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Modulation of the CXCL12-CXCR4 signaling axis is of the utmost importance due to its central involvement in several pathological disorders, including inflammatory diseases and cancer. Among the different currently available drugs that inhibit CXCR4 activation, motixafortide-a best-in-class antagonist of this GPCR receptor-has exhibited promising results in preclinical studies of pancreatic, breast, and lung cancers. However, detailed information on the interaction mechanism of motixafortide is still lacking. Here, we characterize the motixafortide/CXCR4 and CXCL12/CXCR4 protein complexes by using computational techniques including unbiased all-atom molecular dynamics simulations. Our microsecond-long simulations of the protein systems indicate that the agonist triggers changes associated with active-like GPCR conformations, while the antagonist favors inactive conformations of CXCR4. Detailed ligand-protein analysis indicates the importance of motixafortide's six cationic residues, all of which established charge-charge interactions with acidic CXCR4 residues. Furthermore, two synthetic bulky chemical moieties of motixafortide work in tandem to restrict the conformations of important residues associated with CXCR4 activation. Our results not only elucidate the molecular mechanism by which motixafortide interacts with the CXCR4 receptor and stabilizes its inactive states, but also provide essential information to rationally design CXCR4 inhibitors that preserve the outstanding pharmacological features of motixafortide.

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Knowledge of glycogen synthase kinase 3ß (GSK3ß) activity and the molecules identified that regulate its function in infections caused by pathogenic microorganisms is crucial to understanding how the intensity of the inflammatory response can be controlled in the course of infections. In recent years many reports have described small molecular weight synthetic and natural compounds, proteins, and interference RNA with the potential to regulate the GSK3ß activity and reduce the deleterious effects of the inflammatory response. Our goal in this review is to summarize the most recent advances on the role of GSK3ß in the inflammatory response caused by bacteria, bacterial virulence factors (i.e. LPS and others), viruses, and parasites and how the regulation of its activity, mainly its inhibition by different type of molecules, modulates the inflammation.

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The biological activity of the enzyme glycogen synthase kinase-3 (GSK3) is fulfilled by two paralogs named GSK3α and GSK3ß, which possess both redundancy and specific functions. The upregulated activity of these proteins is linked to the development of disorders such as neurodegenerative disorders (ND) and cancer. Although various chemical inhibitors of these enzymes restore the brain functions in models of ND such as Alzheimer's disease (AD), and reduce the proliferation and survival of cancer cells, the particular contribution of each paralog to these effects remains unclear as these molecules downregulate the activity of both paralogs with a similar efficacy. Moreover, given that GSK3 paralogs phosphorylate more than 100 substrates, the simultaneous inhibition of both enzymes has detrimental effects during long-term inhibition. Although the GSK3ß kinase function has usually been taken as the global GSK3 activity, in the last few years, a growing interest in the study of GSK3α has emerged because several studies have recognized it as the main GSK3 paralog involved in a variety of diseases. This review summarizes the current biological evidence on the role of GSK3α in AD and various types of cancer. We also provide a discussion on some strategies that may lead to the design of the paralog-specific inhibition of GSK3α.

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The Wnt/ß-catenin signaling pathway is crucial to regulate cell proliferation and polarity, cell determination, and tissue homeostasis. The activation of Wnt/ß-catenin signaling is based on the interaction between Wnt glycoproteins and seven transmembrane receptors-Frizzled (Fzd). This binding promotes recruitment of the scaffolding protein Disheveled (Dvl), which results in the phosphorylation of the co-receptor LRP5/6. The resultant molecular complex Wnt-Fzd-LRP5/6-Dvl forms a structural region for Axin interaction that disrupts Axin-mediated phosphorylation/degradation of the transcriptional co-activator ß-catenin, thereby allowing it to stabilize and accumulate in the nucleus where it activates the expression of Wnt-dependent genes. Due to the prominent physiological function, the Wnt/ß-catenin signaling must be strictly controlled because its dysregulation, which is caused by different stimuli, may lead to alterations in cell proliferation, apoptosis, and inflammation-associated cancer. The virulence factors from pathogenic bacteria such as Salmonella enterica sv Typhimurium, Helicobacter pylori, Mycobacterium tuberculosis, Pseudomonas aeruginosa, Citrobacter rodentium, Clostridium difficile, Bacteroides fragilis, Escherichia coli, Haemophilus parasuis, Lawsonia intracellularis, Shigella dysenteriae, and Staphylococcus epidermidis employ a variety of molecular strategies to alter the appropriate functioning of diverse signaling pathways. Among these, Wnt/ß-catenin has recently emerged as an important target of several virulence factors produced by bacteria. The mechanisms used by these factors to interfere with the activity of Wnt/ß-catenin is diverse and include the repression of Wnt inhibitors' expression by the epigenetic modification of histones, blocking Wnt-Fzd ligand binding, activation or inhibition of ß-catenin nuclear translocation, down- or up-regulation of Wnt family members, and inhibition of Axin-1 expression that promotes ß-catenin activity. Such a variety of mechanisms illustrate an evolutionary co-adaptation of eukaryotic molecular signaling to a battery of soluble or structural components synthesized by pathogenic bacteria. This review gathers the recent efforts to elucidate the mechanistic details through which bacterial virulence factors modulate Wnt/ß-catenin signaling and its physiological consequences concerning the inflammatory response and cancer.

