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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 grampositive 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 [[DELTA]dlt.sub.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 Spodopterafrugiperda. [[DELTA]dlt.sub.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. meUonella 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 [[DELTA]dlt.sub.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. doi: 10.1128/JB.00892-09

Abstract. Two colonies of Culex quinquefasciatus Say (Diptera: Culicidae) were selected with Bacillus sphaericus strains C3-41 and IAB59 in the laboratory for 13 and 18 generations; they attained 145 000- and 48.3-fold resistance, respectively, in comparison with a susceptible laboratory colony (SLCq) and showed very high levels of cross-resistance (8500- to 145 000-fold) to B. sphaericus strains C3-41, 1593, 2297 and 2362. They were relatively susceptible to B. sphaericus strains LP1-G and 47-6B (only 0.8- to 2.8-fold tolerance), with 24.8- to 48.3-fold cross-resistance to strain IAB59. B. sphaericus -resistant mosquito colonies remained highly susceptible to B. thuringiensis israelensis , suggesting that B.t.i. would be of value in the management of B. sphaericus -resistant Cx. quinquefasciatus colonies. The demonstration of low or no cross-resistance of two selected resistant Cx. quinquefasciatus colonies to IAB59, LP1-G and 47-6B strains of B. sphaericus and the finding of a major 49 kDa protein in these strains suggest that there is likely to be another mosquitocidal factor in the three strains. [ABSTRACT FROM AUTHOR]

Abstract Background Most extracellular virulence factors produced by Bacillus cereus are regulated by the pleiotropic transcriptional activator PlcR. Among strains belonging to the B. cereus group, the plcR gene is always located in the vicinity of genes encoding the YvfTU two-component system. The putative role of YvfTU in the expression of the PlcR regulon was therefore investigated. Results Expression of the plcR gene was monitored using a transcriptional fusion with a lacZ reporter gene in a yvfTU mutant and in its B. cereus ATCC 14579 parental strain. Two hours after the onset of the stationary phase, a stage at which the PlcR regulon is highly expressed, the plcR expression in the yvfTU mutant was only 50% of that of its parental strain. In addition to the reduced plcR expression in the yvfTU mutant, a few members of the PlcR regulon showed a differential expression, as revealed by transcriptomic and proteomic analyses. The virulence of the yvfTU mutant in a Galleria mellonella insect model was slightly lower than that of the parental strain. Conclusion The YvfTU two-component system is not required for the expression of most of the virulence factors belonging to the PlcR regulon. However, YvfTU is involved in expression of plcR, a major regulator of virulence in B. cereus.

The industrial rearing of the yellow mealworm ( Tenebrio molitor ) for feed and food purposes on agricultural by-products may expose larvae and adults to entomopathogens used as biocontrol agents in crop production. Bacterial spores/toxins or fungal conidia from species such as Bacillus thuringiensis or Metarhizium brunneum could affect the survival and growth of insects. Therefore, the aim of this study was to investigate the potential benefits of a wheat bran diet supplemented with probiotic bacteria and dried egg white on larval development and survival and its effects on the gut microbiome composition. Two probiotic bacterial species, Pediococcus pentosaceus KVL B19-01 and Lactiplantibacillus plantarum WJB, were added to wheat bran feed with and without dried egg white, as an additional protein source, directly from neonate larval hatching until reaching a body mass of 20 mg. Subsequently, larvae from the various diets were exposed for 72 h to B. thuringiensis , M. brunneum , or their combination. Larval survival and growth were recorded for 14 days, and the bacterial microbiota composition was analyzed using 16S rDNA sequencing prior to pathogen exposure and on days 3 and 11 after inoculation with the pathogens. The results showed increased survival for T. molitor larvae reared on feed supplemented with P. pentosaceus in the case of co-infection. Larval growth was also impacted in the co-infection treatment. No significant impact of egg white or of P. pentosaceus on larval growth was recorded, while the addition of Lb. plantarum resulted in a minor increase in individual mass gain compared with infected larvae without the latter probiotic. On day 14, B. thuringiensis was no longer detected and the overall bacterial community composition of the larvae was similar in all treatments. On the other hand, the relative operational taxonomic unit (OTU) abundance was dependent on day, diet, and probiotic. Interestingly, P. pentosaceus was present throughout the experiments, while Lb. plantarum was not found at a detectable level, although its transient presence slightly improved larval performance. Overall, this study confirms the potential benefits of some probiotics during the development of T. molitor while underlining the complexity of the relationship between the host and its microbiome., Competing Interests: ARi is employed by Ynsect, company that is rearing Tenebrio molitor on a mass rearing scale. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Savio, Herren, Rejasse, Rios, Bourelle, Bruun-Jensen, Lecocq, van Loon and Nielsen-LeRoux.)

