The NF-κB factor Relish is essential for the epithelial defenses protecting against δ-endotoxin dependent effects of Bacillus thuringiensis israelensis infection in the Drosophila model.

Bacillus thuringiensis israelensis is largely regarded as the most selective, safe and ecofriendly biopesticide used for the control of insect vectors of human diseases. Bti enthomopathogenicity relies on the Cry and Cyt δ-endotoxins, produced as crystalline inclusions during sporulation. Insecticidal selectivity of Bti is mainly ascribed to the binding of the Cry toxins to receptors in the gut of target insects. However, the contribution of epithelial defenses in limiting Bti side effects in non-target species remains largely unexplored. Here, taking advantage of the genetically tractable Drosophila melanogaster model and its amenability for deciphering highly conserved innate immune defenses, we unravel a central role of the NF-κB factor Relish in the protection against the effects of ingested Bti spores in a non-susceptible host. Intriguingly, our data indicate that the Bti-induced Relish response is independent of its canonical activation downstream of peptidoglycan sensing and does not involve its longstanding role in the regulation of antimicrobial peptides encoding genes. In contrast, our data highlight a novel enterocyte specific function of Relish that is essential for preventing general septicemia following Bti oral infections strictly when producing δ-endotoxins. Altogether, our data provide novel insights into Bti-hosts interactions of prominent interest for the optimization and sustainability of insects' biocontrol strategies.

Protein Phosphatase 4 Negatively Regulates the Immune Deficiency-NF-κB Pathway during the Drosophila Immune Response.

The evolutionarily conserved immune deficiency (IMD) signaling pathway shields Drosophila against bacterial infections. It regulates the expression of antimicrobial peptides encoding genes through the activation of the NF-κB transcription factor Relish. Tight regulation of the signaling cascade ensures a balanced immune response, which is otherwise highly harmful. Several phosphorylation events mediate intracellular progression of the IMD pathway. However, signal termination by dephosphorylation remains largely elusive. Here, we identify the highly conserved protein phosphatase 4 (PP4) complex as a bona fide negative regulator of the IMD pathway. RNA interference-mediated gene silencing of PP4-19c, PP4R2, and Falafel, which encode the catalytic and regulatory subunits of the phosphatase complex, respectively, caused a marked upregulation of bacterial-induced antimicrobial peptide gene expression in both Drosophila melanogaster S2 cells and adult flies. Deregulated IMD signaling is associated with reduced lifespan of PP4-deficient flies in the absence of any infection. In contrast, flies overexpressing this phosphatase are highly sensitive to bacterial infections. Altogether, our results highlight an evolutionarily conserved function of PP4c in the regulation of NF-κB signaling from Drosophila to mammals.

A novel anti-dipteran Bacillus thuringiensis strain: Unusual Cry toxin genes in a highly dynamic plasmid environment.

Bacillus thuringiensis emerged as a major bioinsecticide on the global market. It offers a valuable alternative to chemical products classically utilized to control pest insects. Despite the efficiency of several strains and products available on the market, the scientific community is always on the lookout for novel toxins that can replace or supplement the existing products. In this study, H3, a novel B. thuringiensis strain showing mosquitocidal activity, was isolated from Lebanese soil and characterized at an in vivo, genomic and proteomic levels. H3 parasporal crystal is toxic on its own but displays an unusual killing profile with a higher LC50 than the reference B. thuringiensis serovar israelensis crystal proteins. In addition, H3 has a different toxicity order: it is more toxic to Aedes albopictus and Anopheles gambiae than to Culex pipiens Whole genome sequencing and crystal analysis revealed that H3 can produce eleven novel Cry proteins, eight of which are assembled in genes with an orf1-gap-orf2 organization, where orf2 is a potential Cry4-type crystallization domain. Moreover, pH3-180, the toxin-carrying plasmid, holds a wide repertoire of mobile genetic elements that amount to ca 22% of its size., including novel insertion sequences and class II transposable elements Two other large plasmids present in H3 carry genetic determinants for the production of many interesting molecules - such as chitinase, cellulase and bacitracin - that may add up to H3 bioactive properties. This study therefore reports a novel mosquitocidal Bacillus thuringiensis strain with unusual Cry toxin genes in a rich mobile DNA environment.IMPORTANCE Bacillus thuringiensis, a soil entomopathogenic bacteria, is at the base of many sustainable eco-friendly bio-insecticides. Hence stems the need to continually characterize insecticidal toxins. H3 is an anti-dipteran B. thuringiensis strain, isolated from Lebanese soil, whose parasporal crystal contains eleven novel Cry toxins and no Cyt toxins. In addition to its individual activity, H3 showed potential as a co-formulant with classic commercialized B. thuringiensis products, to delay the emergence of resistance and to shorten the time required for killing. On a genomic level, H3 holds three large plasmids, one of which carries the toxin-coding genes, with four occurrences of the distinct orf1-gap-orf2 organization. Moreover, this plasmid is extremely rich in mobile genetic elements, unlike its two co-residents. This highlights the important underlying evolutionary traits between toxin-carrying plasmids and the adaptation of a B. thuringiensis strain to its environment and insect host spectrum.

