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In JoVE (1)
Other Publications (22)
- Proceedings of the National Academy of Sciences of the United States of America
- Applied and Environmental Microbiology
- Journal of Invertebrate Pathology
- Journal of Medical Entomology
- Journal of Medical Entomology
- FEMS Microbiology Letters
- Microbial Ecology
- Applied and Environmental Microbiology
- FEMS Microbiology Letters
- Molecular Microbiology
- FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
- BMC Microbiology
- Microbiology (Reading, England)
- Journal of Food Protection
- PLoS Pathogens
- Trends in Microbiology
- FEMS Microbiology Letters
- PloS One
- PLoS Pathogens
- FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology
- Current Opinion in Microbiology
Articles by Christina Nielsen-Leroux in JoVE
The Insect Galleria mellonella as a Powerful Infection Model to Investigate Bacterial Pathogenesis
Nalini Ramarao1, Christina Nielsen-Leroux1, Didier Lereclus1
1INRA, Micalis UMR1319, France
Oral and intra haemocolic infection of larvae of the greater wax moth Galleria mellonella is described. This insect can be used to study virulence factors of entomopathogenic as well as mammalian opportunistic bacteria. Rearing of the insects, methods of infection and examples of in vivo analysis are described.
Other articles by Christina Nielsen-Leroux on PubMed
Proceedings of the National Academy of Sciences of the United States of America. Apr, 2002 | Pubmed ID: 11983886
The mosquitocidal activity of Bacillus sphaericus is because of a binary toxin (Bin), which binds to Culex pipiens maltase 1 (Cpm1), an alpha-glucosidase present in the midgut of Culex pipiens larvae. In this work, we studied the molecular basis of the resistance to Bin developed by a strain (GEO) of C. pipiens. Immunohistochemical and in situ hybridization experiments showed that Cpm1 was undetectable in the midgut of GEO larvae, although the gene was correctly transcribed. The sequence of the cpm1(GEO) cDNA differs from the sequence we previously reported for a susceptible strain (cpm1(IP)) by seven mutations: six missense mutations and a mutation leading to the premature termination of translation. When produced in insect cells, Cpm1(IP) was attached to the membrane by a glycosylphosphatidylinositol (GPI). In contrast, the premature termination of translation of Cpm1(GEO) resulted in the targeting of the protein to the extracellular compartment because of truncation of the GPI-anchoring site. The interaction between Bin and Cpm1(GEO) and the enzyme activity of the receptor were not affected. Thus, Bin is not toxic to GEO larvae because it cannot interact with the midgut cell membrane, even though its receptor site is unaffected. This mechanism contrasts with other known resistance mechanisms in which point mutations decrease the affinity of binding between the receptor and the toxin.
Applied and Environmental Microbiology. Jun, 2002 | Pubmed ID: 12039761
Two field-collected Culex quinquefasciatus colonies were subjected to selection pressure by three strains of Bacillus sphaericus, C3-41, 2362, and IAB59, under laboratory conditions. After 13 and 18 generations of exposure to high concentrations of C3-41 and IAB59, a field-collected low-level-resistant colony developed >144,000- and 46.3-fold resistance to strains C3-41 and IAB59, respectively. A field-collected susceptible colony was selected with 2362 and IAB59 for 46 and 12 generations and attained >162,000- and 5.7-fold resistance to the two agents, respectively. The pattern of resistance evolution in mosquitoes depended on continuous selection pressure, and the stronger the selection pressure, the more quickly resistance developed. The resistant colonies obtained after selection with B. sphaericus C3-41 and 2362 showed very high levels of cross-resistance to B. sphaericus 2362 and C3-41, respectively, but they displayed only low-level cross-resistance to IAB59. On the other hand, the IAB59-selected colonies had high cross-resistance to both strains C3-41 and 2362. Additionally, the slower evolution of resistance against strain IAB59 may be explained by the presence of another larvicidal factor. This is in agreement with the nontoxicity of the cloned and purified binary toxin (Bin1) of IAB59 for 2362-resistant larvae. We also verified that all the B. sphaericus-selected colonies showed no cross-resistance to Bacillus thuringiensis subsp. israelensis, suggesting that it would be a promising alternative in managing resistance to B. sphaericus in C. quinquefasciatus larvae.