Understanding the intricate interplay between the gut microbiota and the immune response in insects is crucial, given its diverse impact on the pathogenesis of various microbial species. The microbiota's modulation of the host immune system is one such mechanism, although its complete impact on immune responses remains elusive. This study investigated the tripartite interaction between the gut microbiota, pathogens, and the host's response in Galleria mellonella larvae reared under axenic (sterile) and conventional (non-sterile) conditions. The influence of the microbiota on host fitness during infections was evaluated via two different routes: oral infection induced by Bacillus thuringiensis subsp. galleriae ( Btg ), and topical infection induced by Metarhizium robertsii ( Mr ). We observed that larvae without a microbiota can successfully fulfill their life cycle, albeit with more variation in their developmental time. We subsequently performed survival assays on final-instar larvae, using the median lethal dose (LD 50 ) of Btg and Mr . Our findings indicated that axenic larvae were more vulnerable to an oral infection of Btg ; specifically, a dose that was calculated to be half-lethal for the conventional group resulted in a 90%-100% mortality rate in the axenic group. Through a dual-analysis experimental design, we could identify the status of the gut microbiota using 16S rRNA sequencing and assess the level of immune-related gene expression in the same group of larvae at basal conditions and during infection. This analysis revealed that the microbiota of our conventionally reared population was dominated entirely by four Enterococcus species, and these species potentially stimulated the immune response in the gut, due to the increased basal expression of two antimicrobial peptides (AMPs)-gallerimycin and gloverin-in the conventional larvae compared with the axenic larvae. Furthermore, Enterococcus mundtii , isolated from the gut of conventional larvae, showed inhibition activity against Btg in vitro . Lastly, other immune effectors, namely, phenoloxidase activity in the hemolymph and total reactive oxygen/nitrogen species (ROS/RNS) in the gut, were tested to further investigate the extent of the stimulation of the microbiota on the immune response. These findings highlight the immune-modulatory role of the Enterococcus- dominated gut microbiota, an increasingly reported microbiota assemblage of laboratory populations of Lepidoptera, and its influence on the host's response to oral and topical infections., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Upfold, Rejasse, Nielsen-Leroux, Jensen and Sanchis-Borja.)

Industrial insect mass rearing aims to produce quality insects under safe sanitary conditions which can be compromised by pathogens and abiotic stressors. Therefore, knowledge on pathogen persistence, virulence and means of detection is of importance. This study focuses on the opportunistic pathogen Serratia marcescens ( Sm ) as a possible candidate to reveal sanitary issues in Tenebrio molitor ( Tm ) breeding. A screening test was performed to assess the impact of abiotic stressors (starvation, density and sieving) in presence and absence of Sm . Two Sm detection methods were conducted, and the kinetics of Sm persistence were investigated. Our results show that (i) the presence of Sm had a low but significant effect on Tm mortality, (ii) a short temporary starvation period had a negative impact on larval growth, (iii) the detection of Sm by q-PCR was sensitive but less convenient than a specific Sm growth media, (iv) the kinetics of persistence showed that Sm declined but survived for nine days in the feed and in the feces for three weeks. Both the relatively low virulence and the persistence in the environment suggest that Sm could be used as an indicator for the sanitary status of mealworm production.

Environmental pollution by the nearly nonbiodegradable polyethylene (PE) plastics is of major concern; thus, organisms capable of biodegrading PE are required. The larvae of the Greater Wax Moth, Galleria mellonella (Gm), were identified as a potential candidate to digest PE. In this study, we tested whether PE was metabolized by Gm larvae and could be found in their tissues. We examined the implication of the larval gut microbiota by using conventional and axenic reared insects. First, our study showed that neither beeswax nor LDPE alone favor the growth of young larvae. We then used Fourier transform infrared microspectroscopy (μFTIR) to detect deuterium in larvae fed with isotopically labeled food. Deuterated molecules were found in tissues of larvae fed with deuterium labeled oil for 24 and 72 h, proving that μFTIR can detect metabolization of 1 to 2 mg of deuterated food. Then, Gm larvae were fed with deuterated PE (821 kDa). No bioassimilation was detected in the tissues of larvae that had ingested 1 to 5 mg of deuterated PE in 72 h or in 19 days, but micrometer sized PE particles were found in the larval digestive tract cavities. We evidenced weak biodegradation of 641 kDa PE films in contact for 24 h with the dissected gut of conventional larvae and in the PED4 particles from excreted larval frass. Our study confirms that Gm larvae can biodegrade HDPE but cannot necessarily metabolize it.

