In vivo characterization of an Hfq protein encoded by the Bacillus anthracis virulence plasmid pXO1.

BACKGROUND: Bacterial Hfq proteins post-transcriptionally regulate gene expression, primarily by mediating the interaction between sRNAs (small RNAs) and their target mRNAs. The role of Hfq-based regulation has been well defined in Gram-negative bacteria, but comparatively less is known about the impact of Hfq proteins in Gram-positive species. The Gram-positive pathogen Bacillus anthracis (causative agent of anthrax) is distinct in that it expresses three homologs of Hfq: Hfq1 and Hfq2 from the chromosome, and Hfq3 from the pXO1 virulence plasmid. RESULTS: In this study, we utilized overexpression as a strategy to examine the impact of Hfq3 on B. anthracis physiology. The increase in Hfq3 protein levels led to anomalous cell shape and chain formation, which manifested as a severe growth defect. This phenotype was specific to B. anthracis, as Hfq3 expression in B. subtilis at similar levels was not toxic. Toxicity was dependent on residues on the distal face of Hfq3 that are involved in mRNA binding in other bacterial species. CONCLUSIONS: Thus, we hypothesize that Hfq3 interacts with RNA(s) involved in essential functions in the B. anthracis cell, leading to increased binding upon overexpression that either sequesters or accelerates degradation of RNAs important for growth. These results not only aid in elucidating the role of Hfq proteins in B. anthracis, but also contribute to our current understanding of Hfq in Gram-positive bacteria.

Milk-originated Bacillus cereus sensu lato strains harbouring Bacillus anthracis-like plasmids are genetically and phenotypically diverse.

Bacillus cereus sensu lato is widely distributed in food products, including raw and processed milk. Plasmids often determine bacterial virulence and toxicity, but their role in the evolution of B. cereus sensu lato is only partly known. Here, we observed that nearly 8% of B. cereus sensu lato isolates were positive for pXO1-like plasmids and 12% for pXO2-like plasmids in raw and ultra-heat-treated (UHT) milk from one dairy plant. However, pXO1-like plasmids were significantly more frequent in raw milk, while pXO2-like plasmids were more frequent in processed milk. Strains from raw and UHT milk were enterotoxigenic, with up to one-fifth of the isolates being psychrotolerant. Phylogenetic assessment using multi-locus sequence typing revealed a polyphyletic structure for these bacilli, with distinct groups of cold-adapted isolates and pathogenic strains (including emetic B. cereus). Populations corresponding to both sampling sites exhibited significant linkage disequilibrium and the presence of purifying selection. The far-from-clonal population structure indicated the presence of sequence types or ecotypes adapted to specific conditions in the dairy industry. A high recombination-to-mutation ratio suggested an important role for horizontal gene transfer among B. cereus sensu lato isolates in milk.

Modeling gastrointestinal anthrax disease.

Bacillus anthracis is a spore-forming microbe that persists in soil and causes anthrax disease. The most natural route of infection is ingestion by grazing animals. Gastrointestinal (GI) anthrax also occurs in their monogastric predators, including humans. Exposure of carcasses to oxygen triggers sporulation and contamination of the surrounding soil completing the unusual life cycle of this microbe. The pathogenesis of GI anthrax is poorly characterized. Here, we use B. anthracis carrying the virulence plasmids pXO1 and pXO2, to model gastrointestinal disease in Guinea pigs and mice. We find that spores germinate in the GI tract and precipitate disease in a dose-dependent manner. Inoculation of vegetative bacilli also results in GI anthrax. Virulence is impacted severely by the loss of capsule (pXO2-encoded) but only moderately in absence of toxins (pXO1-encoded). Nonetheless, the lack of toxins leads to reduced bacterial replication in infected hosts. B. cereus Elc4, a strain isolated from a fatal case of inhalational anthrax-like disease, was also found to cause GI anthrax. Because transmission to new hosts depends on the release of large numbers of spores in the environment, we propose that the acquisition of pXO1- and pXO2-like plasmids may promote the successful expansion of members of the Bacillus cereus sensu lato group able to cause anthrax-like disease.

Ecological Niche Model of Bacillus cereus Group Isolates Containing a Homologue of the pXO1 Anthrax Toxin Genes Infecting Metalworkers in the United States.

While Bacillus cereus typically causes opportunistic infections in humans, within the last three decades, severe and fatal infections caused by isolates of the B. cereus group harboring anthrax toxin genes have been reported in the United States. From 1994 to 2020, seven cases of anthrax-like illness resulting from these isolates have been identified. With one exception, the cases have occurred in the Gulf States region of the United States among metalworkers. We aimed to develop an ecological niche model (ENM) to estimate a spatial area conducive to the survival of these organisms based on the presence of known human infections and environmental variables. The estimated ecological niche for B. cereus was modeled with the maximum entropy algorithm (Maxent). Environmental variables contributing most to the model were soil characteristics (cation exchange capacity, carbon content, soil pH), temperature, enhanced vegetation index (EVI), and land surface temperature (LST). Much of the suitable environments were located throughout the Gulf Coast Plain, Texas Backland Prairies, East Central Texas Plains, Edwards Plateau, Cross Timbers, Mississippi Alluvial Plain, and Central Great Plains. These findings may provide additional guidance to narrow potential risk areas to efficiently communicate messages to metalworkers and potentially identify individuals who may benefit from the anthrax vaccine.

