Abstract
The Gram-positive, spore-forming bacterium Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a globally occurring, deathly epizootic of honey bee brood. AFB outbreaks are predominantly caused by two genotypes of P. larvae, ERIC I and ERIC II, with P. larvae ERIC II being the more virulent genotype on larval level. Recently, comparative proteome analyses have revealed that P. larvae ERIC II but not ERIC I might harbour a functional S-layer protein, named SplA. We here determine the genomic sequence of splA in both genotypes and demonstrate by in vitro self-assembly studies of recombinant and purified SplA protein in combination with electron-microscopy that SplA is a true S-layer protein self-assembling into a square 2D lattice. The existence of a functional S-layer protein is novel for this bacterial species. For elucidating the biological function of P. larvae SplA, a genetic system for disruption of gene expression in this important honey bee pathogen was developed. Subsequent analyses of in vivo biological functions of SplA were based on comparing a wild-type strain of P. larvae ERIC II with the newly constructed splA-knockout mutant of this strain. Differences in cell and colony morphology suggest that SplA is a shape-determining factor. Marked differences between P. larvae ERIC II wild-type and mutant cells with regard to (i) adhesion to primary pupal midgut cells and (ii) larval mortality as measured in exposure bioassays corroborate the assumption that the S-layer of P. larvae ERIC II is an important virulence factor. Since SplA is the first functionally proven virulence factor for this species, our data extend the knowledge of the molecular differences between these two genotypes of P. larvae and contribute to explaining the observed differences in virulence. These results present an immense advancement in our understanding of P. larvae pathogenesis.
Highlights
Due to their role as pollinators in natural and agricultural ecosystems, managed honey bees (Apis mellifera) are among the most important productive livestock [1,2]
By comparative proteome analysis between P. larvae enterobacterial repetitive intergenic consensus (ERIC) I and ERIC II we recently demonstrated the expression of a putative Slayer protein SplA in ERIC II but not in ERIC I strains of P. larvae [25]
In P. larvae ERIC I strains, the gene was interrupted by a frameshift mutation due to an inserted adenine (A) resulting in a premature stop of translation due to a stop codon TAA; the insertion and resulting frameshift mutation was missing in P. larvae
Summary
Due to their role as pollinators in natural and agricultural ecosystems, managed honey bees (Apis mellifera) are among the most important productive livestock [1,2]. Paenibacillus larvae (P. larvae), a Gram-positive, rod-shaped, sporeforming bacterium, is the most devastating bacterial pathogen of honey bees. It is the etiological agent of the epizootic American Foulbrood (AFB), a non-rare, globally occurring brood disease which is classified as notifiable disease in most countries [5]. Larvae become infected by consuming spore contaminated larval diet. The ropy mass dries down to a hard scale which consists of billions of spores. These spores drive disease transmission within the colony as well as between colonies when either fed to young larvae or when distributed by contaminated adult bees. P. larvae is an obligate killer because transmission depends on the death of the host (for recent reviews see [7,8,9])
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