Bacillus cereus  is a Gram-positive opportunistic pathogen closely related to the entomopathogen,  Bacillus thuringiensis , both of which are involved in intestinal infections. Iron is an essential micronutrient for full growth and virulence of pathogens during infection. However, little is known about iron homeostasis during gut infection. Therefore, we aimed to assess the expression of  B. cereus  genes related to bacterial iron homeostasis, virulence and oxidative stress. The hypothesis is that the expression of such genes would vary between early and later stage colonization in correlation to gut cell damage. To perform the study, a germ-free  Galleria mellonella  model was set up in order to adapt the use of Laser-capture microdissection (LCM), to select precise areas in the gut lumen from frozen whole larval cryo-sections. Analyses were performed from alive larvae and the expression of targeted genes was assessed byspecific pre-amplification of mRNA followed by quantitative PCR. Firstly, the results reinforce the reliability of LCM, despite a low amount of bacterial RNA recovered. Secondly, bacterial genes involved in iron homeostasis are expressed in the lumen at both 3 and 16 hours post force-feeding. Thirdly, iron gene expression is slightly modulated during gut infection, and lastly, the mRNA of  G. mellonella  encoding for ferritin and transferrin iron storage and transport are recovered too. Therefore, iron homeostasis should play a role in  B. cereus  gut colonization. Furthermore, we demonstrate for the first time the value of using LCM for specific  in situ  gene expression analysis of extracellular bacteria in a whole animal.

Due to massive production, inefficient waste collection, and long lives, plastics have become a source of persistent pollution. Biodegradation is explored as an environmentally friendly remediation method for removing plastics from the environment. Microbial and animal biodegradation methods have been reported in the literature for various plastics. Levels of plastic oxidation are often used as an evidence of degradation and can be measured with great sensitivity by Fourier Transform Infrared (FTIR) spectroscopy. FTIR is highly sensitive to the creation of new CO, CO and OH bonds during oxidation. However, many studies reporting the use of FTIR spectroscopy to evidence plastic oxidation confused the spectral signatures of biomass contamination (CO and CO from lipids, CONH from proteins, O-H from polysaccharides) with plastic oxidation. Here, based on spectra of oxidized plastic and of probable contaminants, we make recommendations for performing and analyzing FTIR measurements properly., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Flagellar motility is considered an important virulence factor in different pathogenic bacteria. In Listeria monocytogenes the transcriptional repressor MogR regulates motility in a temperature-dependent manner, directly repressing flagellar- and chemotaxis genes. The only other bacteria known to carry a mogR homolog are members of the Bacillus cereus group, which includes motile species such as B. cereus and Bacillus thuringiensis as well as the non-motile species Bacillus anthracis , Bacillus mycoides and Bacillus pseudomycoides. Furthermore, the main motility locus in B. cereus group bacteria, carrying the genes for flagellar synthesis, appears to be more closely related to L. monocytogenes than to Bacillus subtilis , which belongs to a separate phylogenetic group of Bacilli and does not carry a mogR ortholog. Here, we show that in B. thuringiensis , MogR overexpression results in non-motile cells devoid of flagella. Global gene expression profiling showed that 110 genes were differentially regulated by MogR overexpression, including flagellar motility genes, but also genes associated with virulence, stress response and biofilm lifestyle. Accordingly, phenotypic assays showed that MogR also affects cytotoxicity and biofilm formation in B. thuringiensis . Overexpression of a MogR variant mutated in two amino acids within the putative DNA binding domain restored phenotypes to those of an empty vector control. In accordance, introduction of these mutations resulted in complete loss in MogR binding to its candidate flagellar locus target site in vitro . In contrast to L. monocytogenes , MogR appears to be regulated in a growth-phase dependent and temperature-independent manner in B. thuringiensis 407. Interestingly, mogR was found to be conserved also in non-motile B. cereus group species such as B. mycoides and B. pseudomycoides , which both carry major gene deletions in the flagellar motility locus and where in B. pseudomycoides mogR is the only gene retained. Furthermore, mogR is expressed in non-motile B. anthracis. Altogether this provides indications of an expanded set of functions for MogR in B. cereus group species, beyond motility regulation. In conclusion, MogR constitutes a novel B. thuringiensis pleiotropic transcriptional regulator, acting as a repressor of motility genes, and affecting the expression of a variety of additional genes involved in biofilm formation and virulence., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Smith, Josefsen, Lindbäck, Hegna, Finke, Tourasse, Nielsen-LeRoux, Økstad and Fagerlund.)

