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Inflammatory bowel disease (IBD) constitutes a substantial risk factor for colorectal cancer. Connexin 43 (Cx43) is a protein that forms gap junction (GJ) complexes involved in intercellular communication, and its expression is altered under pathological conditions, such as IBD and cancer. Recent studies have implicated epigenetic processes modulating DNA methylation in the pathogenesis of diverse inflammatory and malignant diseases. The ten-eleven translocation-2 (TET-2) enzyme catalyzes the demethylation, hence, regulating the activity of various cancer-promoting and tumor-suppressor genes.To investigate Cx43 and TET-2 expression levels and presence of 5-hydroxymethylcytosine (5-hmC) marks under inflammatory conditions bothTET-2 expression was evaluated in parental HT-29 cells and in HT-29 cells expressing low or high levels of Cx43, a putative tumor-suppressor gene whose expression varies in IBD and colorectal cancer, and which has been implicated in the inflammatory process and in tumor onset. The dextran sulfate sodium-induced colitis model was reproduced in BALB/c mice to evaluate the expression of TET-2 and Cx43 under inflammatory conditionsUnder inflammatory conditions, Cx43 and TET-2 expression levels increased compared to non-inflammatory conditions. TET-2 upregulation was more pronounced in Cx43-deficient cells. Moreover, colon tissue sections from normal, ulcerative colitis, and sporadic colon adenocarcinoma patients corroborated that Cx43 expression increased in IBD and decreased in adenocarcinoma, compared to tissues from non-IBD subjects. However, TET-2 expression and 5-hmC mark levels decreased in samples from patients with ulcerative colitis or cancer. Cx43 and TET-2 expression levels were also investigated in an experimental colitis mouse model. Interestingly, mice exposed to carbenoxolone (CBX), a GJ inhibitor, had upregulated TET-2 levels. Collectively, these results show that TET-2 levels and activity increased under inflammatory conditions, in cells downregulating gap junctional protein Cx43, and in colon tissues from mice exposed to CBX.These results suggest that TET-2 expression levels, as well as Cx43 expression levels, are modulated in models of intestinal inflammation. We hypothesize that TET-2 may demethylate genes involved in inflammation and tumorigenesis, such as Cx43, potentially contributing to intestinal inflammation and associated carcinogenesis.

Objective/Background Natural products (NPs) derived from microorganisms are the basis of a plethora of clinically utilized medications, namely, antimicrobial remedies. Although these secondary metabolites have been extensively explored all over the planet, they remain understudied in the Middle East and North Africa (MENA) region. Methods A literature search was conducted to first find NPs that were isolated from environmental fungi and bacteria that inhabit the soils and seawater of the MENA region. Then, purified molecules with biological activity against pathogenic bacteria, biofilms, fungi, and parasites were described in terms of structure, function, and location. Moreover, the methods that could be used to ameliorate the discovery of novel NPs from this region were investigated. Results A multitude of antimicrobial molecules from various chemical classes were found to be derived from the environmental microbes of MENA. Although many were rediscovered, some represented novel structural scaffolds for novel families of antimicrobial agents. Additionally, the geographical distribution showed a high number of these NPs were unraveled in a restricted area leaving much of MENA untapped. Furthermore, as relatively traditional and low-efficiency methods were typically used in the discovery process, advanced high-throughput techniques were suggested to enhance this practice at the regional level. Conclusion MENA represents a fairly unexploited region where antimicrobial drug discovery could be performed comprehensively through the concomitant exploration of untouched geographical locations and advanced molecular techniques.

Biopesticides, vectors for the development of the bioeconomy, represent a realistic alternative to petroleum-based chemical pesticides and their known harmfulness to humans and the environment. Bacillus thuringiensis is a gram-positive, spore-forming bacterium used for the production of biocontrol agents in agriculture. This insecticidal activity is mainly due to the production of parasporal δ-endotoxins consisting of an assembly of crystalline proteins called Cry. The European project IPM-4-Citrus (MSCA RISE, No. 734921, 2017-2023) aims to optimise these bioproductions from 3 strains of Bacillus thuringiensis subsp kurstaki (Lip, BLB1 and HD1) in a complex medium (wheat bran). The biochemical composition and nutritional limitations induced by the medium were identified. The physical properties of these heterogeneous suspensions are necessary for the definition of operating conditions favourable to scale-up. In this study, the morpho-granulometry of substrates, the ability to separate solid-liquid suspension and to clarify fermentation broth, and their rheological behaviour were studied as a function of substrate concentration and granulometry.

