Bacillus anthracis in South Africa, 1975-2013: are some lineages vanishing?

The anthrax-causing bacterium Bacillus anthracis comprises the genetic clades A, B, and C. In the northernmost part (Pafuri) of Kruger National Park (KNP), South Africa, both the common A and rare B strains clades occur. The B clade strains were reported to be dominant in Pafuri before 1991, while A clade strains occurred towards the central parts of KNP. The prevalence of B clade strains is currently much lower as only A clade strains have been isolated from 1992 onwards in KNP. In this study 319 B. anthracis strains were characterized with 31-loci multiple-locus variable-number tandem repeat analysis (MLVA-31). B clade strains from soil (n = 9) and a Tragelaphus strepsiceros carcass (n = 1) were further characterised by whole genome sequencing and compared to publicly available genomes. The KNP strains clustered in the B clade before 1991 into two dominant genotypes. South African strains cluster into a dominant genotype A.Br.005/006 consisting of KNP as well as the other anthrax endemic region, Northern Cape Province (NCP), South Africa. A few A.Br.001/002 strains from both endemic areas were also identified. Subclade A.Br.101 belonging to the A.Br.Aust94 lineage was reported in the NCP. The B-clade strains seems to be vanishing, while outbreaks in South Africa are caused mainly by the A.Br.005/006 genotypes as well as a few minor clades such as A.Br.001/002 and A.Br.101 present in NCP. This work confirmed the existence of the rare and vanishing B-clade strains that group in B.Br.001 branch with KrugerB and A0991 KNP strains.

Rapid in vitro activity of telavancin against Bacillus anthracis and in vivo protection against inhalation anthrax infection in the rabbit model.

Inhalation anthrax is the most severe form of Bacillus anthracis infection, often progressing to fatal conditions if left untreated. While recommended antibiotics can effectively treat anthrax when promptly administered, strains engineered for antibiotic resistance could render these drugs ineffective. Telavancin, a semisynthetic lipoglycopeptide antibiotic, was evaluated in this study as a novel therapeutic against anthrax disease. Specifically, the aims were to (i) assess in vitro potency of telavancin against 17 B. anthracis isolates by minimum inhibitory concentration (MIC) testing and (ii) evaluate protective efficacy in rabbits infected with a lethal dose of aerosolized anthrax spores and treated with human-equivalent intravenous telavancin doses (30 mg/kg every 12 hours) for 5 days post-antigen detection versus a humanized dose of levofloxacin and vehicle control. Blood samples were collected at various times post-infection to assess the level of bacteremia and antibody production, and tissues were collected to determine bacterial load. The animals' body temperatures were also recorded. Telavancin demonstrated potent bactericidal activity against all strains tested (MICs 0.06-0.125 µg/mL). Further, telavancin conveyed 100% survival in this model and cleared B. anthracis from the bloodstream and organ tissues more effectively than a humanized dose of levofloxacin. Collectively, the low MICs against all strains tested and rapid bactericidal in vivo activity demonstrate that telavancin has the potential to be an effective alternative for the treatment or prophylaxis of anthrax infection.

Efficacy of therapeutically administered gepotidacin in a rabbit model of inhalational anthrax.

Bacillus anthracis is a Gram-positive Centers for Disease Control and Prevention category "A" biothreat pathogen. Without early treatment, inhalation of anthrax spores with progression to inhalational anthrax disease is associated with high fatality rates. Gepotidacin is a novel first-in-class triazaacenaphthylene antibiotic that inhibits bacterial DNA replication by a distinct mechanism of action and is being evaluated for use against biothreat and conventional pathogens. Gepotidacin selectively inhibits bacterial DNA replication via a unique binding mode and has in vitro activity against a collection of B. anthracis isolates including antibacterial-resistant strains, with the MIC90 ranging from 0.5 to 1 µg/mL. In vivo activity of gepotidacin was also evaluated in the New Zealand White rabbit model of inhalational anthrax. The primary endpoint was survival, with survival duration and bacterial clearance as secondary endpoints. The trigger for treatment was the presence of anthrax protective antigen in serum. New Zealand White rabbits were dosed intravenously for 5 days with saline or gepotidacin at 114 mg/kg/d to simulate a dosing regimen of 1,000 mg intravenous (i.v.) three times a day (TID) in humans. Gepotidacin provided a survival benefit compared to saline control, with 91% survival (P-value: 0.0001). All control animals succumbed to anthrax and were found to be blood- and organ culture-positive for B. anthracis. The novel mode of action, in vitro microbiology, preclinical safety, and animal model efficacy data, which were generated in line with Food and Drug Administration Animal Rule, support gepotidacin as a potential treatment for anthrax in an emergency biothreat situation.