Lactobacillus sakei is a nonpathogenic lactic acid bacterium and a natural inhabitant of meat ecosystems. Although red meat is a heme-rich environment, L. sakei does not need iron or heme for growth, although it possesses a heme-dependent catalase. Iron incorporation into L. sakei from myoglobin and hemoglobin was previously shown by microscopy and the L. sakei genome reveals the complete equipment for iron and heme transport. Here, we report the characterization of a five-gene cluster (from lsa1836 to lsa1840 [ lsa1836-1840 ]) encoding a putative metal iron ABC transporter. Interestingly, this cluster, together with a heme-dependent catalase gene, is also conserved in other species from the meat ecosystem. Our bioinformatic analyses revealed that the locus might correspond to a complete machinery of an energy coupling factor (ECF) transport system. We quantified in vitro the intracellular heme in the wild type (WT) and in our Δ lsa1836-1840 deletion mutant using an intracellular heme sensor and inductively coupled plasma mass spectrometry for quantifying incorporated 57 Fe heme. We showed that in the WT L. sakei , heme accumulation occurs rapidly and massively in the presence of hemin, while the deletion mutant was impaired in heme uptake; this ability was restored by in trans complementation. Our results establish the main role of the L. sakei Lsa1836-1840 ECF-like system in heme uptake. Therefore, this research outcome sheds new light on other possible functions of ECF-like systems. IMPORTANCE Lactobacillus sakei is a nonpathogenic bacterial species exhibiting high fitness in heme-rich environments such as meat products, although it does not need iron or heme for growth. Heme capture and utilization capacities are often associated with pathogenic species and are considered virulence-associated factors in the infected hosts. For these reasons, iron acquisition systems have been deeply studied in such species, while for nonpathogenic bacteria the information is scarce. Genomic data revealed that several putative iron transporters are present in the genome of the lactic acid bacterium L. sakei In this study, we demonstrate that one of them is an ECF-like ABC transporter with a functional role in heme transport. Such evidence has not yet been brought for an ECF; therefore, our study reveals a new class of heme transport system., (Copyright © 2020 American Society for Microbiology.)

Bacillus thuringiensis (Bt) is used for insect pest control, and its larvicidal activity is primarily attributed to Cry toxins. Other factors participate in infection, and limited information is available regarding factors acting on the peritrophic matrix (PM). This study aimed to investigate the role of a Bt chitin-binding protein (CBPA) that had been previously shown to be expressed at pH 9 in vitro and could therefore be expressed in the alkaline gut of lepidopteron larvae. A ∆cbpA mutant was generated that was 10-fold less virulent than wild-type Bt HD73 towards Ostrinia furnacalis neonate larvae, indicating its important role in infection. Purified recombinant Escherichia coli CBPA was shown to have a chitin affinity, thus indicating a possible interaction with the chitin-rich PM. A translational GFP-CBPA fusion elucidated the localization of CBPA on the bacterial surface, and the transcriptional activity of the promoter P cbpA was immediately induced and confirmed at pH 9. Next, in order to connect surface expression and possible in vivo gut activity , last instar Galleria mellonella (Gm) larvae (not susceptible to Bt HD-73) were used as a model to follow CBPA in gut expression, bacterial transit, and PM adhesion. CBPA-GFP was quickly expressed in the Gm gut lumen, and more Bt HD73 strain bacteria adhered to the PM than those of the ∆cbpA mutant strain. Therefore, CBPA may help to retain the bacteria, via the PM binding, close to the gut surface and thus takes part in the early steps of Bt gut interactions.

Cyclic diguanylate (c-di-GMP) signalling affects several cellular processes in Bacillus cereus group bacteria including biofilm formation and motility, and CdgF was previously identified as a diguanylate cyclase promoting biofilm formation in B. thuringiensis. C-di-GMP can exert its function as a second messenger via riboswitch binding, and a functional c-di-GMP-responsive riboswitch has been found upstream of cbpA in various B. cereus group strains. Protein signature recognition predicted CbpA to be a cell wall-anchored surface protein with a fibrinogen or collagen binding domain. The aim of this study was to identify the binding ligand of CbpA and the function of CbpA in cellular processes that are part of the B. cereus group c-di-GMP regulatory network. By global gene expression profiling cbpA was found to be down-regulated in a cdgF deletion mutant, and cbpA exhibited maximum expression in early exponential growth. Contrary to the wild type, a ΔcbpA deletion mutant showed no binding to collagen in a cell adhesion assay, while a CbpA overexpression strain exhibited slightly increased collagen binding compared to the control. For both fibrinogen and fibronectin there was however no change in binding activity compared to controls, and CbpA did not appear to contribute to binding to abiotic surfaces (polystyrene, glass, steel). Also, the CbpA overexpression strain appeared to be less motile and showed a decrease in biofilm formation compared to the control. This study provides the first experimental proof that the binding ligand of the c-di-GMP regulated adhesin CbpA is collagen., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2019 Published by Elsevier B.V.)

The extracellular biofilm matrix often contains a network of amyloid fibers which, in the human opportunistic pathogen Bacillus cereus, includes the two homologous proteins TasA and CalY. We show here, in the closely related entomopathogenic species Bacillus thuringiensis, that CalY also displays a second function. In the early stationary phase of planktonic cultures, CalY was located at the bacterial cell-surface, as shown by immunodetection. Deletion of calY revealed that this protein plays a major role in adhesion to HeLa epithelial cells, to the insect Galleria mellonella hemocytes and in the bacterial virulence against larvae of this insect, suggesting that CalY is a cell-surface adhesin. In mid-stationary phase and in biofilms, the location of CalY shifted from the cell surface to the extracellular medium, where it was found as fibers. The transcription study and the deletion of sipW suggested that CalY change of location is due to a delayed activity of the SipW signal peptidase. Using purified CalY, we found that the protein polymerization occurred only in the presence of cell-surface components. CalY is, therefore, a bifunctional protein, which switches from a cell-surface adhesin activity in early stationary phase, to the production of fibers in mid-stationary phase and in biofilms., (© 2018 John Wiley & Sons Ltd.)