Genomic Characterization and Copy Number Variation of Bacillus anthracis Plasmids pXO1 and pXO2 in a Historical Collection of 412 Strains.

Bacillus anthracis plasmids pXO1 and pXO2 carry the main virulence factors responsible for anthrax. However, the extent of copy number variation within the species and how the plasmids are related to pXO1/pXO2-like plasmids in other species of the Bacillus cereus sensu lato group remain unclear. To gain new insights into these issues, we sequenced 412 B. anthracis strains representing the total phylogenetic and ecological diversity of the species. Our results revealed that B. anthracis genomes carried, on average, 3.86 and 2.29 copies of pXO1 and pXO2, respectively, and also revealed a positive linear correlation between the copy numbers of pXO1 and pXO2. No correlation between the plasmid copy number and the phylogenetic relatedness of the strains was observed. However, genomes of strains isolated from animal tissues generally maintained a higher plasmid copy number than genomes of strains from environmental sources (P < 0.05 [Welch two-sample t test]). Comparisons against B. cereus genomes carrying complete or partial pXO1-like and pXO2-like plasmids showed that the plasmid-based phylogeny recapitulated that of the main chromosome, indicating limited plasmid horizontal transfer between or within these species. Comparisons of gene content revealed a closed pXO1 and pXO2 pangenome; e.g., plasmids encode <8 unique genes, on average, and a single large fragment deletion of pXO1 in one B. anthracis strain (2000031682) was detected. Collectively, our results provide a more complete view of the genomic diversity of B. anthracis plasmids, their copy number variation, and the virulence potential of other Bacillus species carrying pXO1/pXO2-like plasmids. IMPORTANCE Bacillus anthracis microorganisms are of historical and epidemiological importance and are among the most homogenous bacterial groups known, even though the B. anthracis genome is rich in mobile elements. Mobile elements can trigger the diversification of lineages; therefore, characterizing the extent of genomic variation in a large collection of strains is critical for a complete understanding of the diversity and evolution of the species. Here, we sequenced a large collection of B. anthracis strains (>400) that were recovered from human, animal, and environmental sources around the world. Our results confirmed the remarkable stability of gene content and synteny of the anthrax plasmids and revealed no signal of plasmid exchange between B. anthracis and pathogenic B. cereus isolates but rather predominantly vertical descent. These findings advance our understanding of the biology and pathogenomic evolution of B. anthracis and its plasmids.

The pag Gene of pXO1 Is Involved in Capsule Biosynthesis of Bacillus anthracis Pasteur II Strain.

The poly-γ-D-glutamic acid capsule and anthrax toxins are major virulence factors of Bacillus anthracis. Genes responsible for capsule biosynthesis are located on pXO2, whereas genes encoding the toxins, which are composed of edema factors, lethal factors, and protective antigens (PA), are located on pXO1. In this study, we found that the pag null mutation not only eliminated the production of the protective antigen, it also eliminated the ability of the B. anthracis Pasteur II strain to form capsules. qPCR analysis revealed that the deletion of pag decreased the transcription levels of the capABCD operon and its regulatory genes acpA and acpB. The introduction of the acpA or acpB plasmid complemented the effect of the pag null mutation on capsule formation. Taken together, the above results suggest that PA probably affects capsule biosynthesis by altering the expression of acpA and acpB. In addition, we found that the deletion mutation of pag remarkably attenuated bacterial pathogenicity in a mouse model of infection. Our results indicate that besides encoding the protective antigen, the pag gene of pXO1 is also involved in the modulation of capsule biosynthesis. Our findings provide new insight into the regulation mechanisms of capsule formation in B. anthracis Pasteur II strain.

Identification of the pXO1 plasmid in attenuated Bacillus anthracis vaccine strains.