Doxycycline, levofloxacin, and moxifloxacin are superior to ciprofloxacin in treating anthrax meningitis in rabbits and NHP.

Efficient treatment of anthrax-related meningitis in patients poses a significant therapeutic challenge. Previously, we demonstrated in our anthrax meningitis rabbit model that ciprofloxacin treatment is ineffective with most of the treated animals succumbing to the infection. Herein we tested the efficacy of doxycycline in our rabbit model and found it highly effective. Since all of our findings are based on a rabbit model, we test the efficacy of ciprofloxacin or doxycycline in a specific central nervous system (CNS) model developed in non-human primates (NHPs). Similar to rabbits, ciprofloxacin treatment was ineffective, while doxycycline protected the infected rhesus macaques (n = 2) from the lethal CNS Bacillus anthracis infection. To test whether the low efficacy of Ciprofloxacin is an example of low efficacy of all fluoroquinolones or only this substance, we treated rabbits that were inoculated intracisterna magna (ICM) with levofloxacin or moxifloxacin. We found that in contrast to ciprofloxacin, levofloxacin and moxifloxacin were highly efficacious in treating lethal anthrax-related meningitis in rabbits and NHP (levofloxacin). We demonstrated (in naïve rabbits) that this difference probably results from variances in blood-brain-barrier penetration of the different fluoroquinolones. The combined treatment of doxycycline and any one of the tested fluoroquinolones was highly effective in the rabbit CNS infection model. The combined treatment of doxycycline and levofloxacin was effective in an inhalation rabbit model, as good as the doxycycline mono-therapy. These findings imply that while ciprofloxacin is highly effective as a post-exposure prophylactic drug, using this drug to treat symptomatic patients should be reconsidered.

Season of death, pathogen persistence and wildlife behaviour alter number of anthrax secondary infections from environmental reservoirs.

An important part of infectious disease management is predicting factors that influence disease outbreaks, such as R, the number of secondary infections arising from an infected individual. Estimating R is particularly challenging for environmentally transmitted pathogens given time lags between cases and subsequent infections. Here, we calculated R for Bacillus anthracis infections arising from anthrax carcass sites in Etosha National Park, Namibia. Combining host behavioural data, pathogen concentrations and simulation models, we show that R is spatially and temporally variable, driven by spore concentrations at death, host visitation rates and early preference for foraging at infectious sites. While spores were detected up to a decade after death, most secondary infections occurred within 2 years. Transmission simulations under scenarios combining site infectiousness and host exposure risk under different environmental conditions led to dramatically different outbreak dynamics, from pathogen extinction (R < 1) to explosive outbreaks (R > 10). These transmission heterogeneities may explain variation in anthrax outbreak dynamics observed globally, and more generally, the critical importance of environmental variation underlying host-pathogen interactions. Notably, our approach allowed us to estimate the lethal dose of a highly virulent pathogen non-invasively from observational studies and epidemiological data, useful when experiments on wildlife are undesirable or impractical.

Lysins as a powerful alternative to combat Bacillus anthracis.