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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.

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Early sensing of pathogenic bacteria by the host immune system is important to develop effective mechanisms to kill the invader. Microbial recognition, activation of signaling pathways, and effector mechanisms are sequential events that must be highly controlled to successfully eliminate the pathogen. Host recognizes pathogens through pattern-recognition receptors (PRRs) that sense pathogen-associated molecular patterns (PAMPs). Some of these PRRs include Toll-like receptors (TLRs), nucleotide-binding oligomerization domain-like receptors (NLRs), retinoic acid-inducible gene-I- (RIG-I-) like receptors (RLRs), and C-type lectin receptors (CLRs). TLRs and NLRs are PRRs that play a key role in recognition of extracellular and intracellular bacteria and control the inflammatory response. The activation of TLRs and NLRs by their respective ligands activates downstream signaling pathways that converge on activation of transcription factors, such as nuclear factor-kappaB (NF-κB), activator protein-1 (AP-1) or interferon regulatory factors (IRFs), leading to expression of inflammatory cytokines and antimicrobial molecules. The goal of this review is to discuss how the TLRs and NRLs signaling pathways collaborate in a cooperative or synergistic manner to counteract the infectious agents. A deep knowledge of the biochemical events initiated by each of these receptors will undoubtedly have a high impact in the design of more effective strategies to control inflammation.

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Innate immunity against pathogenic bacteria is critical to protect host cells from invasion and infection as well as to develop an appropriate adaptive immune response. During bacterial infection, different signaling transduction pathways control the expression of a wide range of genes that orchestrate a number of molecular and cellular events to eliminate the invading microorganisms and regulate inflammation. The inflammatory response must be tightly regulated because uncontrolled inflammation may lead to tissue injury. Among the many signaling pathways activated, the canonical Wnt/ß-catenin has been recently shown to play an important role in the expression of several inflammatory molecules during bacterial infections. Our main goal in this review is to discuss the mechanism used by several pathogenic bacteria to modulate the inflammatory response through the Wnt/ß-catenin signaling pathway. We think that a deep insight into the role of Wnt/ß-catenin signaling in the inflammation may open new venues for biotechnological approaches designed to control bacterial infectious diseases.

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Glycogen synthase kinase 3ß (GSK3ß) plays a fundamental role during the inflammatory response induced by bacteria. Depending on the pathogen and its virulence factors, the type of cell and probably the context in which the interaction between host cells and bacteria takes place, GSK3ß may promote or inhibit inflammation. The goal of this review is to discuss recent findings on the role of the inhibition or activation of GSK3ß and its modulation of the inflammatory signaling in monocytes/macrophages and epithelial cells at the transcriptional level, mainly through the regulation of nuclear factor-kappaB (NF-κB) activity. Also included is a brief overview on the importance of GSK3 in non-inflammatory processes during bacterial infection.

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Internalization of Staphylococcus aureus in bovine endothelial cells (BEC) is increased by tumor necrosis factor alpha stimulation and NF-κB activation. Because the phosphoinositide-3-kinase (PI3K)-Akt signaling pathway also modulates NF-κB activity, we considered whether the internalization of S. aureus by BEC is associated with the activity of PI3K and Akt. We found a time- and multiplicity of infection-dependent phosphorylation of Akt on Ser473 in BEC infected with S. aureus. This phosphorylation was inhibited by LY294002 (LY), indicating the participation of PI3K. Inhibition of either PI3K with LY or wortmannin, or Akt with SH-5, strongly reduced the internalization of S. aureus. Transfection of BEC with a dominant-negative form of the Akt gene significantly decreased S. aureus internalization, whereas transfection with the constitutively active mutant increased the number of internalized bacterium. Inhibition of PDK1 activity with OSU-03012 did not affect the level of S. aureus internalization, demonstrating that phosphorylation of Akt on Thr308 is not important for this process. Compared to the untreated control, the adherence of S. aureus to the surface of BEC was unaltered when cells were transfected or incubated with the pharmacological inhibitors. Furthermore, Akt activation by internalized S. aureus triggered a time-dependent phosphorylation of glycogen synthase kinase-3α (GSK-3α) on Ser21 and GSK-3ß on Ser9 that was partially inhibited with SH-5. Finally, treatment of BEC with LY prior to S. aureus infection inhibited the NF-κB p65 subunit phosphorylation on Ser536, indicating the involvement of PI3K. These results suggest that PI3K-Akt activity is important for the internalization of S. aureus and phosphorylation of GSK-3α, GSK-3ß, and NF-κB.