Anthrax toxins and capsule are the major virulence factors of Bacillus anthracis. They are encoded by genes located on the plasmids pXO1 and pXO2, respectively. The vaccine strain Pasteur II was produced from high temperature subcultures of B. anthracis, which resulted in virulence attenuation through the loss of the plasmid pXO1. However, it is unclear whether the high temperature culture completely abolishes the plasmid DNA or affects the replication of the plasmid pXO1. In this study, we tested 3 B. anthracis vaccine strains, including Pasteur II from France, Qiankefusiji II from Russia, and Rentian II from Japan, which were all generated from subcultures at high temperatures. Surprisingly, we detected the presence of pXO1 plasmid DNA using overlap PCR in all these vaccine strains. DNA sequencing analysis of overlap PCR products further confirmed the presence of pXO1. Moreover, the expression of the protective antigen (PA) encoded on pXO1 was determined by using SDS-PAGE and western blotting. In addition, we mimicked Pasteur's method and exposed the A16R vaccine strain, which lacks the pXO2 plasmid, to high temperature, and identified the pXO1 plasmid in the subcultures at high temperatures. This indicated that the high temperature treatment at 42.5°C was unable to eliminate pXO1 plasmid DNA from B. anthracis. Our results suggest that the attenuation of the Pasteur II vaccine strain is likely due to the impact of high temperature stress on plasmid replication, which in turn limits the copy number of pXO1. Our data provide new insights into the mechanisms of the remaining immunogenicity and toxicity of the vaccine strains.

Curing Both Virulent Mega-Plasmids from Bacillus anthracis Wild-Type Strain A16 Simultaneously Using Plasmid Incompatibility.

Plasmid-cured derivative strains of Bacillus anthracis are frequently used in laboratory studies. Plasmid incompatibility, which does not increase the risk of chromosomal mutation, is a useful method for plasmid curing. However, in bacteria containing multiple plasmids, it often requires the sequential introduction of multiple, specific incompatibility plasmids. This lengthy process renders the traditional plasmid incompatibility method inefficient and mutation-prone. In this study, we successfully cured plasmids pXO1 and pXO2 from B. anthracis A16 simultaneously using only one recombinant incompatible plasmid, pKORT, to obtain a plasmid-free strain, designated A16DD. This method may also be useful for the simultaneous, one-step curing of multiple plasmids from other bacteria, including Bacillus thuringiensis and Yersinia pestis.

Hfqs in Bacillus anthracis: Role of protein sequence variation in the structure and function of proteins in the Hfq family.

Hfq proteins in Gram-negative bacteria play important roles in bacterial physiology and virulence, mediated by binding of the Hfq hexamer to small RNAs and/or mRNAs to post-transcriptionally regulate gene expression. However, the physiological role of Hfqs in Gram-positive bacteria is less clear. Bacillus anthracis, the causative agent of anthrax, uniquely expresses three distinct Hfq proteins, two from the chromosome (Hfq1, Hfq2) and one from its pXO1 virulence plasmid (Hfq3). The protein sequences of Hfq1 and 3 are evolutionarily distinct from those of Hfq2 and of Hfqs found in other Bacilli. Here, the quaternary structure of each B. anthracis Hfq protein, as produced heterologously in Escherichia coli, was characterized. While Hfq2 adopts the expected hexamer structure, Hfq1 does not form similarly stable hexamers in vitro. The impact on the monomer-hexamer equilibrium of varying Hfq C-terminal tail length and other sequence differences among the Hfqs was examined, and a sequence region of the Hfq proteins that was involved in hexamer formation was identified. It was found that, in addition to the distinct higher-order structures of the Hfq homologs, they give rise to different phenotypes. Hfq1 has a disruptive effect on the function of E. coli Hfq in vivo, while Hfq3 expression at high levels is toxic to E. coli but also partially complements Hfq function in E. coli. These results set the stage for future studies of the roles of these proteins in B. anthracis physiology and for the identification of sequence determinants of phenotypic complementation.

The effect of prolonged storage on the virulence of isolates of Bacillus anthracis obtained from environmental and animal sources in the Kars Region of Turkey.

The stability of the plasmid-mediated virulence factors of Bacillus anthracis, a tripartite toxin located on pXO1 and an antiphagocytic capsule encoded by genes located on pXO2, following long-term storage was investigated. A collection of 159 isolates of B. anthracis were collected from the Kars region of Turkey between 2000 and 2013 and stored at -20°C in Brucella broth supplemented with 20% glycerine. A total of 142 isolates were recovered of which one failed to express a capsule upon primary culture. A further 35 isolates yielded a mixture of mucoid and non-mucoid colonies; the majority of which had lost the pXO2 plasmid as determined by PCR analysis. Results would suggest that pXO2 is more unstable than pXO1 and that this instability increases with the length of storage. It is possible that the pXO2-deficient isolates of B. anthracis described here could be developed into a vaccine to treat at risk animals in the Kars region as many animal vaccines are based upon pXO2 deficiency.

Bacillus anthracis pXO1 plasmid encodes a putative membrane-bound bacteriocin.

Evolutionary advantages over cousin cells in bacterial pathogens may decide about the success of a specific cell in its environment. Bacteria use a plethora of methods to defend against other cells and many devices to attack their opponents when competing for resources. Bacteriocins are antibacterial proteins that are used to eliminate competition. We report the discovery of a putative membrane-bound bacteriocin encoded by the Bacillus anthracis pathogenic pXO1 plasmid. We analyze the genomic structure of the bacteriocin operon. The proposed mechanisms of action predestine this operon as a potent competitive advantage over cohabitants of the same niche.