This review gathers all, to the best of our current knowledge, known lysins, mainly bacteriophage-derived, that have demonstrated activity against Bacillus anthracis strains. B. anthracis is a spore-forming, toxin-producing bacteria, naturally dwelling in soil. It is best known as a potential biowarfare threat, an etiological agent of anthrax, and a severe zoonotic disease. Anthrax can be treated with antibiotics (ciprofloxacin, penicillin, doxycycline); however, their administration may take up even to 60 days, and different factors can compromise their effectiveness. Bacterial viruses, bacteriophages (phages), are natural enemies of bacteria and use their lytic enzymes, endolysins (lysins), to specifically kill bacterial cells. Harnessing the potential of lysins to combat bacterial infections holds promise for diminishing antibiotic usage and, consequently, addressing the escalating antibiotic resistance in bacteria. In this context, we list the lysins with the activity against B. anthracis, providing a summary of their lytic properties in vitro and the outcomes observed in animal models. Bacillus cereus strain ATCC 4342/RSVF1, a surrogate for B. anthracis, was also included as a target bacteria. KEY POINTS: • More than a dozen different B. anthracis lysins have been identified and studied. • They fall into three blocks regarding their amino acid sequence similarity and most of them are amidases. • Lysins could be used in treating B. anthracis infections.

New bacteriophage-derived lysins, LysJ and LysF, with the potential to control Bacillus anthracis.

Bacillus anthracis is an etiological agent of anthrax, a severe zoonotic disease that can be transmitted to people and cause high mortalities. Bacteriophages and their lytic enzymes, endolysins, have potential therapeutic value in treating infections caused by this bacterium as alternatives or complements to antibiotic therapy. They can also be used to identify and detect B. anthracis. Endolysins of two B. anthracis Wbetavirus phages, J5a and F16Ba which were described by us recently, differ significantly from the best-known B. anthracis phage endolysin PlyG from Wbetavirus genus bacteriophage Gamma and a few other Wbetavirus genus phages. They are larger than PlyG (351 vs. 233 amino acid residues), contain a signal peptide at their N-termini, and, by prediction, have a different fold of cell binding domain suggesting different structural basis of cell epitope recognition. We purified in a soluble form the modified versions of these endolysins, designated by us LysJ and LysF, respectively, and depleted of signal peptides. Both modified endolysins could lyse the B. anthracis cell wall in zymogram assays. Their activity against the living cells of B. anthracis and other species of Bacillus genus was tested by spotting on the layers of bacteria in soft agar and by assessing the reduction of optical density of bacterial suspensions. Both methods proved the effectiveness of LysJ and LysF in killing the anthrax bacilli, although the results obtained by each method differed. Additionally, the lytic efficiency of both proteins was different, which apparently correlates with differences in their amino acid sequence. KEY POINTS: • LysJ and LysF are B. anthracis-targeting lysins differing from lysins studied so far • LysJ and LysF could be overproduced in E. coli in soluble and active forms • LysJ and LysF are active in killing cells of B. anthracis virulent strains.

Dachshund Homolog 1: Unveiling Its Potential Role in Megakaryopoiesis and Bacillus anthracis Lethal Toxin-Induced Thrombocytopenia.

Lethal toxin (LT) is the critical virulence factor of Bacillus anthracis, the causative agent of anthrax. One common symptom observed in patients with anthrax is thrombocytopenia, which has also been observed in mice injected with LT. Our previous study demonstrated that LT induces thrombocytopenia by suppressing megakaryopoiesis, but the precise molecular mechanisms behind this phenomenon remain unknown. In this study, we utilized 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced megakaryocytic differentiation in human erythroleukemia (HEL) cells to identify genes involved in LT-induced megakaryocytic suppression. Through cDNA microarray analysis, we identified Dachshund homolog 1 (DACH1) as a gene that was upregulated upon TPA treatment but downregulated in the presence of TPA and LT, purified from the culture supernatants of B. anthracis. To investigate the function of DACH1 in megakaryocytic differentiation, we employed short hairpin RNA technology to knock down DACH1 expression in HEL cells and assessed its effect on differentiation. Our data revealed that the knockdown of DACH1 expression suppressed megakaryocytic differentiation, particularly in polyploidization. We demonstrated that one mechanism by which B. anthracis LT induces suppression of polyploidization in HEL cells is through the cleavage of MEK1/2. This cleavage results in the downregulation of the ERK signaling pathway, thereby suppressing DACH1 gene expression and inhibiting polyploidization. Additionally, we found that known megakaryopoiesis-related genes, such as FOSB, ZFP36L1, RUNX1, FLI1, AHR, and GFI1B genes may be positively regulated by DACH1. Furthermore, we observed an upregulation of DACH1 during in vitro differentiation of CD34-megakaryocytes and downregulation of DACH1 in patients with thrombocytopenia. In summary, our findings shed light on one of the molecular mechanisms behind LT-induced thrombocytopenia and unveil a previously unknown role for DACH1 in megakaryopoiesis.