Alternative Method for Preservation of Mosquito Larvae to Study Binding Mechanisms of Bacillus Sphaericus Binary Toxin
Journal of Invertebrate Pathology. Feb, 2002 | Pubmed ID: 12095245
High Resistance to Bacillus Sphaericus Binary Toxin in Culex Pipiens (Diptera: Culicidae): the Complex Situation of West Mediterranean Countries
Journal of Medical Entomology. Sep, 2002 | Pubmed ID: 12349855
This study was aimed at clarifying the nature of the resistance to Bacillus sphaericus Neide (Bs) that Culex pipiens L. has developed in west Mediterranean countries, France, and Tunisia. Two recessive and sex-linked mutants, sp-1R and sp-2R, were previously detected in southern France. Here, the Tunisian resistance was also shown to involve a single recessive and sex-linked gene that was temporarily named sp-T(R). In addition, sp-1R, sp-2R, and sp-T(R) were shown to separately confer a similar high resistance level (> 5,000-fold) in the homozygous state. Knowing that sp-1R resistance does not alter the binding of Bs binary toxin to its specific receptor, we investigated this character in sp-2RR and sp-T(RR) homozygotes. This was performed by in vitro experiments in which larval brush border membrane fractions (BBMF) were exposed to the 125I-Bin2 toxin of B. sphaericus strain 1593. The toxin-receptor binding was found disrupted by sp-2R but not by sp-T(R). Comparing the binding kinetics among nine Culex pipiens strains of diverse origins revealed that the Bs receptors of sp-1RR and Sp-T(RR) homozygous larvae were displaying the highest affinity toward Bs binary toxins. These results are discussed with regard to alternative assumptions on the dynamics of high Bs-resistance and on the emerging possibilities to test them in a near future.
Inheritance and Mechanism of Resistance to Bacillus Sphaericus in Culex Quinquefasciatus (Diptera: Culicidae) from China and Brazil
Journal of Medical Entomology. Jan, 2004 | Pubmed ID: 14989346
Investigations on the inheritance and mechanism of resistance to Bacillus sphaericus Neide in Culex quinquefasciatus Say colonies, selected with strains C3-41 (RLCq1/C3-41) and 2362 (CqRL1/2362), were performed in China and Brazil, respectively. The progeny of reciprocal F1 crosses (susceptible female x resistant male and vice versa) from both resistant colonies responded alike in bioassays, indicating recessive inheritance. Data on larvae susceptibility from the backcross offspring between F1 and their respective susceptible and resistant parental colonies are consistent with a monofactorial and autosomal mode of inheritance. In vitro binding assays between 125I binary (Bin2) toxin and the brush border membrane fractions (BBMF) from CqRL1/2362 and RLCq1/C3-41 larvae showed that resistance, in both colonies, is caused by a failure in the binding step of the B. sphaericus Bin2 toxin to its specific midgut receptor. The specific and saturable binding of Bin2 toxin to BBMF from F1 larvae (CqRL1/2362 X susceptible counterpart) confirms the recessive inheritance of the resistance gene. Further studies are needed to advance understanding of B. sphaericus resistance.
Two Bacillus Sphaericus Binary Toxins Share the Midgut Receptor Binding Site: Implications for Resistance of Culex Pipiens Complex (Diptera: Culicidae) Larvae
FEMS Microbiology Letters. Dec, 2004 | Pubmed ID: 15598531
This work demonstrates that Bin1 and Bin2 toxins, produced by Bacillus sphaericus strains IAB59 and 2362, respectively, share a binding site in midgut brush border membranes (BBMF) from Culex pipiens complex larvae. However, a colony selected with strain IAB59, displaying a resistance ratio of only 42-fold to IAB59, but a 162,000-fold resistance to strain 2362, was found to miss receptors for Bin2 in the BBMF. This correlates with results showing that Bin1, produced in strain IAB59, failed to bind specifically to BBMF from other colony highly resistant to strain 2362. Data indicate the loss of the BBMF bound receptor as a general mechanism of resistance to binary toxins in mosquito.
