anti-Pet antibodies and by phenylmethylsulfonyl fluoride. A site-directed Pet mutant, which had been shown to abolish the enterotoxic and cytotoxic effects of Pet, was unable to degrade spectrin in erythrocyte membranes or purified spectrin or fodrin in epithelial cell assays. This is a new system of cellular damage identified in bacterial toxins which includes the internalization of the protease, induction of some unknown intermediate signaling steps, and finally the fodrin degradation to destroy the cell.

Involvement of the enteroaggregative Escherichia coli plasmid-encoded toxin in causing human intestinal damage.

Enteroaggregative Escherichia coli (EAEC) strains have been shown to adhere to human intestinal tissue in an in vitro organ culture (IVOC) model, and certain strains manifest mucosal toxicity. We have recently described the EAEC plasmid-encoded toxin (Pet), a member of a specific serine protease subclass of the autotransporter proteins. When injected into rat ileal loops, Pet both elicited fluid accumulation and had cytotoxic effects on the mucosa. Furthermore, the Pet protein caused rises in short circuit current from rat jejunal tissue mounted in a Ussing chamber and rounding of intestinal epithelial cells in culture. We therefore hypothesized that the mucosal pathology induced by EAEC strains in the IVOC model was related to expression of the Pet protein. Here, we have examined the effects of EAEC strain 042 and its isogenic pet mutant in the IVOC model. 042-infected colonic explants exhibited dilation of crypt openings, increased cell rounding, development of prominent intercrypt crevices, and absence of apical mucus plugs. Colonic tissue incubated with the pet mutant exhibited significantly fewer mucosal abnormalities both subjectively and as quantitated morphometrically by measurement of crypt aperture diameter. Mucosal effects were restored upon complementation of the pet mutation in trans. Interestingly, we found that the ability of 042 to damage T84 cells was not dependent upon Pet. The data suggest that the Pet toxin is active on the human intestinal mucosa but that EAEC may have other mechanisms of eliciting mucosal damage.

Cytoskeletal effects induced by pet, the serine protease enterotoxin of enteroaggregative Escherichia coli.

We have previously described enteroaggregative Escherichia coli (EAEC) strains that induce cytotoxic effects on T84 cells, ligated rat ileal loops, and human intestine in culture. Such strains secrete a 104-kDa protein termed Pet (for plasmid-encoded toxin). We have also shown previously that the Pet toxin induces rises in short-circuit current and decreases the electrical resistance in rat jejunum mounted in an Ussing chamber. The nucleotide sequence of the pet gene revealed that Pet is a member of the autotransporter class of secreted proteins. Here we show that a concentrated supernatant of E. coli HB101 harboring the minimal pet clone pCEFN1 induces temperature-, time- and dose-dependent cytopathic effects on HEp-2 cells and HT29 C1 cells in culture. The effects were characterized by release of the cellular focal contacts from the glass substratum, followed by complete rounding of the cells and detachment from the glass. Staining of the Pet-treated cells with Live/Dead viability stain revealed that >90% of rounded cells were viable. Pet-intoxicated HEp-2 and HT29 cells stained with fluorescein-labeled phalloidin revealed contraction of the cytoskeleton and loss of actin stress fibers. However, the effects of Pet were not inhibited by cytoskeleton-altering drugs, including colchicine, taxol, cytochalasin D, and phallicidin. The Pet protein induced proteolysis in zymogram gels, and preincubation with the serine protease inhibitor phenylmethylsulfonyl fluoride resulted in complete abrogation of Pet cytopathic effects. We introduced a mutation in a predicted catalytic serine residue and found that the mutant (Pet S260I) was deficient in protease activity and did not produce cytopathic effects, cytoskeletal damage, or enterotoxic effects in Ussing chambers. These data suggest that Pet is a cytoskeleton-altering toxin and that its protease activity is involved in each of the observed phenotypes.

Role of the mitogen-activated protein kinase Hog1p in morphogenesis and virulence of Candida albicans.

The relevance of the mitogen-activated protein (MAP) kinase Hog1p in Candida albicans was addressed through the characterization of C. albicans strains without a functional HOG1 gene. Analysis of the phenotype of hog1 mutants under osmostressing conditions revealed that this mutant displays a set of morphological alterations as the result of a failure to complete the final stages of cytokinesis, with parallel defects in the budding pattern. Even under permissive conditions, hog1 mutants displayed a different susceptibility to some compounds such as nikkomycin Z or Congo red, which interfere with cell wall functionality. In addition, the hog1 mutant displayed a colony morphology different from that of the wild-type strain on some media which promote morphological transitions in C. albicans. We show that C. albicans hog1 mutants are derepressed in the serum-induced hyphal formation and, consistently with this behavior, that HOG1 overexpression in Saccharomyces cerevisiae represses the pseudodimorphic transition. Most interestingly, deletion of HOG1 resulted in a drastic increase in the mean survival time of systemically infected mice, supporting a role for this MAP kinase pathway in virulence of pathogenic fungi. This finding has potential implications in antifungal therapy.

Phylogenetic analysis of enteroaggregative and diffusely adherent Escherichia coli.

The phylogenetics of the various pathotypes of diarrheagenic Escherichia coli are not completely understood. In this study, we identified several plasmid and chromosomal genes in the pathogenic enteroaggregative E. coli (EAEC) prototype strain 042 and determined the prevalence of these loci among EAEC and diffusely adherent E. coli strains. The distribution of these genes is analyzed within an evolutionary framework provided by the characterization of allelic variation in housekeeping genes via multilocus enzyme electrophoresis. Our data reveal that EAEC strains are heterogeneous with respect to chromosomal and plasmid-borne genes but that the majority harbor a member of a conserved family of virulence plasmids. Comparison of plasmid and chromosomal relatedness of strains suggests clonality of chromosomal markers and a limited transfer model of plasmid distribution.

The great escape: structure and function of the autotransporter proteins.

The autotransporters, a family of secreted proteins from Gram-negative bacteria, possess an overall unifying structure comprising three functional domains: the amino-terminal leader sequence, the secreted mature protein (passenger domain) and a carboxy-terminal (beta-) domain that forms a beta-barrel pore to allow secretion of the passenger protein. Members of this family have been implicated as important or putative virulence factors in many Gram-negative pathogens.

Cloning and sequence of a 3.835 kbp DNA fragment containing the HIS4 gene and a fragment of a PEX5-like gene from Candida albicans.

We have isolated the Candida albicans HIS4 (CaHIS4) gene by complementation of a his4-34 Saccharomyces cerevisiae mutant. The sequenced DNA fragment contains a putative ORF of 2514 bp, whose translation product shares a global identity of 44% and 55% to the His4 protein homologs of S. cerevisiae and Kluyveromyces lactis, respectively. Analysis of CaHIS4 sequence suggests that, similarly to S. cerevisiae HIS4, it codes for a polypeptide having three separate enzymatic activities (phosphoribosyl-AMP cyclohydrolase, phosphoribosyl-ATP pyrophosphohydrolase and histidinol dehydrogenase) which reside in different domains of the protein. A C. albicans his4 strain is complemented with this gene when using a C. albicans-S. cerevisiae-Escherichia coli shuttle vector, thus enabling the construction of a host system for C. albicans genetic manipulation. In addition, upstream of the sequenced CaHIS4 sequence, we have found the 3'-terminal half of a gene encoding a PEX5-like protein.