Comparison of Illumina and Oxford Nanopore Technology for genome analysis of Francisella tularensis, Bacillus anthracis, and Brucella suis.

BACKGROUND: Bacterial epidemiology needs to understand the spread and dissemination of strains in a One Health context. This is important for highly pathogenic bacteria such as Bacillus anthracis, Brucella species, and Francisella tularensis. Whole genome sequencing (WGS) has paved the way for genetic marker detection and high-resolution genotyping. While such tasks are established for Illumina short-read sequencing, Oxford Nanopore Technology (ONT) long-read sequencing has yet to be evaluated for such highly pathogenic bacteria with little genomic variations between strains. In this study, three independent sequencing runs were performed using Illumina, ONT flow cell version 9.4.1, and 10.4 for six strains of each of Ba. anthracis, Br. suis and F. tularensis. Data from ONT sequencing alone, Illumina sequencing alone and two hybrid assembly approaches were compared. RESULTS: As previously shown, ONT produces ultra-long reads, while Illumina produces short reads with higher sequencing accuracy. Flow cell version 10.4 improved sequencing accuracy over version 9.4.1. The correct (sub-)species were inferred from all tested technologies, individually. Moreover, the sets of genetic markers for virulence, were almost identical for the respective species. The long reads of ONT allowed to assemble not only chromosomes of all species to near closure, but also virulence plasmids of Ba. anthracis. Assemblies based on nanopore data alone, Illumina data alone, and both hybrid assemblies correctly detected canonical (sub-)clades for Ba. anthracis and F. tularensis as well as multilocus sequence types for Br. suis. For F. tularensis, high-resolution genotyping using core-genome MLST (cgMLST) and core-genome Single-Nucleotide-Polymorphism (cgSNP) typing produced highly comparable results between data from Illumina and both ONT flow cell versions. For Ba. anthracis, only data from flow cell version 10.4 produced similar results to Illumina for both high-resolution typing methods. However, for Br. suis, high-resolution genotyping yielded larger differences comparing Illumina data to data from both ONT flow cell versions. CONCLUSIONS: In summary, combining data from ONT and Illumina for high-resolution genotyping might be feasible for F. tularensis and Ba. anthracis, but not yet for Br. suis. The ongoing improvement of nanopore technology and subsequent data analysis may facilitate high-resolution genotyping for all bacteria with highly stable genomes in future.

Role of novel polysaccharide layers in assembly of the exosporium, the outermost protein layer of the Bacillus anthracis spore.

A fundamental question in cell biology is how cells assemble their outer layers. The bacterial endospore is a well-established model for cell layer assembly. However, the assembly of the exosporium, a complex protein shell comprising the outermost layer in the pathogen Bacillus anthracis, remains poorly understood. Exosporium assembly begins with the deposition of proteins at one side of the spore surface, followed by the progressive encirclement of the spore. We seek to resolve a major open question: the mechanism directing exosporium assembly to the spore, and then into a closed shell. We hypothesized that material directly underneath the exosporium (the interspace) directs exosporium assembly to the spore and drives encirclement. In support of this, we show that the interspace possesses at least two distinct layers of polysaccharide. Secondly, we show that putative polysaccharide biosynthetic genes are required for exosporium encirclement, suggesting a direct role for the interspace. These results not only significantly clarify the mechanism of assembly of the exosporium, an especially widespread bacterial outer layer, but also suggest a novel mechanism in which polysaccharide layers drive the assembly of a protein shell.

Phosphorylation-mediated regulation of the Bacillus anthracis phosphoglycerate mutase by the Ser/Thr protein kinase PrkC.