Low Persistence of Bacillus Thuringiensis Serovar Israelensis Spores in Four Mosquito Biotopes of a Salt Marsh in Southern France
Microbial Ecology. Nov, 2005 | Pubmed ID: 16328650
We studied the persistence of Bacillus thuringiensis serovar israelensis (Bti) in a typical breeding site of the mosquito Ochlerotatus caspius in a particularly sensitive salt marsh ecosystem following two Bti-based larvicidal applications (Vectobac 12AS, 1.95 L/ha). The treated area was composed of four larval biotopes that differed in terms of the most representative plant species (Sarcocornia fruticosa, Bolboschoenus maritimus, Phragmites australis, and Juncus maritimus) and the physical and chemical characteristics of the soil. We sampled water, soil, and plants at various times before and after the applications (from spring to autumn, 2001) and quantified the spores of B. thuringiensis (Bt) and Bacillus species. The B. cereus group accounted for between 0% and 20% of all Bacillus spp. before application depending on the larval biotope. No Bti were found before application. The variation in the quantity of bacilli during the mosquito breeding season depended more on the larval biotope than on the season or the larvicidal application. More bacilli were found in soil (10(4)-10(6) spores/g) than on plant samples (10(2)-10(4) spores/g). The abundance in water (10(5) to 10(7) spores/L) appeared to be correlated to the water level of the breeding site. The number of Bti spores increased just after application, after declining; no spores were detected in soil or water 3 months after application. However, low numbers of Bti spores were present on foliage from three of the four studied plant strata. In conclusion, the larvicidal application has very little impact on Bacillus spp. flora after one breeding season (two applications).
FlhA Influences Bacillus Thuringiensis PlcR-regulated Gene Transcription, Protein Production, and Virulence
Applied and Environmental Microbiology. Dec, 2005 | Pubmed ID: 16332888
Bacillus thuringiensis and Bacillus cereus are closely related. B. thuringiensis is well known for its entomopathogenic properties, principally due to the synthesis of plasmid-encoded crystal toxins. B. cereus appears to be an emerging opportunistic human pathogen. B. thuringiensis and B. cereus produce many putative virulence factors which are positively controlled by the pleiotropic transcriptional regulator PlcR. The inactivation of plcR decreases but does not abolish virulence, indicating that additional factors like flagella may contribute to pathogenicity. Therefore, we further analyzed a mutant (B. thuringiensis 407 Cry(-) DeltaflhA) previously described as being defective in flagellar apparatus assembly and in motility as well as in the production of hemolysin BL and phospholipases. A large picture of secreted proteins was obtained by two-dimensional electrophoresis analysis, which revealed that flagellar proteins are not secreted and that production of several virulence-associated factors is reduced in the flhA mutant. Moreover, we quantified the effect of FlhA on plcA and hblC gene transcription. The results show that the flhA mutation results in a significant reduction of plcA and hblC transcription. These results indicate that the transcription of several PlcR-regulated virulence factors is coordinated with the flagellar apparatus. Consistently, the flhA mutant also shows a strong decrease in cytotoxicity towards HeLa cells and in virulence against Galleria mellonella larvae following oral and intrahemocoelic inoculation. The decrease in virulence may be due to both a lack of flagella and a lower production of secreted factors. Hence, FlhA appears to be an essential virulence factor with a pleiotropic role.
