The influence of extrachromosomal elements in the anthrax “cross-over” strain Bacillus cereus G9241

Bacillus cereus G9241 was isolated from a welder who survived a pulmonary anthrax-like disease. Strain G9241 carries two virulence plasmids, pBCX01 and pBC210, as well as an extrachromosomal prophage, pBFH_1. pBCX01 has 99.6% sequence identity to pXO1 carried by Bacillus anthracis and encodes the tripartite anthrax toxin genes and atxA, a mammalian virulence transcriptional regulator. This work looks at how the presence of pBCX01 and temperature may affect the lifestyle of B. cereus G9241 using a transcriptomic analysis and by studying spore formation, an important part of the B. anthracis lifecycle. Here we report that pBCX01 has a stronger effect on gene transcription at the mammalian infection relevant temperature of 37°C in comparison to 25°C. At 37°C, the presence of pBCX01 appears to have a negative effect on genes involved in cell metabolism, including biosynthesis of amino acids, whilst positively affecting the transcription of many transmembrane proteins. The study of spore formation showed B. cereus G9241 sporulated rapidly in comparison to the B. cereus sensu stricto type strain ATCC 14579, particularly at 37°C. The carriage of pBCX01 did not affect this phenotype suggesting that other genetic elements were driving rapid sporulation. An unexpected finding of this study was that pBFH_1 is highly expressed at 37°C in comparison to 25°C and pBFH_1 expression leads to the production of Siphoviridae-like phage particles in the supernatant of B. cereus G9241. This study provides an insight on how the extrachromosomal genetic elements in B. cereus G9241 has an influence in bacterial phenotypes

From cereus to anthrax and back again : the role of the PlcR regulator in the “cross-over” strain Bacillus cereus G9241

The plcR gene, which encodes the pleiotropic transcriptional regulator of secreted proteins found in most members of the Bacillus cereus group, is truncated in all Bacillus anthracis isolates. The current dogma suggests this truncation was evolved to accommodate the acquisition of the anthrax toxin regulator, AtxA. However, the B. cereus-B. anthracis “cross-over” strain Bacillus cereus G9241, isolated from a Louisiana welder suffering from an anthrax-like infection, appears to contradict the proposed dogma as it encodes intact copies of both regulators. Here we report that when cultured at 25 °C, cell free B. cereus G9241 culture supernatants are cytotoxic and haemolytic to various eukaryotic cells in addition to insect haemocytes from Manduca sexta. However, this cytotoxic and haemolytic activity of the culture supernatant is lost when the bacteria are grown at 37 °C, behaving much like the supernatants generated by B. anthracis. Using a combination of genetic and proteomic approaches, we identified several PlcR-regulated toxins secreted at 25 °C. We demonstrate that a limiting step for the production of these virulence factors at 37 °C exists within the PlcR-PapR regulation circuit in strain G9241, giving rise to the temperature-dependent haemolytic and cytotoxic activity of the culture supernatants. Differential expression of the protease responsible in processing the PlcR quorum sensing activator PapR appears to be responsible for this phenotype. This study confirms that B. cereus G9241 is able to ‘switch’ between B. cereus and B. anthracis–like phenotypes in a temperature-dependent manner, potentially accommodating the activities of both PlcR and AtxA

From cereus to anthrax and back again: Assessment of the temperature-dependent phenotypic switching in the "cross-over" strain Bacillus cereus G9241

Bacillus cereus G9241 was isolated from a Louisiana welder suffering from an anthrax-like infection. The organism carries two transcriptional regulators that have previously been proposed to be incompatible with each other in Bacillus anthracis: the pleiotropic transcriptional regulator PlcR found in most members of the Bacillus cereus group but truncated in all B. anthracis isolates, and the anthrax toxin regulator AtxA found in all B. anthracis strains and a few B. cereus sensu stricto strains. Here we report cytotoxic and hemolytic activity of cell free B. cereus G9241 culture supernatants cultured at 25°C to various eukaryotic cells. However, this is not observed at the mammalian infection relevant temperature 37°C, behaving much like the supernatants generated by B. anthracis. Using a combination of genetic and proteomic approaches to understand this unique phenotype, we identified several PlcR-regulated toxins to be secreted highly at 25°C compared to 37°C. Furthermore, results suggest that differential expression of the protease involved in processing the PlcR quorum sensing activator molecule PapR appears to be the limiting step for the production of PlcR-regulated toxins at 37°C, giving rise to the temperature-dependent hemolytic and cytotoxic activity of the culture supernatants. This study provides an insight on how B. cereus G9241 is able to “switch” between B. cereus and B. anthracis–like phenotypes in a temperature-dependent manner, potentially accommodating the activities of both PlcR and AtxA

Temperature Dependent Toxin Expression in Bacillus Cereus G9241, the Causative Agent of Anthrax like Illness