Bacillus anthracis Ser/Thr protein kinase PrkC is necessary for phenotypic memory and spore germination, and the loss of PrkC-dependent phosphorylation events affect the spore development. During sporulation, Bacillus sp. can store 3-Phosphoglycerate (3-PGA) that will be required at the onset of germination when ATP will be necessary. The Phosphoglycerate mutase (Pgm) catalyzes the isomerization of 2-PGA and 3-PGA and is important for spore germination as a key metabolic enzyme that maintains 3-PGA pool at later events. Therefore, regulation of Pgm is important for an efficient spore germination process and metabolic switching. While the increased expression of Pgm in B. anthracis decreases spore germination efficiency, it remains unexplored if PrkC could directly influence Pgm activity. Here, we report the phosphorylation and regulation of Pgm by PrkC and its impact on Pgm stability and catalytic activity. Mass spectrometry revealed Pgm phosphorylation on seven threonine residues. In silico mutational analysis highlighted the role of Thr459 residue towards metal and substrate binding. Altogether, we demonstrated that PrkC-mediated Pgm phosphorylation negatively regulates its activity that is essential to maintain Pgm in its apo-like isoform before germination. This study advances the role of Pgm regulation that represents an important switch for B. anthracis resumption of metabolism and spore germination.

Bacillus anthracis spores are internalized in human lung epithelial cells by Rab GTPase-supported macropinocytosis.

Inhalation anthrax, the deadliest form of the disease, requires inhaled B. anthracis spores to escape from the alveolar space and travel to the mediastinal lymph nodes, from where the vegetative form of the pathogen disseminates, resulting in a rapidly fatal outcome. The role of epithelia in alveolar escape is unclear, but previous work suggests these epithelial cells are involved in this process. Using confocal microscopy, we found that B. anthracis spores are internalized more rapidly by A549 type II alveolar epithelial cells compared to hAELVi type I alveolar epithelial cells. Internalization of spores by alveolar epithelial cells requires cytoskeletal rearrangement evidenced by significant inhibition by cytochalasin D, an actin inhibitor. Chemical inhibitors of macropinocytosis significantly downregulated B. anthracis spore internalization in human alveolar cells, while inhibitors of other endocytosis pathways had minimal effects. Additional studies using a macropinosome marker and electron microscopy confirmed the role of macropinocytosis in spore uptake. By colocalization of B. anthracis spores with four endocytic Rab proteins, we demonstrated that Rab31 played a role in B. anthracis spore macropinocytosis. Finally, we confirmed that Rab31 is involved in B. anthracis spore internalization by enhanced spore uptake in Rab31-overexpressing A549 cells. This is the first report that shows B. anthracis spore internalization by macropinocytosis in human epithelial cells. Several Rab GTPases are involved in the process.

Passenger vehicle interior decontamination by low concentration hydrogen peroxide vapor following a wide area biological contamination incident.

AIMS: To assess low concentration hydrogen peroxide (LCHP) (H2O2) vapor dispersed with a home humidifier for its ability to decontaminate vehicle interiors contaminated with Bacillus anthracis surrogate Bacillus atrophaeus spores. METHODS AND RESULTS: Efficacy of a vaporized 3% H2O2 solution was evaluated for liquid volumes, on/off vehicle heating, ventilation, and air conditioning (HVAC) system operations, and temperatures that ranged from 5 to 27°C. Survival of the spores was assessed by quantification of remaining viable spores with efficacy quantified in terms of mean log10 reduction. Decontamination efficacy after the 6-day dwell time increased when the 3% H2O2 liquid volume was doubled, increasing from 4-of-10 to 10-of-10 nondetects (zero colonies counted using standard dilution and filter plating) inside the vehicle cabin. Recirculating cabin air through the HVAC system during decontamination decreased efficacy to 6-of-10 non-detects. While no 6-log10 reduction in viable spores was observed on the cabin filter with the cabin filter kept in place, a 6-log10 reduction was achieved after its removal and placement in the cabin during treatment. CONCLUSIONS: Results from this study allow for informed decisions on the use of LCHP vapor as an effective decontamination approach for vehicle interiors.

