title: Identification and functional analysis of the S-layer protein SplA of Paenibacillus larvae, the causative agent of American Foulbrood of honey bees.
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.
authors.affiliation: Institute for Bee Research, Department of Molecular Bee Pathology, Hohen Neuendorf, Germany.
authors.forename: Lena
authors.initials: L
authors.lastname: Poppinga
authors.forename: Bettina
authors.initials: B
authors.lastname: Janesch
authors.forename: Anne
authors.initials: A
authors.lastname: Fünfhaus
authors.forename: Gerhard
authors.initials: G
authors.lastname: Sekot
authors.forename: Eva
authors.initials: E
authors.lastname: Garcia-Gonzalez
authors.forename: Gillian
authors.initials: G
authors.lastname: Hertlein
authors.forename: Kati
authors.initials: K
authors.lastname: Hedtke
authors.forename: Christina
authors.initials: C
authors.lastname: Schäffer
authors.forename: Elke
authors.initials: E
authors.lastname: Genersch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001422:Bacterial Adhesion; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D001516:Bees / Q000382:microbiology*; D002478:Cells, Cultured; D055786:Gene Knockout Techniques; D005838:Genotype; D007814:Larva / Q000382:microbiology; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D056507:Paenibacillus / Q000472:pathogenicity*; D016415:Sequence Alignment; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D008562:Membrane Glycoproteins; C055764:S-layer proteins; D037521:Virulence Factors