Bacillus thuringiensis is an invertebrate pathogen that produces insecticidal crystal toxins acting on the intestinal barrier. In the Galleria mellonella larvae infection model, toxins from the PlcR virulence regulon contribute to pathogenicity by the oral route. While B. thuringiensis is principally an oral pathogen, bacteria may also reach the insect haemocoel following injury of the cuticle. Here, we address the question of spore virulence as compared to vegetative cells when the wild-type Bt407cry- strain and its isogenic ∆ plcR mutant are inoculated directly into G. mellonella haemocoel. Mortality dose-response curves were constructed at 25 and 37 °C using spores or vegetative cell inocula, and the 50% lethal dose (LD 50 ) in all infection conditions was determined after 48 h of infection. Our findings show that (i) the LD 50 is lower for spores than for vegetative cells for both strains, while the temperature has no significant influence, and (ii) the ∆ plcR mutant is four to six times less virulent than the wild-type strain in all infection conditions. Our results suggest that the environmental resistant spores are the most infecting form in haemocoel and that the PlcR virulence regulon plays an important role in toxicity when reaching the haemocoel from the cuticle and not only following ingestion.

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.

Bacillus thuringiensis subsp. israelensis is one of the most important microorganisms used against mosquitoes. It was intensively studied following its discovery and became a model bacterium of the B. thuringiensis species. Those studies focused on toxin genes, aggregation-associated conjugation, linear genome phages, etc. Recent announcements of genomic sequences of different strains have not been explicitly related to the biological properties studied. We report data on plasmid content analysis of four strains using ultra-high-throughput sequencing. The strains were commercial product isolates, with their putative ancestor and type B. thuringiensis subsp. israelensis strain sequenced earlier. The assembled contigs corresponding to published and novel data were assigned to plasmids described earlier in B. thuringiensis subsp. israelensis and other B. thuringiensis strains. A new 360 kb plasmid was identified, encoding multiple transporters, also found in most of the earlier sequenced strains. Our genomic data show the presence of two toxin-coding plasmids of 128 and 100 kb instead of the reported 225 kb plasmid, a co-integrate of the former two. In two of the sequenced strains, only a 100 kb plasmid was present. Some heterogeneity exists in the small plasmid content and structure between strains. These data support the perception of active plasmid exchange among B. thuringiensis subsp. israelensis strains in nature., (Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Background: 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: 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)., Results: 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., Conclusions: 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., General Significance: 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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Many microorganisms cooperate by secreting products that are commonly available to neighboring cells. These "public goods" include autoinduced, quorum-sensing (QS) molecules and the virulence factors activated by these signals. Public goods cooperation is exploitable by cheaters, cells that avoid the costs of production but gain an advantage by freeloading on the products of others. QS signals and responses can be cooperative under artificial laboratory conditions, but it remains unclear whether QS is cooperative in nature: little is known about the frequency of cheaters in natural populations, and cheaters may do poorly because of the importance of QS in major transcriptional networks. Here, we investigate the cooperative nature of QS in a natural system: the Gram-positive insect pathogen Bacillus thuringiensis and the larvae of the diamondback moth, Plutella xylostella. Although we find evidence of cooperation, QS null mutants are not effective cheats in vivo and cannot outcompete wild-type strains. We show that spatial structure limits mutant fitness and that well-separated microcolonies occur in vivo because of the strong population bottlenecks occurring during natural infection. We argue that spatial structure and low densities are the norm in early-stage infections, and this can explain why QS cheaters are rare in B. thuringiensis and its relatives. These results contrast with earlier experiments describing the high fitness of Gram-negative QS cheaters and suggest that QS suppression ("quorum quenching") can be clinically effective without having negative impacts on the evolution of virulence., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

The aim of this study was to explore the role of SecDF in protein secretion in Bacillus cereus ATCC 14579 by in-depth characterization of a markerless secDF knock out mutant. Deletion of secDF resulted in pleiotropic effects characterized by a moderately slower growth rate, aberrant cell morphology, enhanced susceptibility to xenobiotics, reduced virulence and motility. Most toxins, including food poisoning-associated enterotoxins Nhe, Hbl, and cytotoxin K, as well as phospholipase C were less abundant in the secretome of the ΔsecDF mutant as determined by label-free mass spectrometry. Global transcriptome studies revealed profound transcriptional changes upon deletion of secDF indicating cell envelope stress. Interestingly, the addition of glucose enhanced the described phenotypes. This study shows that SecDF is an important part of the Sec-translocase mediating efficient secretion of virulence factors in the Gram-positive opportunistic pathogen B. cereus, and further supports the notion that B. cereus enterotoxins are secreted by the Sec-system.