Impact of filter material and holding time on spore sampling efficiency in water.

Bacillus anthracis and other environmentally persistent pathogens pose a significant threat to human and environmental health. If contamination is spread over a wide area (e.g. resulting from a bioterrorism or biowarfare incident), readily deployable and scalable sample collection methods will be necessary for rapidly developing and implementing effective remediation strategies. A recent surge in environmental (eDNA) sampling technologies could prove useful for quantifying the extent and levels of contamination from biological agents in environmental and drinking water. In this study, three commonly used membrane filtration materials (cellulose acetate, cellulose nitrate, and nylon) were evaluated for spore filtration efficiency, yielding recoveries from 17%-68% to 25%-117% for high and low titer samples, respectively, where cellulose nitrate filters generated the highest recoveries. A holding time test revealed no statistically significant differences between spore recoveries when analyzed at the specified timepoints, suggesting that eDNA filter sampling techniques can yield and maintain a relatively high recovery of spores for an extended period of time between filtration and analysis without a detrimental impact on spore recoveries. The results shown here indicate that emerging eDNA technologies could be leveraged for sampling following a wide-area contamination incident and for other microbiological water sampling applications.

Recent Advancements and Novel Approaches Contributing to the Present Arsenal of Prophylaxis and Treatment Strategies Against Category A Bacterial Biothreat Agents.

Bacterial pathogens have always been a part of the ecosystem in which we thrive. Some pathogens have caused deadly outbreaks in the past and have been exploited as an agent of threat. Natural hotspots for these biological pathogens are widely distributed throughout the world and hence they remain clinically important. Technological advancement and change in general lifestyle has driven the evolution of these pathogens into more virulent and resistant variants. There has been a growing concern over the development of multidrug-resistant bacterial strains that could be used as bioweapons. This rapid change in pathogens also propels the field of science to develop and innovate new strategies and methodologies which are superior and safer to the existing ones. Some bacterial agents like-Bacillus anthracis, Yersinia pestis, Francisella tularensis and toxins produced by strains of Clostridium botulinum, have been segregated as Category A substances as they pose imminent threat to public health with a history of life threatening and catastrophic disease. This review highlights some encouraging developments and value additions in the current plan of action for protection against these select biothreat bacterial pathogens.

Overlapping and Distinct Functions of the Paralogous PagR Regulators of Bacillus anthracis.

The Bacillus anthracis pagA gene, encoding the protective antigen component of anthrax toxin, is part of a bicistronic operon on pXO1 that codes for its own repressor, PagR1. In addition to the pagAR1 operon, PagR1 regulates sap and eag, two chromosome genes encoding components of the surface layer, a mounting structure for surface proteins involved in virulence. Genomic studies have revealed a PagR1 paralog, PagR2, encoded by a gene on pXO2. The amino acid sequences of the paralogues are 71% identical and show similarity to the ArsR family of transcription regulators. We determined that the expression of either rPagR1 or rPagR2 in a ΔpagR1 pXO1+/pXO2- (PagR1-PagR2) background repressed the expression of pagA, sap, eag, and a newly discovered target, atxA, encoding virulence activator AtxA. Despite the redundancy in PagR1 and PagR2 function, we determined that purified rPagR1 bound DNA corresponding to the control regions of all four target genes and existed as a dimer in cell lysates, whereas rPagR2 exhibited weak binding to the DNA of the pagA and atxA promoters, did not bind sap or eag promoter DNA, and did not appear as a dimer in cell lysates. A single amino acid change in PagR2, S81Y, designed to match the native Y81 of PagR1, allowed for DNA-binding to the sap and eag promoters. Moreover, the S81Y mutation allowed for the detection of PagR2 homomultimers in coaffinity purification experiments. Our results expand our knowledge of the roles of the paralogues in B. anthracis gene expression and provide a potential mechanistic basis for differences in the functions of these repressors. IMPORTANCE The protective antigen component of the anthrax toxin is essential for the delivery of the enzymatic components of the toxin into host target cells. The toxin genes and other virulence genes of B. anthracis are regulated by multiple trans-acting regulators that respond to a variety of host-related signals. PagR1, one such trans-acting regulator, connects the regulation of plasmid-encoded and chromosome-encoded virulence genes by controlling both protective antigen and surface layer protein expression. Whether PagR2, a paralog of PagR1, also functions as a trans-acting regulator was unknown. This work advances our knowledge of the complex model of virulence regulation in B. anthracis and furthers our understanding of the intriguing evolution of this pathogen.

