Christina Nielsen-LeRoux

Bacillus thuringiensis: an impotent pathogen?

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.

How the insect pathogen bacteria Bacillus thuringiensis and Xenorhabdus/Photorhabdus occupy their hosts

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.

IlsA, A Unique Surface Protein of Bacillus cereus Required for Iron Acquisition from Heme, Hemoglobin and Ferritin

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.

Necrotrophism is a quorum-sensing-regulated lifestyle in Bacillus thuringiensis.

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.

Virulence of Enterococcus faecalis dairy strains in an insect model: the role of fsrB and gelE

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.

The pore-forming protein Cry5B elicits the pathogenicity of Bacillus sp. against Caenorhabditis elegans.

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.

The Social Biology of Quorum Sensing in a Naturalistic Host Pathogen System

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.

The YvfTU Two-component System is involved in plcR expression in Bacillus cereus

BackgroundMost 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.ResultsExpression 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.ConclusionThe 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.

Using an insect model to assess correlation between temperature and virulence in Bacillus weihenstephanensis and Bacillus cereus

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.

SecDF as Part of the Sec-Translocase Facilitates Efficient Secretion of Bacillus cereus Toxins and Cell Wall-Associated Proteins

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.