International audience; Bacillus thuringiensis is a microorganism used for the production of biopesticides worldwide. In the present paper, different kinetic models were analyzed to study and compare three different strains of Bt ssp kurstaki (LIP, BLB1, and HD1). Bioperformances (vegetative cell, spore, substrate, and protein) and successive culture phases (oxidative growth, limitation and sporulation, and protein release) were depicted with an overarching aim to estimate total protein productivity, yield, and titer. In the end, two models were calibrated using experimental dataset (11 batches culture in 3 L bioreactor with semisynthetic medium), subsequently validated, and statistically compared. Both models satisfactorily followed the dynamics of the experimental data. Finally, a dynamic model was selected following the Akaike information criterion (AIC).

Bacillus thuringiensis, a soil entomopathogenic bacterium, is at the base of many sustainable eco-friendly bioinsecticides. Hence stems the need to continually characterize insecticidal toxins., Bacillus thuringiensis has 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 in vivo at genomic and proteomic levels. H3 parasporal crystal is toxic on its own but displays an unusual killing profile with a higher 50% lethal concentration (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 11 novel Cry proteins, 8 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 paper 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 bacterium, is at the base of many sustainable eco-friendly bioinsecticides. Hence stems the need to continually characterize insecticidal toxins. H3 is an antidipteran B. thuringiensis strain, isolated from Lebanese soil, whose parasporal crystal contains 11 novel Cry toxins and no Cyt toxins. In addition to its individual activity, H3 showed potential as a coformulant 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 coresidents. 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 speckle imaging technique has been proven to be a reliable and effective method for real-time monitoring of the growth kinetics of any bacterium in suspension. To understand the interaction between the light and the bacterial density, a simulation of the bacterial growth of Bacillus thuringiensis was performed using calibrated microspheres of different concentrations and sizes. Results show that the decrease of speckle grain size with the increase of the medium scattering coefficient reveals the two essential phases of the bacterial growth: the exponential phase where the number of the bacteria increases and the stationary phase where sporulation and cell lysis occur.

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.

Additional file 3: Table S1. Pairwise distance estimation with Poisson correction between IS982 family elements, ordered according to the relationship dendrogram shown in Fig. 2. Elements belonging to each cluster (indicated on the left) are highlighted. These numbers reflect the possibility of aa substitution in a certain position of the protein, in a pairwise manner. For example, distance estimation values vary between zero and ca. 2.128. The higher the number, protein sequence identity percentage is lower.

Additional file 2: Figure S2. Multiple Sequence alignment of IS982 family transposases, ordered according to the relationship dendrogram (Fig. 2). Alignment was done using Mega-X via a ClustalW algorithm [34]. Only residues conserved at a minimum of 50% are highlighted. Predicted helix-turn-helix and DDE domains are indicated by orange and grey arrows. The catalytic triad DDE and the potential missing K/R residue are indicated by red and green marks, respectively.

Additional file 1: Figure S1. The first (Left End) and complement of the last (Right End) 25 nt of IS982 elements, ordered according to the relationship dendrogram shown in Fig. 2. Each nucleotide is colored differently.

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.

Modification of cell wall components is a prominent mean for pathogens to hinder host immune defenses. Here, using the Drosophila model, we aimed at characterizing the role of D-alanine esterification of teichoic acids (TAs) in the resistance of Bacillus thuringiensis to host defenses in vivo. We show that, by preventing cationic antimicrobial effectors-mediated bacterial lysis, this cell wall modification also limits the release of peptidoglycan immunostimulatory fragments thus impeding their sensing and the subsequent induction of the IMD- NF-κB pathway. Interestingly, we show that this strategy is also adopted by Lactobacillus plantarum, a bona fide commensal, to fine-tune its immunomodulatory potential in the Drosophila gut. Markedly, we show that the D-alanylation of TAs is essential for the resistance of L. plantarum to gut lysozyme. Altogether our data shed light on the mechanism underlying the persistence and the low immunostimulatory potential of L. plantarum in the Drosophila gut.

Table S1A. Accession numbers of the analyzed B. cereus s.l. replicons. B. thuringiensis cry- and B. cereus s.s. cry+ are placed under B. thuringiensis-like. Table S1B. Genomic details of eight B. thuringiensis cry- and four B. cereus s.s. cryâ +Â Table S2. Heat map of the total bcr number in the complete genomes of nine B. cereus s.l. species. The heat map is arranged by species and decreasing bcr1 occurrence. Table S3A. Correlation pairs for the 102 analyzed B. cereus s.l. genomes. Table S3B. Correlation pairs for the 102 analyzed B. cereus s.l. genomes by species. Table S4A. Heat map and distribution of group II intron types in the complete genomes of 9 B. cereus sensu lato species. Table S4B. Heat map and distribution of group II introns in the complete genomes of 9 B. cereus sensu lato species. (DOCX 1276 kb)