ExsY, CotY, and CotE Effects on Bacillus anthracis Outer Spore Layer Architecture.

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are the major pathogens of the spore-forming genus Bacillus and possess an outer spore layer, the exosporium, not found in many of the nonpathogenic species. The exosporium consists of a basal layer with the ExsY, CotY, and BxpB proteins being the major structural components and an exterior nap layer containing the BclA glycoprotein. During the assembly process, the nascent exosporium basal layer is attached to the spore coat by a protein linker that includes the CotO and CotE proteins. Using transmission electron microscopy, Western blotting, immunofluorescence, and fluorescent fusion protein approaches, we examined the impact of single, double, and triple mutants of the major exosporium proteins on exosporium protein content and distribution. Plasmid-based expression of exsY and cotE resulted in increased production of exosporium lacking spores, and the former also resulted in outer spore coat disruptions. The exosporium bottlecap produced by exsY null spores was found to be more stable than previously reported, and its spore association was partially dependent on CotE. Deletion mutants of five putative spore genes (bas1131, bas1142, bas1143, bas2277, and bas3594) were created and shown not to have obvious effects on spore morphology or BclA and BxpB content. The BclC collagen-like glycoprotein was found to be present in the spore and possibly localized to the interspace region. IMPORTANCE B. anthracis is an important zoonotic animal pathogen causing sporadic outbreaks of anthrax worldwide. Spores are the infectious form of the bacterium and can persist in soil for prolonged periods of time. The outermost B. anthracis spore layer is the exosporium, a protein shell that is the site of interactions with both the soil and with the innate immune system of infected hosts. Although much is known regarding the sporulation process among members of the genus Bacillus, significant gaps in our understanding of the exosporium assembly process exist. This study provides evidence for the properties of key exosporium basal layer structural proteins. The results of this work will guide future studies on exosporium protein-protein interactions during the assembly process.

Roles and Organization of BxpB (ExsFA) and ExsFB in the Exosporium Outer Basal Layer of Bacillus anthracis.

BxpB (also known as ExsFA) and ExsFB are an exosporium basal layer structural protein and a putative interspace protein of Bacillus anthracis that are known to be required for proper incorporation of the BclA collagen-like glycoprotein on the spore surface. Despite extensive similarity of the two proteins, their distribution in the spore is markedly different. We utilized a fluorescent fusion approach to examine features of the two genes that affect spore localization. The timing of expression of the bxpB and exsFB genes and their distinct N-terminal sequences were both found to be important for proper assembly into the exosporium basal layer. Results of this study provided evidence that the BclA nap glycoprotein is not covalently attached to BxpB protein despite the key role that the latter plays in BclA incorporation. Assembly of the BxpB- and ExsFB-containing outer basal layer appears not to be completely abolished in mutants lacking the ExsY and CotY basal layer structural proteins despite these spores lacking a visible exosporium. The BxpB and, to a lesser extent, the ExsFB proteins, were found to be capable of self-assembly in vitro into higher-molecular-weight forms that are stable to boiling in SDS under reducing conditions. IMPORTANCE The genus Bacillus consists of spore-forming bacteria. Some species of this genus, especially those that are pathogens of animals or insects, contain an outermost spore layer called the exosporium. The zoonotic pathogen B. anthracis is an example of this group. The exosporium likely contributes to virulence and environmental persistence of these pathogens. This work provides important new insights into the exosporium assembly process and the interplay between BclA and BxpB in this process.

Contribution of TagA-Like Glycosyltransferases to the Assembly of the Secondary Cell Wall Polysaccharide in Bacillus anthracis.