The fliK Gene Is Required for the Resistance of Bacillus thuringiensis to Antimicrobial Peptides and Virulence in Drosophila melanogaster.

Antimicrobial peptides (AMPs) are essential effectors of the host innate immune system and they represent promising molecules for the treatment of multidrug resistant microbes. A better understanding of microbial resistance to these defense peptides is thus prerequisite for the control of infectious diseases. Here, using a random mutagenesis approach, we identify the fliK gene, encoding an internal molecular ruler that controls flagella hook length, as an essential element for Bacillus thuringiensis resistance to AMPs in Drosophila. Unlike its parental strain, that is highly virulent to both wild-type and AMPs deficient mutant flies, the fliK deletion mutant is only lethal to the latter's. In agreement with its conserved function, the fliK mutant is non-flagellated and exhibits highly compromised motility. However, comparative analysis of the fliK mutant phenotype to that of a fla mutant, in which the genes encoding flagella proteins are interrupted, indicate that B. thuringiensis FliK-dependent resistance to AMPs is independent of flagella assembly. As a whole, our results identify FliK as an essential determinant for B. thuringiensis virulence in Drosophila and provide new insights on the mechanisms underlying bacteria resistance to AMPs.

Unusually High Prevalence of Cosecretion of Ambler Class A and B Carbapenemases and Nonenzymatic Mechanisms in Multidrug-Resistant Clinical Isolates of Pseudomonas aeruginosa in Lebanon.

The opportunistic pathogen, Pseudomonas aeruginosa, is a main cause of nosocomial infections in Lebanese hospitals. This pathogen is highly threatening due to its ability to develop multiresistance toward a large variety of antibiotics, including the carbapenem subgroup of ß-lactams. In this study, we surveyed the enzymatic and nonenzymatic mechanisms of carbapenem resistance in several multidrug-resistant (MDR) strains of P. aeruginosa isolated from patients suffering from nosocomial urinary tract infections in a Lebanese hospital. The occurrence of ß-lactamase-encoding genes notably GES, KPC, IMP, VIM, NDM, and OXA, which are characterized by a carbapenemase activity was checked by genomic analyses. Our results provide a first evidence of the occurrence of GES in clinical P. aeruginosa isolates resistant to carbapenems in Lebanon. More interestingly, we showed that almost 40% of the analyzed strains have acquired a dual-carbapenemase secretion of GES-6 and VIM-2 or IMP-15, this being a rare phenomenon among this type of multidrug resistance. Moreover, LC-MS/MS analyses revealed a high prevalence of another enzymatic mechanism of resistance; this is the coexistence of AmpC and Pdc-13 as well as a number of virulence proteins, for instance pilin, lytic transglycosylase, ecotin, chitin-binding protein (Cbp), and TolB-dependent receptor. It is to be noted that a mutation of the oprD2 gene encoding a porin selective for carbapenems has been detected in almost 66% of our strains. All in all, our study reveals by the use of different methods, unusual simultaneous enzymatic (GES, IMP, VIM, pdc13, and AmpC) and nonenzymatic mechanisms of resistance (reduction of OprD2 expression) for MDR Pseudomonas aeruginosa.

Characterization and Whole Genome Sequencing of AR23, a Highly Toxic Bacillus thuringiensis Strain Isolated from Lebanese Soil.

The demand for sustainable and eco-friendly control methods of pests and insects is increasing worldwide. From this came the interest in Bacillus thuringiensis, an entomopathogenic bacterium capable of replacing chemical pesticides. However, the possibility of pests developing resistance to a particular strain may impair its use, and there is a need to identify novel strains of this species as potential commercial biopesticides. B. thuringiensis sv. israelensis is one of the most successful serovars, widely commercialized for its activity against black fly and mosquito larvae. In this study, we isolated, characterized, and sequenced a new Lebanese B. thuringiensis sv. israelensis isolate, strain AR23. Compared to the commercialized reference strain AM65-52 (Vectobac®, Sumitomo), AR23 showed an increased activity against several mosquito species. The genomic analysis revealed that this strain, compared to AM65-52, possesses a simplified plasmid content and an additional functional cry4Ba coding gene that most likely accounts for the increased effectiveness of this strain in mosquito larvae killing.

DltX of Bacillus thuringiensis Is Essential for D-Alanylation of Teichoic Acids and Resistance to Antimicrobial Response in Insects.