Background Bacillus cereus sensu lato s.l.) is a group of bacteria displaying close phylogenetic relationships but a high ecological diversity. The three most studied species are Bacillus anthracis, Bacillus cereus sensu stricto and Bacillus thuringiensis. While some species are pathogenic to mammals or associated with food poisoning, Bacillus thuringiensis is a well-known entomopathogenic bacterium used as biopesticide worldwide. B. cereus s.l. also contains a large variety of mobile genetic elements (MGEs). Results In this study, we detail the occurrence and plasmid vs. chromosome distribution of several MGEs in 102 complete and annotated genomes of B. cereus s.l. These MGEs include 16 Insertion Sequence (IS) families, the Tn3 family, 18 different Bacillus cereus repeats (BCRs) and 30 known group II introns. Conclusions Our analysis not only shows the diversity of these MGEs among strains of the same species and between different species within the B. cereus s.l. group, but also highlights the potential impact of these elements on the plasticity of the plasmid pool, and the TEs (Transposable Elements) - species relationship within B. cereus s.l. Electronic supplementary material The online version of this article (10.1186/s12864-019-5764-4) contains supplementary material, which is available to authorized users.

Countrywide pesticide management activities are resource draining, even for developed countries, which sometimes fall short in achieving the optimum protection against pesticides deleterious effects on humans and environment. Additionally, in Lebanon, basic flaws exist at different levels of pesticide management cycle. In this study, through an extensive review of relevant literature regarding the pesticides impact on humans and environment in Lebanon and adopted policies in existing legislation, several gaps have been identified. Accordingly, recommendations to reduce pesticide risk through a combination of reforms at the policy level and its tools, particularly legislation, are proposed. In our opinion, the starting point is to adopt a minimum list of lower risk pesticides supported by a combination of: “prescriptions” based on a comprehensive registration and an effective implementation systems, a suitable IPM/ICM government-supported credit system, traceability systems of agricultural commodities and pesticides containers, Pesticide stock management system to reduce the quantity of obsolete pesticides, and containers recycling system. For a global sustainability of pesticides risk reduction, a binding global intervention fostered by the UN, based on human rights for safe food, is called upon to ban hazardous pesticides-except those of WHO class IV- trafficking in developing countries scoring low in an international official assessment of their pesticides lifecycle management. At the same time, global funds should support pesticides alternatives and the enhancement of the developing countries capacities for pesticides lifecycle management, which is a part of a larger global matrix in risk reduction., Policy, legislations, reforms, pesticides, prescription, developing countries, risk reduction; Agricultural Policy; Crop Protection; Chemical Pest Control; Environmental Assessment; Legislation

Bacillus thuringiensis is a well-known biopesticide that has been used for more than 80 years. This spore-forming bacterium belongs to the group of Bacillus cereus that also includes, among others, emetic and diarrheic pathotypes of B. cereus, the animal pathogen Bacillus anthracis and the psychrotolerant Bacillus weihenstephanensis. Bacillus thuringiensis is rather unique since it has adapted its lifestyle as an efficient pathogen of specific insect larvae. One of the peculiarities of B. thuringiensis strains is the extent of their extrachromosomal pool, with strains harbouring more than 10 distinct plasmid molecules. Among the numerous serovars of B. thuringiensis, ‘israelensis’ is certainly emblematic since its host spectrum is apparently restricted to dipteran insects like mosquitoes and black flies, vectors of human and animal diseases such as malaria, yellow fever, or river blindness. In this review, the putative role of the mobile gene pool of B. thuringiensis serovar israelensis in its pathogenicity and dedicated lifestyle is reviewed, with specific emphasis on the nature, diversity, and potential mobility of its constituents. Variations among the few related strains of B. thuringiensis serovar israelensis will also be reported and discussed in the scope of this specialised insect pathogen, whose lifestyle in the environment remains largely unknown., This review describes the potential roles of the mobile gene pool of the entomopathogenic Bacillus thuringiensis serovar israelensis to its pathogenicity and dedicated lifestyle.

International audience; 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.

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.

The olive fruit fly Bactrocera oleae is a major olive pest in Algeriaand other Mediterranean countries. Its attacks, in some cases, seriously compromised production. Bacillus thuringiensis is a spore-forming soil bacterium which produces a protein crystal toxic to some insects. The objective of this study was to search for isolates toxic to larvae and adults of B. oleae. Four doses test were performed on neonate larvae, second instar larvae and third instar larvae. Seven Bt strains examined, showed highest toxicity levels against adults. 86% mortality was observed against neonate larvae after 7 days of application by Bt toxin isolate. The mortality of the second instar larvae was 65% mortality at dose of 108 UFC/mL. The third instar larvae was very susceptible to Bt strain with 70% of mortality. This study demonstrated that some local Algerian B. thuringiensis isolates exhibit toxic potential that could be used to control B. oleae.

