Glycogen Synthase Kinase 3α Is the Main Isoform That Regulates the Transcription Factors Nuclear Factor-Kappa B and cAMP Response Element Binding in Bovine Endothelial Cells Infected with Staphylococcus aureus.

Glycogen synthase kinase 3 (GSK3) is a constitutive enzyme implicated in the regulation of cytokine expression and the inflammatory response during bacterial infections. Mammals have two GSK3 isoforms named GSK3α and GSK3ß that plays different but often overlapping functions. Although the role of GSK3ß in cytokine regulation during the inflammatory response caused by bacteria is well described, GSK3α has not been found to participate in this process. Therefore, we tested if GSK3α may act as a regulatory isoform in the cytokine expression by bovine endothelial cells infected with Staphylococcus aureus because this bacterium is one of the major pathogens that cause tissue damage associated with inflammatory dysfunction. Interestingly, although both isoforms were phosphorylated-inactivated, we consistently observed a higher phosphorylation of GSK3α at Ser21 than that of GSK3ß at Ser9 after bacterial challenge. During a temporal course of infection, we characterized a molecular switch from pro-inflammatory cytokine expression (IL-8), promoted by nuclear factor-kappa B (NF-κB), at an early stage (2 h) to an anti-inflammatory cytokine expression (IL-10), promoted by cAMP response element binding (CREB), at a later stage (6 h). We observed an indirect effect of GSK3α activity on NF-κB activation that resulted in a low phosphorylation of CREB at Ser133, a decreased interaction between CREB and the co-activator CREB-binding protein (CBP), and a lower expression level of IL-10. Gene silencing of GSK3α and GSK3ß with siRNA indicated that GSK3α knockout promoted the interaction between CREB and CBP that, in turn, increased the expression of IL-10, reduced the interaction of NF-κB with CBP, and reduced the expression of IL-8. These results indicate that GSK3α functions as the primary isoform that regulates the expression of IL-10 in endothelial cells infected with S. aureus.

Effect of antimicrobial peptides derived from human cathelicidin LL-37 on Entamoeba histolytica trophozoites.

The human cathelicidin hCAP18/LL-37 is an antimicrobial protein consisting of a conserved N-terminal prosequence called the cathelin-like domain and a C-terminal peptide called LL-37. This peptide contains 37 amino acid residues, and several truncated variants obtained from natural sources or by chemical synthesis differ in their capability to damage Gram positive and Gram negative bacteria as well as Candida albicans. KR-12 is the shortest peptide (12 amino acids) of LL-37 that has conserved antibacterial activity. In addition to LL-37, other active cathelicidin-derived peptides have been reported; for instance, the peptides KR-20, a 20-aa derivative of LL-37, and KS-30, a 30-aa derivative of LL-37, have been found in human sweat. Both peptides exhibit an overall increased antibacterial and antifungal activity when compared with LL-37. We investigated the effect of LL-37 and three peptides derived from this antimicrobial molecule, KR-12, KR-20 and KS-30, on the integrity of Entamoeba histolytica trophozoites. The four peptides showed effects on E. histolytica integrity and viability in the concentration range of 10-50 µM. The peptides KR-12, KR-20, KS-30 and LL-37 differed in their capability to damage the parasite integrity, with KR-20 being the most effective and with KR-12 and LL-37 being less active. These results demonstrate the ability of antimicrobial peptides derived from human cathelicidin to damage Entamoeba trophozoites. Moreover, it was shown that the integrity of the peptides is altered in the presence of an ameba soluble fraction with cysteine protease activity.

Degradation of human secretory IgA1 and IgA2 by Entamoeba histolytica surface-associated proteolytic activity.

The protozoan Entamoeba histolytica is the etiological agent of amebiasis, an infection with high prevalence worldwide. The host-ameba relationship outcome depends on parasite and host factors, and among these is secretory IgA. These antibodies reduce mucosal colonization by pathogens and neutralize a variety of toxins and enzymes. The functionality of secretory IgA depends on its integrity. Some bacteria produce IgA proteases that cleave mainly the IgA1 subclass; live E. histolytica trophozoites, and other ameba fractions are also able to degrade human IgA. The aim of this study was to determine if serum and secretory IgA, its subclasses and secretory component, are degraded by cysteine proteases, which are present and active on the surface of glutaraldehyde-fixed amebas. It was observed that secretory IgA1, IgA2, free and IgA-bound secretory component were degraded by E. histolytica surface-associated cysteine proteinases. Secretory IgA2, although it was degraded, conserved its ability to agglutinate live amebas better than IgA1. Therefore, while specificity of known ameba cysteine proteases is cathepsin B-like and is different from bacterial IgA proteases, IgA2 was functionally more resistant than IgA1 to ameba surface-associated cysteine protease degradation, similar to the greater resistance of IgA2 to bacterial IgA-specific proteases.