From cereus to anthrax and back again: The role of the PlcR regulator 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: the pleiotropic transcriptional regulator PlcR found in most members of the Bacillus cereus group but truncated in all Bacillus 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 haemolytic 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, we demonstrate 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 haemolytic 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.

[1]  L. Baillie,et al.  The influence of extrachromosomal elements in the anthrax “cross-over” strain Bacillus cereus G9241 , 2022, bioRxiv.

[2]  Victoria M. Baldwin.  You Can’t B. cereus – A Review of Bacillus cereus Strains That Cause Anthrax-Like Disease , 2020, Frontiers in Microbiology.

[3]  Guowei Yang,et al.  The Photorhabdus asymbiotica virulence cassettes deliver protein effectors directly into target eukaryotic cells , 2019, eLife.

[4]  R. Grunow,et al.  Analysis of a newly discovered antigen of Bacillus cereus biovar anthracis for its suitability in specific serological antibody testing , 2018, Journal of applied microbiology.

[5]  A. Hoffmaster,et al.  Anthrax Toxin-Expressing Bacillus cereus Isolated from an Anthrax-Like Eschar , 2016, PloS one.

[6]  P. Keim,et al.  The Phylogeny of Bacillus cereus sensu lato , 2016, Microbiology spectrum.

[7]  C. Henry,et al.  CodY Regulates the Activity of the Virulence Quorum Sensor PlcR by Controlling the Import of the Signaling Peptide PapR in Bacillus thuringiensis , 2016, Front. Microbiol..

[8]  D. Lereclus,et al.  Quorum Sensing in Bacillus thuringiensis Is Required for Completion of a Full Infectious Cycle in the Insect , 2014, Toxins.

[9]  A. Edwards,et al.  Oxacillin Alters the Toxin Expression Profile of Community-Associated Methicillin-Resistant Staphylococcus aureus , 2013, Antimicrobial Agents and Chemotherapy.

[10]  M. Uyttendaele,et al.  Small Bacillus cereus ATCC 14579 subpopulations are responsible for cytotoxin K production , 2013, Journal of applied microbiology.

[11]  R. Grenha,et al.  Structural basis for the activation mechanism of the PlcR virulence regulator by the quorum-sensing signal peptide PapR , 2012, Proceedings of the National Academy of Sciences.

[12]  A. R. Merrill,et al.  Certhrax Toxin, an Anthrax-related ADP-ribosyltransferase from Bacillus cereus* , 2012, The Journal of Biological Chemistry.

[13]  C. Duport,et al.  Bacillus cereus Fnr binds a [4Fe-4S] cluster and forms a ternary complex with ResD and PlcR , 2012, BMC Microbiology.

[14]  L. S. Arnesen,et al.  Temperature-Dependent Production of Various PlcR-Controlled Virulence Factors in Bacillus weihenstephanensis Strain KBAB4 , 2012, Applied and Environmental Microbiology.

[15]  S. W. Long,et al.  Rapidly progressive, fatal, inhalation anthrax-like infection in a human: case report, pathogen genome sequencing, pathology, and coordinated response. , 2011, Archives of pathology & laboratory medicine.

[16]  A. Keane-Myers,et al.  Bacillus cereus G9241 Makes Anthrax Toxin and Capsule like Highly Virulent B. anthracis Ames but Behaves like Attenuated Toxigenic Nonencapsulated B. anthracis Sterne in Rabbits and Mice , 2011, Infection and Immunity.

[17]  Robert J. Fieldhouse,et al.  Cholera- and Anthrax-Like Toxins Are among Several New ADP-Ribosyltransferases , 2010, PLoS Comput. Biol..

[18]  Christophe Boesch,et al.  The Genome of a Bacillus Isolate Causing Anthrax in Chimpanzees Combines Chromosomal Properties of B. cereus with B. anthracis Virulence Plasmids , 2010, PloS one.

[19]  S. Leppla,et al.  PapR peptide maturation: role of the NprB protease in Bacillus cereus 569 PlcR/PapR global gene regulation. , 2009, FEMS immunology and medical microbiology.

[20]  Karla D. Passalacqua,et al.  Comparative Transcriptional Profiling of Bacillus cereus Sensu Lato Strains during Growth in CO2-Bicarbonate and Aerobic Atmospheres , 2009, PloS one.

[21]  A. Kolstø,et al.  The YvfTU Two-component System is involved in plcR expression in Bacillus cereus , 2008, BMC Microbiology.

[22]  S. Arold,et al.  Molecular basis for group-specific activation of the virulence regulator PlcR by PapR heptapeptides , 2008, Nucleic acids research.

[23]  S. Arold,et al.  Structure of PlcR: Insights into virulence regulation and evolution of quorum sensing in Gram-positive bacteria , 2007, Proceedings of the National Academy of Sciences.

[24]  C. Duport,et al.  The Redox Regulator Fnr Is Required for Fermentative Growth and Enterotoxin Synthesis in Bacillus cereus F4430/73 , 2007, Journal of bacteriology.

[25]  C. Duport,et al.  Control of Enterotoxin Gene Expression in Bacillus cereus F4430/73 Involves the Redox-Sensitive ResDE Signal Transduction System , 2006, Journal of bacteriology.

[26]  T. Popović,et al.  Characterization of Bacillus cereus Isolates Associated with Fatal Pneumonias: Strains Are Closely Related to Bacillus anthracis and Harbor B. anthracis Virulence Genes , 2006, Journal of Clinical Microbiology.