Bacteria survive under various conditions by sensing stimuli triggering specific adaptive physiological responses, which are often based on membrane-integrated sensors connected to a cytoplasmic regulator. Recent studies reveal that mucus glycans may act as signal molecules for two-component systems involved in intestinal colonization. Bacillus cereus, a human and insect opportunistic pathogen was used to identify bacterial factors expressed in an insect gut infection model. The screen revealed a promoter involved in the expression of a gene with so far unknown functions. A search for gut-related compounds, inducing its transcription, identified glucose-6-phosphate as an activation signal. The gene is part of a five-gene cluster, including a two-component system. Interestingly such five gene loci are conserved in the pathogenic Bacillus group as well as in various Clostridia bacteria and are with analogy to other multi-component sensor systems in enteropathogenic bacteria, such as E. coli. Thus our results provide insights into the function of two-component and auxiliary sensor systems in host-microbe interactions and opens up possible investigations of such systems in other gut associated bacteria.

The study of bacterial virulence often requires a suitable animal model. Mammalian models of infection are costly and may raise ethical issues. The use of insects as infection models provides a valuable alternative. Compared to other non-vertebrate model hosts such as nematodes, insects have a relatively advanced system of antimicrobial defenses and are thus more likely to produce information relevant to the mammalian infection process. Like mammals, insects possess a complex innate immune system(1). Cells in the hemolymph are capable of phagocytosing or encapsulating microbial invaders, and humoral responses include the inducible production of lysozyme and small antibacterial peptides(2,3). In addition, analogies are found between the epithelial cells of insect larval midguts and intestinal cells of mammalian digestive systems. Finally, several basic components essential for the bacterial infection process such as cell adhesion, resistance to antimicrobial peptides, tissue degradation and adaptation to oxidative stress are likely to be important in both insects and mammals(1). Thus, insects are polyvalent tools for the identification and characterization of microbial virulence factors involved in mammalian infections. Larvae of the greater wax moth Galleria mellonella have been shown to provide a useful insight into the pathogenesis of a wide range of microbial infections including mammalian fungal (Fusarium oxysporum, Aspergillus fumigatus, Candida albicans) and bacterial pathogens, such as Staphylococcus aureus, Proteus vulgaris, Serratia marcescens Pseudomonas aeruginosa, Listeria monocytogenes or Enterococcus faecalis(4-7). Regardless of the bacterial species, results obtained with Galleria larvae infected by direct injection through the cuticle consistently correlate with those of similar mammalian studies: bacterial strains that are attenuated in mammalian models demonstrate lower virulence in Galleria, and strains causing severe human infections are also highly virulent in the Galleria model(8-11). Oral infection of Galleria is much less used and additional compounds, like specific toxins, are needed to reach mortality. G. mellonella larvae present several technical advantages: they are relatively large (last instar larvae before pupation are about 2 cm long and weight 250 mg), thus enabling the injection of defined doses of bacteria; they can be reared at various temperatures (20 °C to 30 °C) and infection studies can be conducted between 15 °C to above 37 °C(12,13), allowing experiments that mimic a mammalian environment. In addition, insect rearing is easy and relatively cheap. Infection of the larvae allows monitoring bacterial virulence by several means, including calculation of LD50(14), measurement of bacterial survival(15,16) and examination of the infection process(17). Here, we describe the rearing of the insects, covering all life stages of G. mellonella. We provide a detailed protocol of infection by two routes of inoculation: oral and intra haemocoelic. The bacterial model used in this protocol is Bacillus cereus, a Gram positive pathogen implicated in gastrointestinal as well as in other severe local or systemic opportunistic infections(18,19).

Using a previously developed Bacillus cereus in vivo expression technology (IVET) promoter trap system, we showed that spsA, a gene of unknown function, was specifically expressed in the larval gut during infection. Search for gut-related compounds inducing spsA transcription identified glucose-6-phosphate (G6P) as an activation signal. Analysis of the spsA-related 5-gene cluster indicated that SpsA is part of a new sugar phosphate sensor system composed of a 2-component system (TCS) encoded by spsR and spsK, and 2 additional downstream genes, spsB and spsC. In B. cereus, American Type Culture Collection (ATCC) 14579, spsRK, and spsABC are separate transcriptional units, of which only spsABC was activated by extracellular G6P. lacZ transcriptional fusions tested in mutant and complemented strains showed that SpsRK, SpsA, and SpsB are essential for the transcription of spsABC. Deletion mutant analysis showed that SpsC is essential for the G6P uptake. gfp-transcriptional fusions showed that these genes are required for host-activated expression, as well. This sugar phosphate sensor and transport system is found in pathogenic Bacillus group and Clostridia bacteria and may be important for host adaptation. Our findings provide new insights into the function of 2-component sensor systems in host-pathogen interactions, specifically in the gut.