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Staphylococcus epidermidis is an environmental opportunistic pathogen associated with bovine intramammary infections. In bacterial infections, the endothelial tissue plays an important role during inflammation and it is the target of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha). Therefore, this work was designed to explore the effect of TNF-alpha on the interaction of S. epidermidis with bovine endothelial cells (BEC). We show that cell signaling activated by TNF-alpha caused a marked reduction in the number of intracellular S. epidermidis, suggesting that molecules participating in this pathway were involved in the internalization of this bacterium. We also found that S. epidermidis internalization was not associated with basal levels of nuclear factor kappa B (NF-kappaB) activity because the intracellular number of bacteria recovered after treating BEC with the NF-kappaB inhibitors, SN50 or BAY 11-7083, was similar to that of the untreated control. Interestingly, inhibition of the basal activity of JNK with SP600125 and p38 with SB203580 caused a decrease in the number of intracellular S. epidermidis. These results suggest that activation of the signaling pathway initiated by TNF-alpha could play an important role in the phagocytosis of this bacterium. However, the basal activity of NF-kappaB was shown not to be important for the internalization process of S. epidermidis.

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This work describes a novel rolling-circle replicating (RCR) plasmid pBMBt1 from Bacillus thuringiensis subsp. darmstadiensis (INTA Mo14-4) encoding an insecticidal crystal protein-like gene. pBMBt1 (6700 bp) contains three ORFs and their putative transcription initiation sites and Shine-Dalgarno sequences were localized. ORF1 encodes a 34.6 kDa protein which showed identity with the protein CryC53 from B. thuringiensis subsp. cameroun (24.6%), the Cry15Aa insecticidal crystal protein from B. thuringiensis subsp. thompsoni (21.9%) and the Mtx3 protein from Bacillus sphaericus (27.8%). The ORF2 (52.3 kDa) showed a 74% identity with the Mob protein coded by pUIBI-1 from B. thuringiensis subsp. entomocidus and 64% identity with the Mob protein of pBMY1 from Bacillus mycoides; both Mob proteins belong to the pMV158 superfamily. To evaluate the Mob protein, the plasmid pHTMob14-4 was constructed. This plasmid shows transfer frequencies of 9.1x10(-6) in B. thuringiensis subsp. israelensis (4Q7Gm(R)). The ORF3 (23.6 kDa) gene product is homologous to the Rep protein from the plasmid pBMYdx of B. mycoides (37.6%). A putative double-strand origin with significant homology to that of B. thuringiensis plasmids, and an ssoA-type single-strand origin were also identified. Detection of single-stranded pBMBt1 DNA replicating intermediaries suggests that replication occurs via the rolling-circle mechanism.

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This work describes a novel plasmid encoding resistance to lincomycin in a staphylococcal isolate associated with mastitis infection from dairy cows. The cryptic plasmid pBMSa1 (2750 bp) of Staphylococcus aureus SA35 was subcloned and sequenced. Two ORFs (ORF1 and ORF2) were identified, and their putative transcription initiation and Shine-Dalgarno sequence were localized. ORF1 encodes a 334-residue protein almost identical to the putative Rep proteins of previously sequenced S. aureus rolling-circle-replicating plasmids. ORF2 encodes a 162-amino acid protein sharing a high degree of homology with LinA proteins (lincosamide O-nucleotidyltransferases) described in a variety of S. aureus strains. Intracellular single-stranded pBMSa1 DNA replicating intermediaries were detected, suggesting replication via the rolling-circle mechanism. A putative double-strand origin with significant homology to that of pC194 and a ssoA-type single-strand origin homologous sequence were also identified.

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Mitogen activated protein (MAP) kinase-like activity was determined in extracts obtained from transformed Catharanthus roseus hairy roots by the ability to phosphorylate myelin basic protein (MBP). Both in solution and in gel kinase assays showed variation in activity, depending on root developmental stage. In gel kinase assays, using the extract soluble fraction, revealed a 56 kDa polypeptide with phosphorylation activity on MBP. In addition, another 75 kDa polypeptide was observed in the particulate fraction. Immunodetection with monoclonal antibodies against ERK-1, a mammalian MAP kinase, and with anti-phosphotyrosine antibodies cross-reacted with the 56 kDa polypeptide, named SMK56, from the soluble fraction, suggesting that this polypeptide could be related with members of the MAP kinase family. Antibodies against the dually phosphorylated threonine-tyrosine motif, characteristic of active forms of MAP kinases, also cross-reacted with this 56 kDa polypeptide. Changes in the levels of SMK56 were detected within the first 30 min of root exposure to low temperatures or hypo-osmotic shock, suggesting that this protein may be involved in the perception of environmental changes.

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