Angeles Cancino-Rodezno

The mitogen-activated protein kinase p38 is involved in insect defense against Cry toxins from Bacillus thuringiensis

The insecticidal Cry toxins are pore-forming toxins produced by the bacteria Bacillus thuringiensis that disrupt insect-midgut cells. In this work we analyzed the response of two different insect orders, the Lepidopteran Manduca sexta and Dipteran Aedes aegypti to highly specific Cry toxins, Cry1Ab and Cry11Aa, respectively. One pathway activated in different organisms in response to a variety of pore-forming toxins is the mitogen-activated protein kinase p38 pathway (MAPK p38) that activates a complex defense response. We analyzed the MAPK p38 activation by immunodetection of its phosphorylated isoform, and the induction of p38 by RT-PCR, real-time PCR quantitative assays and immunodetection. We show that MAPK p38 is activated at postraductional level after Cry toxin intoxication in both insect orders. We detected the p38 induction at the transcriptional and traductional level, and observed a different response. In these three levels, we found that both insects respond to Cry toxin action but M. sexta responses more strongly than A. aegypti. Gene silencing of MAPK p38 in vivo, resulted in both insect species becoming hypersensitive to Cry toxin action, suggesting that the MAPK p38 pathway is involved in insect defense against Bt Cry toxins. This finding may have biotechnological applications for enhancing the activity of some Bt Cry toxins against specific insect pests.

Role of MAPK p38 in the cellular responses to pore-forming toxins.

Understanding the mechanism of action of pore-forming toxins (PFTs) produced by different bacteria, as well as the host responses to toxin action, would provide ways to deal with these pathogenic bacteria. PFTs affect the permeability of target cells by forming pores in their plasma membrane. Target organisms may overcome these effects by triggering intracellular responses that have evolved as defense mechanisms to PFT. Among them it is well documented that stress-activated protein kinases, and specially MAPK p38 pathway, play a crucial role triggering defense responses to several PFTs in different eukaryotic cells. In this review we describe different intracellular effects induced by PFTs in eukaryotic cells and highlight diverse responses activated by p38 pathway.

Comparative proteomic analysis of Aedes aegypti larval midgut after intoxication with Cry11Aa toxin from Bacillus thuringiensis.

Cry toxins produced by Bacillus thuringiensis bacteria are environmentally safe alternatives to control insect pests. They are pore-forming toxins that specifically affect cell permeability and cellular integrity of insect-midgut cells. In this work we analyzed the defensive response of Aedes aegypti larva to Cry11Aa toxin intoxication by proteomic and functional genomic analyses. Two dimensional differential in-gel electrophoresis (2D-DIGE) was utilized to analyze proteomic differences among A. aegypti larvae intoxicated with different doses of Cry11Aa toxin compared to a buffer treatment. Spots with significant differential expression (p<0.05) were then identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS), revealing 18 up-regulated and seven down-regulated proteins. The most abundant subcategories of differentially expressed proteins were proteins involved in protein turnover and folding, energy production, and cytoskeleton maintenance. We selected three candidate proteins based on their differential expression as representatives of the different functional categories to perform gene silencing by RNA interference and analyze their functional role. The heat shock protein HSP90 was selected from the proteins involved in protein turnover and chaperones; actin, was selected as representative of the cytoskeleton protein group, and ATP synthase subunit beta was selected from the group of proteins involved in energy production. When we affected the expression of ATP synthase subunit beta and actin by silencing with RNAi the larvae became hypersensitive to toxin action. In addition, we found that mosquito larvae displayed a resistant phenotype when the heat shock protein was silenced. These results provide insight into the molecular components influencing the defense to Cry toxin intoxication and facilitate further studies on the roles of identified genes.

Aedes aegypti alkaline phosphatase ALP1 is a functional receptor of Bacillus thuringiensis Cry4Ba and Cry11Aa toxins

Bacillus thuringiensis subs. israelensis produces at least three Cry toxins (Cry4Aa, Cry4Ba, and Cry11Aa) that are active against Aedes aegypti larvae. Previous work characterized a GPI-anchored alkaline phosphatase (ALP1) as a Cry11Aa binding molecule from the gut of A. aegypti larvae. We show here that Cry4Ba binds ALP1, and that the binding and toxicity of Cry4Ba mutants located in loop 2 of domain II is correlated. Also, we analyzed the contribution of ALP1 toward the toxicity of Cry4Ba and Cry11Aa toxins by silencing the expression of this protein though RNAi. Efficient silencing of ALP1 was demonstrated by real-time quantitative PCR (qPCR) and Western blot. ALP1 silenced larvae showed tolerance to both Cry4Ba and Cry11Aa although the silenced larvae were more tolerant to Cry11Aa in comparison to Cry4Ba. Our results demonstrate that ALP1 is a functional receptor that plays an important role in the toxicity of the Cry4Ba and Cry11Aa proteins.

Role of UPR pathway in defense response of Aedes aegypti against Cry11Aa toxin from Bacillus thuringiensis.

The insecticidal Cry toxins are pore-forming toxins produced by the bacteria Bacillus thuringiensis that disrupt insect-midgut cells. Cells can trigger different survival mechanisms to counteract the effects of sub-lytic doses of pore forming toxins. Particularly, two signaling pathways have been demonstrated to play a role in the defense mechanism to other toxins in Caenorhabditis elegans and in mammalian cells. These are the unfolded protein response (UPR) and the sterol regulatory element binding proteins (SREBP) pathways, which are proposed to facilitate membrane repair responses. In this work we analyzed the role of these pathways in Aedes aegypti response to intoxication with Cry11Aa toxin. We show that UPR is activated upon toxin ingestion. The role of these two pathways was analyzed in vivo by using RNA interference. We silenced the expression of specific proteins in A. aegypti larvae. Gene silencing of Ire-1 and Xbp-1 proteins from UPR system, resulted in hypersensitive to Cry11Aa toxin action. In contrast, silencing of Cas-1, Scap and S2P from SREBP pathway had no affect on Cry11Aa toxicity in A. aegypti larvae. However, the role of SREBP pathway requires further studies to be conclusive. Our data indicate that the UPR pathway is involved in the insect defense against Cry toxins.

Defense and death responses to pore forming toxins

Abstract Pore forming toxins (PFT) are important virulence factors produced by bacteria to kill eukaryotic cells by forming holes in the cellular membrane. They represent a diverse group of proteins with a wide range of target cells. Although the amino acid sequence is not conserved among the different PFT, many of them share some aspects of their mechanism of action. In general, the mode of action of PFT involves receptor recognition, activation by proteases, and aggregation into oligomeric-structures that insert into the membrane to form ionic pores. Beside the pore formation activity, PFT may have other effects during its interaction with their target cells such as intra-cellular signaling or transport of other enzymatic components, as in the case of anthrax or diphtheria toxins produced by Bacillus anthracis and Corynebacterium diphtheria, respectively (Parker and Feil, 2005). Although PFT have evolved as a pathogenic mechanism, some of them have great impact in society since they have different applications in biotechnology or are used as therapeutic agents, or as tools in the study of cell biology (Schiavo and van der Goot, 2001). On the other side, their target organisms have evolved different mechanisms to counter toxin action. Understanding the mechanism of action of PFT as well as the host responses to toxin action would provide ways to deal with these pathogens or with emerging pathogens and more importantly to improve the action of toxins that have biotechnological applications. In this review we will describe the intracellular effects induced by some PFT and the cellular responses evolved by eukaryotic cell to overcome PFT action.

Neurotrophin-dependent plasticity of neurotransmitter segregation in the rat superior cervical ganglion in vivo.

Neurons are able to segregate transmitters to different axon endings. Segregation is a plastic neuronal feature; it can be modulated by synaptic environment. We have demonstrated that neurotrophin and other cellular factors regulate segregation in sympathetic neurons in culture. Herein we tested the hypothesis that sympathetic neurons in vivo are also capable to exhibit neurotrophin‐dependent plasticity of segregation. To explore the effect of neurotrophin on segregation, we reduced ganglionic NGF content by the transection of postganglionic nerves (axotomy) of the superior cervical ganglia. By immunohistochemistry, Western blot, and PCR analyses, we explored the effect of axotomy on the NGF and BDNF content of ganglionic neurons, and on the segregation extent of vesicular acetylcholine transporter (VAChT) and methionine enkephalin (mENK) in pre‐ganglionic varicosities. We analyzed NGF‐dependence of the changes found by applying exogenous NGF. Axotomy reduced ganglionic NGF and BDNF content, increased NGF transcripts, and increased VAChT‐mENK segregation. Axotomy also increased the number of VAChT immunopositive varicosities, and caused the appearance of a population of VAChT‐, mENK‐ or SV2‐containing varicosities lacking Synaptophysin (Syn). Administration of NGF prevented changes in NGF content, kept NGF transcripts increased, and counteracted changes in segregation and in the number of cholinergic varicosities. The exogenous NGF did not preclude change in BDNF content or in the occurrence of the VAChT‐ or mENK‐containing varicosities lacking Syn. Data demonstrate that segregation of transmitters in vivo is plastic and it is modulated by environmental signals like NGF. We propose a possible functional correlate of segregation plasticity in the sympathetic ganglia.

Aedes aegypti Mos20 Cells Internalizes Cry Toxins by Endocytosis, and Actin Has a Role in the Defense against Cry11Aa Toxin

Bacillus thuringiensis (Bt) Cry toxins are used to control Aedes aegypti, an important vector of dengue fever and yellow fever. Bt Cry toxin forms pores in the gut cells, provoking larvae death by osmotic shock. Little is known, however, about the endocytic and/or degradative cell processes that may counteract the toxin action at low doses. The purpose of this work is to describe the mechanisms of internalization and detoxification of Cry toxins, at low doses, into Mos20 cells from A. aegypti, following endocytotic and cytoskeletal markers or specific chemical inhibitors. Here, we show that both clathrin-dependent and clathrin-independent endocytosis are involved in the internalization into Mos20 cells of Cry11Aa, a toxin specific for Dipteran, and Cry1Ab, a toxin specific for Lepidoptera. Cry11Aa and Cry1Ab are not directed to secretory lysosomes. Instead, Mos20 cells use the Rab5 and Rab11 pathways as a common mechanism, most probably for the expulsion of Cry11Aa and Cry1Ab toxins. In conclusion, we propose that endocytosis is a mechanism induced by Cry toxins independently of specificity, probably as part of a basal immune response. We found, however, that actin is necessary for defense-specific response to Cry11Aa, because actin-silenced Mos20 cells become more sensitive to the toxic action of Cry11A toxin. Cry toxin internalization analysis in insect cell lines may contribute to a better understanding to Cry resistance in mosquitoes.

Presence of Functional Neurotrophin TrkB Receptors in the Rat Superior Cervical Ganglion

Sympathetic neurons express the neurotrophin receptors TrkA, p75NTR, and a non-functional truncated TrkB isoform (TrkB-Tc), but are not thought to express a functional full-length TrkB receptor (TrkB-Fl). We, and others, have demonstrated that nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) modulate synaptic transmission and synaptic plasticity in neurons of the superior cervical ganglion (SCG) of the rat. To clarify whether TrkB is expressed in sympathetic ganglia and contributes to the effects of BDNF upon sympathetic function, we characterized the presence and activity of the neurotrophin receptors expressed in the adult SCG compared with their presence in neonatal and cultured sympathetic neurons. Here, we expand our previous study regarding the immunodetection of neurotrophin receptors. Immunohistochemical analysis revealed that 19% of adult ganglionic neurons expressed TrkB-Fl immunoreactivity (IR), 82% expressed TrkA-IR, and 51% expressed p75NTR-IR; TrkB-Tc would be expressed in 36% of neurons. In addition, using Western-blotting and reverse transcriptase polymerase chain reaction (RT-PCR) analyses, we confirmed the expression of TrkB-Fl and TrkB-Tc protein and mRNA transcripts in adult SCG. Neonatal neurons expressed significantly more TrkA-IR and TrkB-Fl-IR than p75NTR-IR. Finally, the application of neurotrophin, and high frequency stimulation, induced the activation of Trk receptors and the downstream PI3-kinase (phosphatidyl inositol-3-kinase) signaling pathway, thus evoking the phosphorylation of Trk and Akt. These results demonstrate that SCG neurons express functional TrkA and TrkB-Fl receptors, which may contribute to the differential modulation of synaptic transmission and long-term synaptic plasticity.