Insects are the largest group of animals on earth. Like mammals, virus, fungi, bacteria and parasites infect them. Several tissue barriers and defense mechanisms are common for vertebrates and invertebrates. Therefore some insects, notably the fly Drosophila and the caterpillar Galleria mellonella, have been used as models to study host-pathogen interactions for several insect and mammal pathogens. They are excellent tools to identify pathogen determinants and host tissue cell responses. We focus here on the comparison of effectors used by two different groups of bacterial insect pathogens to accomplish the infection process in their lepidopteran larval host: Bacillus thuringiensis and the nematode-associated bacteria, Photorhabdus and Xenorhabdus. The comparison reveals similarities in function and expression profiles for some genes, which suggest that such factors are conserved during evolution in order to attack the tissue encountered during the infection process., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

How pathogenic bacteria infect and kill their host is currently widely investigated. In comparison, the fate of pathogens after the death of their host receives less attention. We studied Bacillus thuringiensis (Bt) infection of an insect host, and show that NprR, a quorum sensor, is active after death of the insect and allows Bt to survive in the cadavers as vegetative cells. Transcriptomic analysis revealed that NprR regulates at least 41 genes, including many encoding degradative enzymes or proteins involved in the synthesis of a nonribosomal peptide named kurstakin. These degradative enzymes are essential in vitro to degrade several substrates and are specifically expressed after host death suggesting that Bt has an active necrotrophic lifestyle in the cadaver. We show that kurstakin is essential for Bt survival during necrotrophic development. It is required for swarming mobility and biofilm formation, presumably through a pore forming activity. A nprR deficient mutant does not develop necrotrophically and does not sporulate efficiently in the cadaver. We report that necrotrophism is a highly regulated mechanism essential for the Bt infectious cycle, contributing to spore spreading.

The closely related bacterial species Bacillus cereus and Bacillus weihenstephanensis are adapted to the mesophilic and the psychrotrophic temperature range, respectively. While B. cereus strains are associated with foodborne diseases, B. weihenstephanensis strains are so far not, although similar virulence genes are found in both species. Our investigations show that both species were virulent in the insect model, Galleria mellonella, following infection via oral and haemocoel routes. However, virulence of B. weihenstephanensis was much higher at 15°C than at 37°C. Furthermore, a temperature-dependent difference between the species was seen in a cell culture cytotoxicity assay. In summary, our results demonstrate for the first time virulence of B. weihenstephanensis strains in an in vivo model. In addition, we found that G. mellonella is a useful model for studies of the psychrotolerant species of the B. cereus group, suggesting that insects might be an ecological growth niche for several members of this bacterial group., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

The soil bacterium Bacillus thuringiensis is a pathogen of insects and nematodes and is very closely related to, if not the same species as, Bacillus cereus and Bacillus anthracis. The defining characteristic of B. thuringiensis that sets it apart from B. cereus and B. anthracis is the production of crystal (Cry) proteins, which are pore-forming toxins or pore-forming proteins (PFPs). Although it is known that PFPs are important virulence factors since their elimination results in reduced virulence of many pathogenic bacteria, the functions by which PFPs promote virulence are incompletely understood. Here we study the effect of Cry proteins in B. thuringiensis pathogenesis of the nematode Caenorhabditis elegans. We find that whereas B. thuringiensis on its own is not able to infect C. elegans, the addition of the PFP Cry protein, Cry5B, results in a robust lethal infection that consumes the nematode host in 1-2 days, leading to a "Bob" or bag-of-bacteria phenotype. Unlike other infections of C. elegans characterized to date, the infection by B. thuringiensis shows dose-dependency based on bacterial inoculum size and based on PFP concentration. Although the infection process takes 1-2 days, the PFP-instigated infection process is irreversibly established within 15 minutes of initial exposure. Remarkably, treatment of C. elegans with Cry5B PFP is able to instigate many other Bacillus species, including B. anthracis and even "non-pathogenic" Bacillus subtilis, to become lethal and infectious agents to C. elegans. Co-culturing of Cry5B-expressing B. thuringiensis with B. anthracis can result in lethal infection of C. elegans by B. anthracis. Our data demonstrate that one potential property of PFPs is to sensitize the host to bacterial infection and further that C. elegans and probably other roundworms can be common hosts for B. cereus-group bacteria, findings with important ecological and research implications., (© 2011 Kho et al.)