Bacillus anthracis elaborates a secondary cell wall polysaccharide (SCWP) made of 6 to 12 trisaccharide units. Pyruvyl and acetyl substitutions of the distal unit are prerequisites for the noncovalent retention of 22 secreted Bacillus S-layer (Bsl)-associated proteins bearing an S-layer homology (SLH) domain. Surface display of Bsl proteins contributes to cell separation as well as virulence. Earlier work suggested that TagO initiates the synthesis of SCWP while GneY and GneZ, two UDP-GlcNAc 2-epimerases, synthesize ManNAc that is later incorporated in the repeat unit (→4)-ManNAc-(ß1→4)-GlcNAc-(ß1→6)-GlcNAc-(α1→). In organisms that synthesize wall teichoic acid, TagA catalysts have been shown to form the glycosidic bond ManNAc-(ß1→4)-GlcNAc. Here, we show that genes bas2675 and bas5272, predicted to encode glycosyltransferases of the WecB/TagA/CpsF family (PFAM03808; CAZy GT26), are required for B. anthracis SCWP synthesis and S-layer assembly. Similar to tagO or gneY gneZ mutants, B. anthracis strains depleted of tagA1 (bas5272) cannot maintain cell shape, support vegetative growth, or synthesize SCWP. Expression of tagA2 (bas2675), or Staphylococcus aureus tagA on a plasmid, rescues the nonviable tagA1 mutant. We propose that TagA1 and TagA2 fulfill overlapping and key glycosyltransferase functions for the synthesis of repeat units of the SCWP of B. anthracis. IMPORTANCE Glycosyltransferases (GTs) catalyze the transfer of sugar moieties from activated donor molecules to acceptor molecules to form glycosidic bonds using a retaining or inverting mechanism. Based on the structural relatedness of their catalytic and carbohydrate-binding modules, GTs have been grouped into 115 families in the Carbohydrate-Active EnZyme (CAZy) database. For complex products, the functional assignment of GTs remains highly challenging without the knowledge of the chemical structure of the assembled polymer. Here, we propose that two uncharacterized GTs of B. anthracis belonging to the WecB/TagA/CpsF family incorporate ManNAc in repeat units of the secondary cell wall polymer of bacilli species.

Structural and Functional Analysis of Toxin and Small RNA Gene Promoter Regions in Bacillus anthracis.

It was previously demonstrated that anthrax toxin activator (AtxA) binds directly to the σA-like promoter region of pagA (encoding protective antigen, PA) immediately upstream of the RNA polymerase binding site. In this study, using electrophoretic mobility shift assays and in vivo analyses, we identified AtxA-binding sites in the promoter regions of the lef and cya genes (encoding lethal and edema factors, respectively) and of two Bacillus anthracis small RNAs (XrrA and XrrB). Activities of all four newly studied promoters were enhanced in the presence of CO2/bicarbonate and AtxA, as previously seen for the pagA promoter. Notably, the cya promoter was less activated by AtxA and CO2/bicarbonate conditions. The putative promoter of a recently described third small RNA, XrrC, showed a negligible response to AtxA and CO2/bicarbonate. RNA polymerase binding sites of the newly studied promoters show no consensus and differ from the σA-like promoter region of pagA. In silico analysis of the probable AtxA binding sites in the studied promoters revealed several palindromes. All the analyzed palindromes showed very little overlap with the σA-like pagA promoter. It remains unclear as to how AtxA and DNA-dependent RNA-polymerase identify such diverse DNA-sequences and differentially regulate promoter activation of the studied genes. IMPORTANCE Anthrax toxin activator (AtxA) is the major virulence regulator of Bacillus anthracis, the causative agent of anthrax. Understanding AtxA's mechanism of regulation could facilitate the development of therapeutics for B. anthracis infection. We provide evidence that AtxA binds to the promoters of the cya, lef, xrrA, and xrrB genes. In vivo assays confirmed the activities of all four promoters were enhanced in the presence of AtxA and CO2/bicarbonate, as previously seen for the pagA promoter. The cya and lef genes encode important toxin components. The xrrA and xrrB genes encode sRNAs with a suggested function as cell physiology regulators. Our data provides further evidence for the direct regulatory role of AtxA that was previously shown with the pagA promoter.