International audience; Bacillus cereus displays a high diversity of lifestyles and ecological niches and include beneficial as well as pathogenic strains. These strains are widespread in the environment, are found on inert as well as on living surfaces and contaminate persistently the production lines of the food industry. Biofilms are suspected to play a key role in this ubiquitous distribution and in this persistency. Indeed, B. cereus produces a variety of biofilms which differ in their architecture and mechanism of formation, possibly reflecting an adaptation to various environments. Depending on the strain, B. cereus has the ability to grow as immersed or floating biofilms, and to secrete within the biofilm a vast array of metabolites, surfactants, bacteriocins, enzymes, and toxins, all compounds susceptible to act on the biofilm itself and/or on its environment. Within the biofilm, B. cereus exists in different physiological states and is able to generate highly resistant and adhesive spores, which themselves will increase the resistance of the bacterium to antimicrobials or to cleaning procedures. Current researches show that, despite similarities with the regulation processes and effector molecules involved in the initiation and maturation of the extensively studied Bacillus subtilis biofilm, important differences exists between the two species. The present review summarizes the up to date knowledge on biofilms produced by B. cereus and by two closely related pathogens, Bacillus thuringiensis and Bacillus anthracis. Economic issues caused by B. cereus biofilms and management strategies implemented to control these biofilms are included in this review, which also discuss the ecological and functional roles of biofilms in the lifecycle of these bacterial species and explore future developments in this important research area.

International audience; Bacillus thuringiensis can produce a floating biofilm which includes two parts: a ring and a pellicle. The ring is a thick structure which sticks to the culture container, while the pellicle extends over the whole liquid surface and joins the ring. We have followed over time, from 16 to 96 h, sporulation in the two biofilm parts. Sporulation was followed in situ in 48-wells polystyrene microtiterplates with a fluorescence binocular stereomicroscope and a spoIID-yfp transcriptional fusion. Sporulation took place much earlier in the ring than in the pellicle. In 20 h-aged biofilms, spoIID was expressed only in the ring, which could be seen as a green fluorescent circle surrounding the non-fluorescent pellicle. However, after 48 h of culture, the pellicle started to express spoIID in specific area corresponding to protrusions, and after 96 h both the ring and the whole pellicle expressed spoIID. Spore counts and microscopy observations of the ring and the pellicle harvested separately confirmed these results and revealed that sporulation occured 24 h-later in the pellicle comparatively to the ring, although both structures contained nearly 100% spores after 96 h of culture. We hypothesize that two mechanisms, due to microenvironments in the biofilm, can explain this difference. First, the ring experiences a decreased concentration of nutrients earlier than the pellicle, because of a lower exchange area with the culture medium. An second, the ring is exposed to partial dryness. Both reasons could speed up sporulation in this biofilm structure. Our results also suggest that spores in the biofilm display a phenotypic heterogeneity. These observations might be of particular significance for the food industry, since the biofilm part sticking to container walls the ring is likely to contain spores and will therefore resist both to washing and to cleaning procedures, and will be able to restart a new biofilm when food production has resumed.

INRA, MIG, Domaine de Vilvert, F-78352 Jouy en Josas, France : depuis janvier 2015 : INRA, MaIAGE, Domaine de Vilvert, F-78352 Jouy en Josas, France; Iron is an essential element for bacterial growth and virulence. Because of its limited bioavailability in the host, bacteria have adapted several strategies to acquire iron during infection. In the human opportunistic bacteria Bacillus cereus, a surface protein IlsA is shown to be involved in iron acquisition from both ferritin and hemoproteins. IlsA has a modular structure consisting of a NEAT (Near Iron transporter) domain at the N-terminus, several LRR (Leucine Rich Repeat) motifs and a SLH (Surface Layer Homology) domain likely involved in anchoring the protein to the cell surface.METHODS:[b]BACKGROUND:[/b]Isothermal titration calorimetry, UV-Vis spectrophotometry, affinity chromatography and rapid kinetics stopped-flow measurements were employed to probe the binding and transfer of hemin between two different B. cereus surface proteins (IlsA and IsdC).[br/][b]RESULTS:[/b]IlsA binds hemin via the NEAT domain and is able to extract heme from hemoglobin whereas the LRR domain alone is not involved in these processes. A rapid hemin transfer from hemin-containing IlsA (holo-IlsA) to hemin-free IsdC (apo-IsdC) is demonstrated.[br/][b]CONCLUSIONS[/b]:For the first time, it is shown that two different B. cereus surface proteins (IlsA and IsdC) can interact and transfer heme suggesting their involvement in B. cereus heme acquisition.[br/][b]GENERAL SIGNIFICANCE:[/b]An important role for the complete Isd system in heme-associated bacterial growth is demonstrated and new insights into the interplay between an Isd NEAT surface protein and an IlsA-NEAT-LRR protein, both of which appear to be involved in heme-iron acquisition in B. cereus are revealed.