The dlt operon of Gram-positive bacteria is required for the incorporation of D-alanine esters into cell wall-associated teichoic acids (TAs). Addition of D-alanine to TAs reduces the negative charge of the cell envelope thereby preventing cationic antimicrobial peptides (CAMPs) from reaching their target of action on the bacterial surface. In most gram-positive bacteria, this operon consists of five genes dltXABCD but the involvement of the first ORF (dltX) encoding a small protein of unknown function, has never been investigated. The aim of this study was to establish whether this protein is involved in the D-alanylation process in Bacillus thuringiensis. We, therefore constructed an in frame deletion mutant of dltX, without affecting the expression of the other genes of the operon. The growth characteristics of the dltX mutant and those of the wild type strain were similar under standard in vitro conditions. However, disruption of dltX drastically impaired the resistance of B. thuringiensis to CAMPs and significantly attenuated its virulence in two insect species. Moreover, high-performance liquid chromatography studies showed that the dltX mutant was devoid of D-alanine, and electrophoretic mobility measurements indicated that the cells carried a higher negative surface charge. Scanning electron microscopy experiments showed morphological alterations of these mutant bacteria, suggesting that depletion of D-alanine from TAs affects cell wall structure. Our findings suggest that DltX is essential for the incorporation of D-alanyl esters into TAs. Therefore, DltX plays a direct role in the resistance to CAMPs, thus contributing to the survival of B. thuringiensis in insects. To our knowledge, this work is the first report examining the involvement of dltX in the D-alanylation of TAs.

Advances in Myeloid-Like Cell Origins and Functions in the Model Organism Drosophila melanogaster.

Drosophila has long served as a valuable model for deciphering many biological processes, including immune responses. Indeed, the genetic tractability of this organism is particularly suited for large-scale analyses. Studies performed during the last 3 decades have proven that the signaling pathways that regulate the innate immune response are conserved between Drosophila and mammals. This review summarizes the recent advances on Drosophila hematopoiesis and immune cellular responses, with a particular emphasis on phagocytosis.

The multilayered innate immune defense of the gut.

In the wild, the fruit fly Drosophila melanogaster thrives on rotten fruit. The digestive tract maintains a powerful gut immune barrier to regulate the ingested microbiota, including entomopathogenic bacteria. This gut immune barrier includes a chitinous peritrophic matrix that isolates the gut contents from the epithelial cells. In addition, the epithelial cells are tightly sealed by septate junctions and can mount an inducible immune response. This local response can be activated by invasive bacteria, or triggered by commensal bacteria in the gut lumen. As with chronic inflammation in mammals, constitutive activation of the gut innate immune response is detrimental to the health of flies. Accordingly, the Drosophila gut innate immune response is tightly regulated to maintain the endogenous microbiota, while preventing infections by pathogenic microorganisms.

Sensing of 'danger signals' and pathogen-associated molecular patterns defines binary signaling pathways 'upstream' of Toll.

In drosophila, molecular determinants from fungi and Gram-positive bacteria are detected by circulating pattern-recognition receptors. Published findings suggest that such pattern-recognition receptors activate as-yet-unidentified serine-protease cascades that culminate in the cleavage of Spätzle, the endogenous Toll receptor ligand, and trigger the immune response. We demonstrate here that the protease Grass defines a common activation cascade for the detection of fungi and Gram-positive bacteria mediated by pattern-recognition receptors. The serine protease Persephone, shown before to be specific for fungal detection in a cascade activated by secreted fungal proteases, was also required for the sensing of proteases elicited by bacteria in the hemolymph. Hence, Persephone defines a parallel proteolytic cascade activated by 'danger signals' such as abnormal proteolytic activities.

Akirins are highly conserved nuclear proteins required for NF-kappaB-dependent gene expression in drosophila and mice.

During a genome-wide screen with RNA-mediated interference, we isolated CG8580 as a gene involved in the innate immune response of Drosophila melanogaster. CG8580, which we called Akirin, encoded a protein that acted in parallel with the NF-kappaB transcription factor downstream of the Imd pathway and was required for defense against Gram-negative bacteria. Akirin is highly conserved, and the human genome contains two homologs, one of which was able to rescue the loss-of-function phenotype in drosophila cells. Akirins were strictly localized to the nucleus. Knockout of both Akirin homologs in mice showed that one had an essential function downstream of the Toll-like receptor, tumor necrosis factor and interleukin (IL)-1beta signaling pathways leading to the production of IL-6. Thus, Akirin is a conserved nuclear factor required for innate immune responses.

Prophenoloxidase activation is not required for survival to microbial infections in Drosophila.

The antimicrobial defence of Drosophila relies on cellular and humoral processes, of which the inducible synthesis of antimicrobial peptides has attracted interest in recent years. Another potential line of defence is the activation, by a proteolytic cascade, of phenoloxidase, which leads to the production of quinones and melanin. However, in spite of several publications on this subject, the contribution of phenoloxidase activation to resistance to infections has not been established under appropriate in vivo conditions. Here, we have isolated the first Drosophila mutant for a prophenoloxidase-activating enzyme (PAE1). In contrast to wild-type flies, PAE1 mutants fail to activate phenoloxidase in the haemolymph following microbial challenge. Surprisingly, we find that these mutants are as resistant to infections as wild-type flies, in the total absence of circulating phenoloxidase activity. This raises the question with regard to the precise function of phenoloxidase activation in defence, if any.