[27]  T. Popović,et al.  Capsule Production in Bacillus cereus Strains Associated with Severe Pneumonia , 2006, Journal of Clinical Microbiology.

[28]  I. Swiecicka,et al.  Hemolytic and Nonhemolytic Enterotoxin Genes are Broadly Distributed among Bacillus thuringiensis Isolated from Wild Mammals , 2006, Microbial Ecology.

[29]  C. Boesch,et al.  Anthrax in Western and Central African great apes , 2006, American journal of primatology.

[30]  Yun Luo,et al.  Analysis of the Life Cycle of the Soil Saprophyte Bacillus cereus in Liquid Soil Extract and in Soil , 2006, Applied and Environmental Microbiology.

[31]  I. Swiecicka,et al.  Diversity of commensal Bacillus cereus sensu lato isolated from the common sow bug (Porcellio scaber, Isopoda). , 2006, FEMS microbiology ecology.

[32]  C. Boesch,et al.  A New Bacillus anthracis Found in Wild Chimpanzees and a Gorilla from West and Central Africa , 2006, PLoS pathogens.

[33]  D. Lereclus,et al.  Specificity and Polymorphism of the PlcR-PapR Quorum-Sensing System in the Bacillus cereus Group , 2005, Journal of bacteriology.

[34]  Christophe Boesch,et al.  Anthrax kills wild chimpanzees in a tropical rainforest , 2004, Nature.

[35]  David A Rasko,et al.  Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[36]  M. Mock,et al.  Distinct Mutations in PlcR Explain Why Some Strains of the Bacillus cereus Group Are Nonhemolytic , 2004, Journal of bacteriology.

[37]  D. Lereclus,et al.  A cell–cell signaling peptide activates the PlcR virulence regulon in bacteria of the Bacillus cereus group , 2002, The EMBO journal.

[38]  O. Økstad,et al.  Two‐dimensional electrophoresis analysis of the extracellular proteome of Bacillus cereus reveals the importance of the PlcR regulon , 2002, Proteomics.

[39]  R. ffrench-Constant,et al.  Bacterial infection of a model insect: Photorhabdus luminescens and Manduca sexta , 2002, Cellular microbiology.

[40]  M. Mock,et al.  The incompatibility between the PlcR‐ and AtxA‐controlled regulons may have selected a nonsense mutation in Bacillus anthracis , 2001, Molecular microbiology.

[41]  M. Rose,et al.  Oligopeptide permease is required for expression of the Bacillus thuringiensis plcR regulon and for virulence , 2001, Molecular microbiology.

[42]  M. Brehélin,et al.  The plcR regulon is involved in the opportunistic properties of Bacillus thuringiensis and Bacillus cereus in mice and insects. , 2000, Microbiology.

[43]  H. Agaisse,et al.  Regulation of toxin and virulence gene transcription in Bacillus thuringiensis. , 2000, International journal of medical microbiology : IJMM.

[44]  P. E. Granum,et al.  A new cytotoxin from Bacillus cereus that may cause necrotic enteritis , 2000, Molecular microbiology.

[45]  P. E. Granum,et al.  Common occurrence of enterotoxin genes and enterotoxicity in Bacillus thuringiensis. , 2000, FEMS microbiology letters.

[46]  B. Purnelle,et al.  Sequence analysis of three Bacillus cereus loci carrying PIcR-regulated genes encoding degradative enzymes and enterotoxin. , 1999, Microbiology.

[47]  H. Agaisse,et al.  PlcR is a pleiotropic regulator of extracellular virulence factor gene expression in Bacillus thuringiensis , 1999, Molecular microbiology.

[48]  P. E. Granum,et al.  Bacillus cereus and its food poisoning toxins. , 1997, FEMS microbiology letters.

[49]  T. Koehler,et al.  Regulation of anthrax toxin activator gene (atxA) expression in Bacillus anthracis: temperature, not CO2/bicarbonate, affects AtxA synthesis , 1997, Infection and immunity.

[50]  J M Miller,et al.  Fulminating bacteremia and pneumonia due to Bacillus cereus , 1997, Journal of clinical microbiology.

[51]  P. E. Granum,et al.  Characterisation of a non-haemolytic enterotoxin complex from Bacillus cereus isolated after a foodborne outbreak. , 1996, FEMS microbiology letters.

[52]  H. Agaisse,et al.  Identification of a Bacillus thuringiensis gene that positively regulates transcription of the phosphatidylinositol-specific phospholipase C gene at the onset of the stationary phase , 1996, Journal of bacteriology.

[53]  D. Macneil,et al.  Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. , 1992, Gene.

[54]  D. Lereclus,et al.  Construction of cloning vectors for Bacillus thuringiensis. , 1991, Gene.

[55]  D. Lane.  16S/23S rRNA sequencing , 1991 .

[56]  W. Goebel,et al.  A Bacillus cereus cytolytic determinant, cereolysin AB, which comprises the phospholipase C and sphingomyelinase genes: nucleotide sequence and genetic linkage , 1989, Journal of bacteriology.

[57]  P. Cresswell,et al.  Impaired assembly and transport of HLA‐A and ‐B antigens in a mutant TxB cell hybrid. , 1986, The EMBO journal.

[58]  K. C. Carter,et al.  Koch's postulates in relation to the work of Jacob Henle and Edwin Klebs. , 1985, Medical History.