Bacillus cereus G9241, a member of the Bacillus cereus sensu lato complex, was isolated from a Louisiana welder with a pulmonary anthrax-like illness and is closely related to B. anthracis. Most members of the B. cereus group express PlcR, a pleiotropic transcriptional regulator of secreted toxins and enzymes allowing insect infection, which is activated by the peptide PapR at stationary phase of growth. However, in all B. anthracis isolates, the plcR gene contains a point mutation, which frame-shifts the gene, thus inactivating it. It has been proposed that the acquisition of AtxA, the mammalian responsive transcriptional regulator, was incompatible with the activity of PlcR, leading to selection for PlcR inactivation. Interestingly, G9241 encodes intact copies of both atxA and plcR. Preliminary data has shown that B. cereus G9241 has a temperature dependent haemolytic activity, possibly caused by haemolysins that are regulated by PlcR. We hypothesise that a change in the PlcR-PapR regulatory network in G9241 has allowed the co-existence of plcR and atxA through temperature dependent suppression of the PlcR-PapR circuit at the time AtxA becomes active.Here we investigated whether the activity of the PlcR-PapR circuit and PlcR regulated toxins in B. cereus G9241 are temperature dependent (25 °C and 37 °C), in order to accommodate the activity of AtxA. Plasmid based transcription-translation GFP reporter of PlcR, PapR and PlcR-regulated toxins have been used to study translational activity.Warwick Collaborative Postgraduate Research Scholarship Dst

Investigation into the temperature-dependent regulation of virulence factors in Bacillus cereus G9241, the causative agent of an anthrax-like disease

Bacillus cereus G9241 was isolated from a Louisiana welder with an anthrax-like illness and is closely related to Bacillus anthracis. Strain G9241 carries the extrachromosomal plasmids pBCX01 and pBC210, each encoding a gene for the mammalian responsive transcriptional regulator AtxA, as well as the linear phagemid pBFH_1. Like most members of the Bacillus cereus group, B. cereus G9241 encodes an intact copy of the gene for PlcR, a pleiotropic transcriptional regulator of secreted toxins and enzymes. Yet all B. anthracis isolates have a point mutation in the plcR gene. It has been proposed that the acquisition of AtxA was incompatible with the activity of PlcR, leading to selection for PlcR inactivation. Interestingly, strain G9241 retains intact copies of both regulators. Work prior to this study has shown that B. cereus G9241 has a temperature-dependent haemolytic activity, with transcriptomic and proteomic analyses revealing the expression and secretion of haemolysins and toxins regulated by PlcR. Further potential virulence factors were identified; growth phase-dependent elongated cell morphology of B. cereus G9241 was observed during exponential phase growth, similar to the B. anthracis cell shape when evading phagocytes. This study focussed on investigating the virulence factors that allow strain G9241 to act either as a typical B. cereus strain or an anthrax-causing B. anthracis. Reporter strains for B. cereus G9241 were constructed to identify the expression pattern of plcR-papR and PlcR-regulated toxins. While PlcR demonstrated a highly heterogeneous expression, PlcR-regulated enterotoxins were highly expressed at 25 °C compared to 37 °C. Multiple approaches were used to identify the limiting step within the PlcR-PapR circuit causing the temperature-dependent haemolytic and cytolytic activity in B. cereus G9241. We propose that B. cereus G9241 is able to partially suppress the PlcR regulon in a temperature-dependent manner, potentially to allow the coexistence of plcR and atxA. Inhibitors against cell elongation and cell wall biosynthesis were used to identify the potential mechanism involved in the elongated cell morphology of B. cereus G9241 and its potential function when interacting with immune cells

Characterisation and Immunogenicity of Neisseria cinerea outer membrane vesicles displaying NadA, NHBA and fHbp from Neisseria meningitidis serogroup B

More affordable and effective vaccines against bacterial meningitis caused by Neisseria meningitidis serogroup B are still required for global prevention. We have previously shown that modified outer membrane vesicles (mOMVs) from commensal Neisseria cinerea can be used as a platform to induce immune responses against meningococcal antigens. The aim of the present study was to use a combination of two genetically engineered mOMVs to express multiple antigens from N. meningitidis known to be involved in protective immunity to meningococcal meningitis (different variants of factor H binding protein (fHbp), Neisseria Heparin Binding Antigen (NHBA) and Neisseria Adhesin A (NadA)). Antigen expression in the mOMVs was confirmed by Western blotting; detoxification of the lipooligosaccharide (LOS) was confirmed by measuring human toll like receptor 4 (hTLR4) activation using in vitro cell assays. Mice immunised with a combination of two mOMVs expressing fHbp, NHBA and NadA produced antibodies to all the antigens. Furthermore, serum bactericidal activity (SBA) was induced by the immunisation, with mOMVs expressing NadA displaying high SBA titres against a nadA+ MenB strain. The work highlights the potential of mOMVs from N. cinerea to induce functional immune responses against multiple antigens involved in the protective immune response to meningococcal disease

Commensal Neisseria cinerea outer membrane vesicles as a platform for the delivery of meningococcal and gonococcal antigens to the immune system.

An affordable, accessible, and broadly protective vaccine is required to tackle the re-occurring bacterial meningococcal epidemics in Sub-Saharan Africa as well as an effective control of multi-drug resistant strains of gonococcus. Outer membrane vesicles (OMVs) secreted from Gram-negative bacteria represent an attractive platform for antigen delivery to the immune system and therefore for development of multi-component vaccines. In this study, we describe the generation of modified OMVs (mOMVs) from commensal biosafety-level 1 (BSL-1) Neisseria cinerea ATCC® 14685TM, which is phylogenetically close to the pathogenic bacteria Neisseria meningitidis and Neisseria gonorrhoeae. mOMVs were prepared from N. cinerea engineered to express heterologous antigens from N. meningitidis (factor H binding protein (fHbp) and Neisseria Heparin Binding Antigen (NHBA-2)) and from N. gonorrhoeae (NHBA-542). Mice immunised with the mOMVs produced antibodies against fHbp and NHBA. The work indicates that mOMV from N. cinerea can be used as a platform to induce immune responses against antigens involved in the protective immune response against meningococcal and gonococcal diseases