The Enhancin-like Metalloprotease from the Bacillus Cereus Group is Regulated by the Pleiotropic Transcriptional Activator PlcR but is Not Essential for Larvicidal Activity
FEMS Microbiology Letters. Jul, 2006 | Pubmed ID: 16790012
Bacillus cereus group bacteria produce virulence factors. Many of these are regulated by the pleiotropic transcriptional activator PlcR, which is implicated in insect virulence. In silico analysis of the B. cereus strain ATCC14579 genome showed an enhancin-like gene preceded by a typical PlcR binding sequence. The gene is predicted to encode a polypeptide showing 23-25% identity with enhancins from several baculoviruses and 31% with that of Yersinia pestis. Viral enhancin acts after oral infection and degrades the peritrophic matrix of various Lepidopteran larvae. To rule out a possible implication of Bacillus enhancin in insect virulence, we sequenced the enhancin gene from the Bacillus thuringiensis 407-crystal minus strain and investigated its gene regulation and larvicidal activity. A typical metalloprotease zinc-binding domain (HEIAH) was detected and the gene was named mpbE (metalloprotease bacillus enhancin). An mpbE'-lacZ transcriptional fusion demonstrated that mpbE belongs to the PlcR regulon. The mpbE mutant was fed to Galleria mellonella larvae, and no significant reduction in virulence was observed. However, this may not exclude MpbE from a role in pathogenesis.
Identification of Bacillus Cereus Internalin and Other Candidate Virulence Genes Specifically Induced During Oral Infection in Insects
Molecular Microbiology. Oct, 2006 | Pubmed ID: 16978259
Bacillus cereus is an opportunistic bacterium frequently associated with food-borne infections causing gastroenteritis. We developed an in vivo expression technology (IVET), with an insect host, for identification of the B. cereus genes specifically expressed during infection. This IVET-based approach uses site-specific recombinase TnpI to identify transient promoter activation. We constructed a genomic library of B. cereus ATCC14579 by cloning DNA fragments upstream from tnpI. The library was screened in vivo by oral infection of the insect Galleria mellonella. We selected 100 clones from dead larvae. Sequencing of the inserts followed by a second screen for specific in vivo induction led to the identification of 20 in vivo-induced genes (ivi genes). They belonged to several different functional classes: regulation, metabolism, DNA repair and replication, cell division, transport, virulence and adaptation. A strongly induced gene, ivi29, was further analysed. It encodes an internalin-like protein with four distinct domains: an N-terminal signal peptide for export, a NEAT domain thought to be involved in iron transport, a leucine-rich repeat domain that may interact with host cells, and a C-terminal SLH domain presumably binding the protein to the peptidoglycan. As suggested by a Fur box in the promoter, transcriptional analysis showed ivi29 expression to be repressed by iron, suggesting that expression was induced in vivo due to iron deprivation in the host. This iron-regulated, leucine-rich surface protein was designated IlsA. Disruption of ilsA reduced the virulence of the bacteria to the insect larvae indicating its role in the overall pathogenesis of B. cereus.
Proteomics. May, 2007 | Pubmed ID: 17486558
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.
FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Dec, 2007 | Pubmed ID: 17646596
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.
BMC Microbiology. 2008 | Pubmed ID: 18925929
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.
Microbiology (Reading, England). Nov, 2009 | Pubmed ID: 19696101
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.
Journal of Food Protection. Nov, 2009 | Pubmed ID: 19903384
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.
IlsA, a Unique Surface Protein of Bacillus Cereus Required for Iron Acquisition from Heme, Hemoglobin and Ferritin
PLoS Pathogens. Nov, 2009 | Pubmed ID: 19956654
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.
Trends in Microbiology. May, 2010 | Pubmed ID: 20338765
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.
Using an Insect Model to Assess Correlation Between Temperature and Virulence in Bacillus Weihenstephanensis and Bacillus Cereus
FEMS Microbiology Letters. Apr, 2011 | Pubmed ID: 21276046
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 15Â°C than at 37Â°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.
The Pore-forming Protein Cry5B Elicits the Pathogenicity of Bacillus Sp. Against Caenorhabditis Elegans
PloS One. 2011 | Pubmed ID: 22216181
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.
PLoS Pathogens. 2012 | Pubmed ID: 22511867
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.
A Multicomponent Sugar Phosphate Sensor System Specifically Induced in Bacillus Cereus During Infection of the Insect Gut
FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology. Aug, 2012 | Pubmed ID: 22611084
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.
How the Insect Pathogen Bacteria Bacillus Thuringiensis and Xenorhabdus/Photorhabdus Occupy Their Hosts
Current Opinion in Microbiology. Jun, 2012 | Pubmed ID: 22633889
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.