Convergent evolution of diverse Bacillus anthracis outbreak strains toward altered surface oligosaccharides that modulate anthrax pathogenesis

Abstract

Bacillus anthracis, a spore-forming gram-positive bacterium, causes anthrax. The external surface of the exosporium is coated with glycosylated proteins. The sugar additions are capped with the unique monosaccharide anthrose. The West African Group (WAG) B. anthracis have mutations rendering them anthrose deficient. Through genome sequencing, we identified 2 different large chromosomal deletions within the anthrose biosynthetic operon of B. anthracis strains from Chile and Poland. In silico analysis identified an anthrose-deficient strain in the anthrax outbreak among European heroin users. Anthrose-deficient strains are no longer restricted to West Africa so the role of anthrose in physiology and pathogenesis was investigated in B. anthracis Sterne. Loss of anthrose delayed spore germination and enhanced sporulation. Spores without anthrose were phagocytized at higher rates than spores with anthrose, indicating that anthrose may serve an antiphagocytic function on the spore surface. The anthrose mutant had half the LD50 and decreased time to death (TTD) of wild type and complement B. anthracis Sterne in the A/J mouse model. Following infection, anthrose mutant bacteria were more abundant in the spleen, indicating enhanced dissemination of Sterne anthrose mutant. At low sample sizes in the A/J mouse model, the mortality of ΔantC-infected mice challenged by intranasal or subcutaneous routes was 20% greater than wild type. Competitive index (CI) studies indicated that spores without anthrose disseminated to organs more extensively than a complemented mutant. Death process modeling using mouse mortality dynamics suggested that larger sample sizes would lead to significantly higher deaths in anthrose-negative infected animals. The model was tested by infecting Galleria mellonella with spores and confirmed the anthrose mutant was significantly more lethal. Vaccination studies in the A/J mouse model showed that the human vaccine protected against high-dose challenges of the nonencapsulated Sterne-based anthrose mutant. This work begins to identify the physiologic and pathogenic consequences of convergent anthrose mutations in B. anthracis.

Bacteriostatic Potential of Melatonin: Therapeutic Standing and Mechanistic Insights

“…These are partially detectable by gram staining ( 14 ), according to which they are categorized as gram positive bacteria (GPB), gram negative bacteria (GNB), and others ( 15 , 16 ). Examples of pathogenic GPB (PGPB) comprise Staphylococcus aureus ( S. aureus ) ( 17 ), Streptococcus pneumoniae ( S. pneumoniae ) ( 18 ) and Bacillus anthracis ( B. anthracis ) ( 19 ). Pathogenic GNB (PGNB) include Escherichia coli ( E. coli ) ( 20 ), Helicobacter pylori ( H. pylori ) ( 21 ), Pseudomonas aeruginosa ( P. aeruginosa ) ( 22 ), Acinetobacter baumannii (A. baumannii) ( 23 ), and Klebsiella pneumoniae ( K. pneumoniae ) ( 24 ).…”

Beyond the spore, the exosporium sugar anthrose impacts vegetative Bacillus anthracis gene regulation in cis and trans

“…Besides physiological changes, anthrose negative spores had half the LD 50 in a subcutaneous mouse challenge model leading to a more rapid time to death and faster dissemination in host organs. Increase in lethality was also observed in a second animal model by challenging Galleria mellonella larvae with spores 18 .…”

“…The precise function of anthrose and its contribution to pathogenesis remained unclear, with evidence supporting interaction with the soil environment and cells of the immune system. Previously, we found removing anthrose from the spore surface reduced germination efficiency and increased sporulation rates in a heterologous B. anthracis Sterne model 18 . Besides physiological changes, anthrose negative spores had half the LD 50 in a subcutaneous mouse challenge model leading to a more rapid time to death and faster dissemination in host organs.…”

“…Five luminescent reporter plasmids were generated to allow expression analysis from the anthrose operon promoter (P ant ) the atxA promoter (P atxA ), the lethal factor promoter (P lef ), the pagA promoter (P pagA ), and the sigF promoter (P sigF aka the spoIIAA - spoIIAB - sigF promoter). These plasmids were conjugated into B.anthracis Sterne and B.anthracis Sterne Δ antC that we previously verified were unable to produce anthrose 18 . The resulting strains were grown in triplicate in Heart Infusion Broth (HIB) a high protein content medium devoid of sugars and spotted on solid HIB agar.…”

“…These strains have a conserved SNP and nucleotide triplication event that renders them ant − . We previously identified two strains of B. anthracis genetically ant— via chromosomal deletions encompassing the entirety of the anthrose biosynthetic operon, one from Chile and another from Poland, in our B. anthracis global collection 18 , 21 . A search of publicly available sequence records indicated B. anthracis strain Ba4599 Heroin, which was isolated from a European anthrax case linked to B. anthracis spore-contaminated heroin, had a novel SNP linked to the ant − genotype.…”