Bacillus thuringiensis (Bt) is an insecticidal bacterium that has successfully been used as a biopesticide for many years. It is usually referred to as a soil-dwelling organism, as a result of the prevalence of its spores in this environment, but one that can act as an opportunistic pathogen under appropriate conditions. Our understanding of the biology of this organism has been challenged further by the recent publication of two reports that claim that Bt requires the co-operation of commensal bacteria within the gut of a susceptible insect for its virulence. It is our opinion that Bt is not primarily a saprophyte and does not require the assistance of commensal bacteria but is a true pathogen in its own right and furthermore that its primary means of reproduction is in an insect cadaver., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Despite the existence of various virulence factors in the Enterococcus genus, enterococcal virulence is still a debated issue. A main consideration is the detection of the same virulence genes in strains isolated from nosocomial or community-acquired infections, and from food products. The goal of this study was to evaluate the roles of two well-characterized enterococcal virulence factors, Fsr and gelatinase, in the potential virulence of Enterococcus faecalis food strains. Virulence of unrelated Enterococcus isolates, including dairy strains carrying fsr and gelE operons, was compared in the Galleria mellonella insect model. E. faecalis dairy strains were able to kill larvae and were as virulent as strain OG1RF, one of the most widely used for virulence studies. In contrast, Enterococcus durans and Enterococcus faecium strains were avirulent or poorly virulent for G. mellonella. To evaluate the role of fsrB and gelE in virulence of E. faecalis dairy strains, both genes were deleted independently in two strains. The Delta fsrB and Delta gelE deletion mutants both produced a gelatinase-negative phenotype. Although both mutations significantly attenuated virulence in G. mellonella, the Delta fsrB strains were more strongly attenuated. These results agree with previous findings suggesting the involvement of fsrB in the control of other cell functions relevant to virulence. Our work demonstrates that the presence of functional fsrB, and to a lesser extent gelE, in dairy enterococci should be considered with caution.

Food packaging papers are not sterile, as the manufacturing is an open process, and the raw materials contain bacteria. We modeled the potential transfer of the Bacillus cereus spores from packaging paper to food by using a green fluorescent protein-expressing construct of Bacillus thuringiensis Bt 407Cry(-) [pHT315Omega(papha3-gfp)], abbreviated BT-1. Paper (260 g m(-2)) containing BT-1 was manufactured with equipment that allowed fiber formation similar to that of full-scale manufactured paper. BT-1 adhered to pulp during papermaking and survived similar to an authentic B. cereus. Rice and chocolate were exposed to the BT-1-containing paper for 10 or 30 days at 40 or 20 degrees C at relative air humidity of 10 to 60%. The majority of the spores remained immobilized inside the fiber web; only 0.001 to 0.03% transferred to the foods. This amount is low compared with the process hygiene criteria and densities commonly found in food, and it does not endanger food safety. To measure this, we introduced BT-1 spores into the paper in densities of 100 to 1,000 times higher than the amounts of the B. cereus group bacteria found in commercial paper. Of BT-1 spores, 0.03 to 0.1% transferred from the paper to fresh agar surface within 5 min of contact, which is more than to food during 10 to 30 days of exposure. The findings indicate that transfer from paper to dry food is restricted to those microbes that are exposed on the paper surface and readily detectable with a contact agar method.

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.

Highly pathogenic strains of Bacillus sphaericus produce the mosquitocidal Bin proteins, but resistance to this toxin can be produced under laboratory and field conditions. Analysis of strains able to overcome this resistance revealed the presence of a previously undescribed type of two-component toxin. One subunit, Cry48Aa1, is related to the 3-domain crystal toxins of Bacillus thuringiensis. Uniquely for this type of protein, insect toxicity is only achieved in the presence of a second, accessory protein, Cry49Aa1. This protein is itself related to both the binary toxin of B. sphaericus and to Cry35 and Cry36 of B. thuringiensis, none of which require interaction with Cry48Aa1-like proteins for their activity. The necessity for both Cry48Aa1 and Cry49Aa1 components for pathogenicity, therefore, indicates an unprecedented interaction to generate toxicity. Despite high potency for purified Cry48Aa1/Cry49Aa1 proteins (LC50 for third instar Culex quinquefasciatus larvae: 15.9 ng/ml and 6.3 ng/ml respectively), bacteria producing them show suboptimal mosquitocidal activity due to low-level Cry48Aa1 production. This new toxin combination may indicate a fortuitous combination of members of the gene families that encode 3-domain Cry toxins and Binary-like toxins, permitting the "mix-and-match" evolution of a new component in the mosquitocidal armoury.

Using 2-DE, transcriptional gene fusions and cell cytotoxicity assays, we followed changes in the Bacillus cereus strain ATCC14579 secretome, gene expression and culture supernatant cytotoxicity from the end of the vegetative phase up to 5 h after entry into the stationary phase. The concentration of each of the 22 proteins in the culture supernatant was determined at various times. In addition, the stability of the proteins was studied. Fifteen of these proteins, including 14 members of the virulence regulon PlcR, were known or predicted to be secreted. All of the secreted proteins reached a maximum concentration during early stationary phase, but there were significant differences in the kinetics of their concentrations. The time courses of protein concentrations were in agreement with gene expression data, except for cytotoxin CytK, which was unstable, and for the metalloprotease InhA1. Supernatant cytoxicity also peaked in early stationary phase, and the kinetics of cytotoxicity paralleled the time course of concentration of the PlcR-controlled toxin, CytK. Our concomitant study of the time course of protein concentrations, gene expression and supernatant cytotoxicity reveals that the pathogenic potential of B. cereus peaks during the transition state. It also suggests that there is diversity in the regulation of gene expression within the PlcR regulon.