The Bacillus cereus pathogenic spectrum ranges from strains used as probiotics to human-lethal strains. However, prediction of the pathogenic potential of a strain remains difficult. Here, we show that food poisoning and clinical strains can be differentiated from harmless strains on the basis of host colonization phenotypes.

Zearalenone (ZEA) is a mycotoxin produced by some species of Fusarium, especially by Fusarium graminearum and F. culmorum. ZEA induces hyperoestrogenic responses in mammals and can result in reproductive disorders in farm animals. In the present study, a real-time PCR (qPCR) assay has been successfully developed for the detection and quantification of Fusarium graminearum based on primers targeting the gene PKS13 involved in ZEA biosynthesis. A standard curve was developed by plotting the logarithm of known concentrations of F. graminearum DNA against the cycle threshold (Ct) value. The developed real time PCR system was also used to analyze the occurrence of zearalenone producing F. graminearum strains on maize. In this context, DNA extractions were performed from thirty-two maize samples, and subjected to real time PCR. Maize samples also were analyzed for zearalenone content by HPLC. F. graminearum DNA content (pg DNA/ mg of maize) was then plotted against ZEA content (ppb) in maize samples. The regression curve showed a positive and good correlation (R2=0.760) allowing for the estimation of the potential risk from ZEA contamination. Consequently, this work offers a quick alternative to conventional methods of ZEA quantification and mycological detection and quantification of F. graminearum in maize.

The human opportunistic pathogen Bacillus cereus belongs to the B. cereus group that includes bacteria with a broad host spectrum. The ability of these bacteria to colonize diverse hosts is reliant on the presence of adaptation factors. Previously, an IVET strategy led to the identification of a novel B. cereus protein (IlsA, Iron-regulated leucine rich surface protein), which is specifically expressed in the insect host or under iron restrictive conditions in vitro. Here, we show that IlsA is localized on the surface of B. cereus and hence has the potential to interact with host proteins. We report that B. cereus uses hemoglobin, heme and ferritin, but not transferrin and lactoferrin. In addition, affinity tests revealed that IlsA interacts with both hemoglobin and ferritin. Furthermore, IlsA directly binds heme probably through the NEAT domain. Inactivation of ilsA drastically decreases the ability of B. cereus to grow in the presence of hemoglobin, heme and ferritin, indicating that IlsA is essential for iron acquisition from these iron sources. In addition, the ilsA mutant displays a reduction in growth and virulence in an insect model. Hence, our results indicate that IlsA is a key factor within a new iron acquisition system, playing an important role in the general virulence strategy adapted by B. cereus to colonize susceptible hosts., Author Summary Iron is an essential compound for almost all living organisms, taking part in basic cellular homeostasis. Preventing access to iron sources for invading pathogens is one of the defense systems used by hosts to avoid pathogen colonization. To counteract this, pathogens have developed mechanisms to acquire nutrient iron during infection. Bacillus cereus is an opportunistic bacterium able to infect both insects and mammals; thus, it should have systems enabling iron uptake from these hosts. Here we describe, for the first time, a unique surface protein, called IlsA, which is essential for iron uptake from two very different iron binding molecules: ferritin and hemoglobin. IlsA is only produced in iron limited environments. We show that during insect infection, its expression is specific to insect hemocoel (blood), where ferritin is the major iron-binding molecule. Interestingly, the IlsA mutant has reduced survival in in vivo infection and in vitro when heme, hemoglobin and ferritin are the sole iron sources available. Thus, as IlsA is important for iron uptake from the major iron rich molecules in insects and mammals, we suggest that this new iron acquisition system may be a key factor that is evolutionary adapted to infection of such diverse hosts.

The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus , which is both an opportunistic human pathogen and an insect pathogen. The Δ dlt Bc mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda . Δ dlt Bc was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis . We detected the dlt operon in three gram-negative genera: Erwinia ( Erwinia carotovora ), Bordetella ( Bordetella pertussis , Bordetella parapertussis , and Bordetella bronchiseptica ), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in Δ dlt Bc ). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans.