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BackgroundBacillus thuringiensis var. israelensis (Bti) is a natural larval mosquito pathogen producing pore-forming toxins targeting the midgut of Diptera larvae. It is used worldwide for mosquito control. Resistance mechanisms of an Aedes aegypti laboratory strain selected for 30 generations with field-collected leaf litter containing Bti toxins were investigated in larval midguts at two levels: 1. gene transcription using DNA microarray and RT-qPCR and 2. differential expression of brush border membrane proteins using DIGE (Differential In Gel Electrophoresis).ResultsSeveral Bti Cry toxin receptors including alkaline phosphatases and N-aminopeptidases and toxin-binding V-ATPases exhibited altered expression levels in the resistant strain. The under-expression of putative Bti-receptors is consistent with Bt-resistance mechanisms previously described in Lepidoptera. Four soluble metalloproteinases were found under-transcribed together with a drastic decrease of metalloproteinases activity in the resistant strain, suggesting a role in resistance by decreasing the amount of activated Cry toxins in the larval midgut.ConclusionsBy combining transcriptomic and proteomic approaches, we detected expression changes at nearly each step of the ingestion-to-infection process, providing a short list of genes and proteins potentially involved in Bti-resistance whose implication needs to be validated. Collectively, these results open the way to further functional analyses to better characterize Bti-resistance mechanisms in mosquitoes.

Alkaline phosphatases (ALPs, EC 3.1.3.1) isolated from lepidopteran and dipteran species are identified as receptors for Cry1Ac and Cry11Aa toxins, respectively [Jurat-Fuentes, J. L., and Adang, M. J. (2004) Eur. J. Biochem. 7, 3127-3135; Fernandez, L. E., et al. (2006) Biochem. J. 396, 77-84]. In our study, an alkaline phosphatase cDNA (AgALP1) was cloned from the midgut of Anopheles gambiae larvae. The encoded 63 kDa protein has a predicted glycosylphosphatidylinositol (GPI) anchor omega-site ((526)Asp), an N-glycosylation site ((239)Asn-Leu-Thr), and an O-glycosylation site ((312)Ser). AgALP1(t) was expressed in Escherichia coli and used to prepare antiserum and to analyze the interaction of AgALP with mosquitocidal Cry11Ba toxin. Anti-AgALP serum localized AgALP to the apical brush border in the anterior and posterior midgut of larvae and detected a 65 kDa species on a blot of brush border membrane vesicles (BBMVs) protein prepared from larvae. ALP activity was released from larval BBMVs prepared by phosphatidylinositol-specific phospholipase C (PIPLC) treatment, and after separation by two-dimensional gel electrophoresis and blotting, a chain of doublet spots at 65 kDa was detected by anti-AgALP. A subset of these doublet spots bound Cry11Ba on a reprobed blot. Heterologously expressed AgALP1(t) bound [(125)I]Cry11Ba on dot blots and reduced the level of binding of [(125)I]Cry11Ba to brush border membrane vesicles by 41%, a percentage comparable to that of unlabeled Cry11Ba and aminopeptidase AgAPN2(t1) peptide. AgALP1(t) binds Cry11Ba toxin with a high affinity (23.9 nM) and shares a binding site on Cry11Ba with AgAPN2(t1). In bioassays against An. gambiae larvae, the presence of AgALP1(t) reduced larval mortality from 78 to 8%. We conclude that AgALP1 is a binding protein and a functional receptor for Cry11Ba toxin.

Lipid rafts are microdomains in the plasma membrane of eukaryotic cells. Among their many functions, lipid rafts are involved in cell toxicity caused by pore forming bacterial toxins including Bacillus thuringiensis (Bt) Cry toxins. We isolated lipid rafts from brush border membrane vesicles (BBMV) of Aedes aegypti larvae as a detergent resistant membrane (DRM) fraction on density gradients. Cholesterol, aminopeptidase (APN), alkaline phosphatase (ALP) and the raft marker flotillin were preferentially partitioned into the lipid raft fraction. When mosquitocidal Cry4Ba toxin was pre-incubated with BBMV, Cry4Ba localized to lipid rafts. A proteomic approach based on one dimensional gel electrophoresis, in-gel trypsin digestion, followed by liquid chromatography-mass spectrometry (geLC-MS/MS) identified a total of 386 proteins. Of which many are typical lipid raft marker proteins including flotillins and glycosylphosphatidylinositol (GPI)-anchored proteins. Identified raft proteins were annotated in silico for functional and physicochemical characteristics. Parameters such as distribution of isoelectric point, molecular mass, and predicted post-translational modifications relevant to lipid raft proteins (GPI anchorage and myristoylation or palmitoylation) were analyzed for identified proteins in the DRM fraction. From a functional point of view, this study identified proteins implicated in Cry toxin interactions as well as membrane-associated proteins expressed in the mosquito midgut that have potential relevance to mosquito biology and vector management.

A molecular hydrogen (H 2 )-stimulated, chemolithoautotrophic growth mode for the gastric pathogen Helicobacter pylori is reported. In a culture medium containing peptides and amino acids, H 2 -supplied cells consistently achieved 40 to 60% greater growth yield in 16 h and accumulated 3-fold more carbon from [ 14 C]bicarbonate (on a per cell basis) in a 10-h period than cells without H 2 . Global proteomic comparisons of cells supplied with different atmospheric conditions revealed that addition of H 2 led to increased amounts of hydrogenase and the biotin carboxylase subunit of acetyl coenzyme A (acetyl-CoA) carboxylase (ACC), as well as other proteins involved in various cellular functions, including amino acid metabolism, heme synthesis, or protein degradation. In agreement with this result, H 2 -supplied cells contained 3-fold more ACC activity than cells without H 2 . Other possible carbon dioxide (CO 2 ) fixation enzymes were not up-expressed under the H 2 -containing atmosphere. As the gastric mucus is limited in carbon and energy sources and the bacterium lacks mucinase, this new growth mode may contribute to the persistence of the pathogen in vivo. This is the first time that chemolithoautotrophic growth is described for a pathogen. IMPORTANCEMany pathogens must survive within host areas that are poorly supplied with carbon and energy sources, and the gastric pathogen Helicobacter pylori resides almost exclusively in the nutritionally stringent mucus barrier of its host. Although this bacterium is already known to be highly adaptable to gastric niches, a new aspect of its metabolic flexibility, whereby molecular hydrogen use (energy) is coupled to carbon dioxide fixation (carbon acquisition) via a described carbon fixation enzyme, is shown here. This growth mode, which supplements heterotrophy, is termed chemolithoautotrophy and has not been previously reported for a pathogen. Helicobacter pylori is the causative agent of gastric ulcers and chronic gastritis (1). It infects about 50% of the world population (2), and if the infection persists without treatment, it can lead to the development of gastric cancers (3). H. pylori inhabits the gastric mucosa, an environment in which energy and carbon sources are limited. Most of the colonizing organisms within the host stomach are not associated with gastric epithelial cells; rather, they are found in the mucosa, where oxidizable carbon substrates are known to be scarce (4, 5). While the highly diverse glycans present in mucus can be accessed by certain microbiota (6), this requires specialized enzymes that seem to be lacking in Helicobacter species (7). Furthermore, the bacterium lacks a mucinase that would liberate utilizable substrates (8). Still, H. pylori seems to thrive in the mucosal environment, achieving typical viable numbers of 10 2 to 10 4 CFU per g or ml of stomach mucus (9). Carbon sources usable by H. pylori include small organic acids, amino acids, and peptides (8, 10-13), but the extent of this metabolism in vivo is not well understood...