Schlüsselwort: immunofluorescence

One of the most devastating diseases of honey bees worldwide is the epizootic American Foulbrood (AFB) caused by Paenibacillus larvae, which exclusively damages the brood. Honey bee larvae become infected by ingesting spore contaminated food. After germination of the spores in the midgut lumen, P. lavae follows a non-invasive, commensal lifestyle and vegetative bacteria start to massively proliferate. After degradation of the peritrophic membrane P. larvae switches to an invasive, destructive lifestyle and attacks the midgut epithelium of the larva. After breaching the midgut epithelium, P. larvae enters the hemocoel leading to a generalized infection and the death of the host. The aim of this thesis was the further elucidation of molecular details of the host-pathogen interaction during pathogenesis of an infection of bee larvae with P. larvae. For this purpose two confirmed virulence factors were further investigated: PlCBP49 secreted by P. larvae genotypes ERIC I and ERIC II, as well as the ERIC II specific S-layer protein SplA. At first a further characterization of PlCBP49, a virulence factor with chitin-binding and -degrading activity, was performed. This general virulence factor is used by P. larvae genotypes ERIC I and ERIC II to degrade the peritrophic membrane (PM). Since the degradation of the PM marks the transition from non-invasive to invasive lifestyle and PlCBP49 is secreted from both P. larvae genotypes, this could be a possible intervention point in the control of AFB. In context of this work additional similarities of PlCBP49 and CBP21 of Serratia marcescens, a well described member of the AA10 family of lytic polysaccharide monooxygenases (LPMOs), could be demonstrated. Therefor cbp49 was successfully expressed in E. coli and assays were performed to further functionally and biochemically characterize PlCBP49. Besides the preference for β-chitin as substrate, the best substrate turnover was achieved after two hours of incubation at pH 7. These results and the first product profile of PlCBP49 illustrate many similarities with CBP21. The assumption that PlCBP49 degrades chitin substrates like CBP21 over a metal ion-dependent, oxidative mechanism could be sustained and the results of this work describe basic parameters for future in-depth studies. In this study the ERIC II-specific S-layer Protein SplA was expressed in the naturally SplAdeficient ERIC I strain ATCC9545. The heterologous expression could be verified in vivo by fluorescence-labelled monoclonal antibodies and fluorescence microscopy. In exposure bioassays with the ERIC I+splA mutant no hyper-virulence in comparison with the wild type could be detected. Cloning of only one virulence factor is not sufficient to initiate the complete pathogenetic strategy of P. larvae ERIC II. Additional not yet known factors are presumably necessary to trigger the complex pathogenetic strategy. The investigation of the immune response of larva during an infection with P. larvae was already subject of several studies with contradicting results. The influence of the infecting P. larvae genotype has been utterly neglected. Through the comparative transcriptome analysis, via RNA-Seq, it could be demonstrated that an infection of larvae with the different P. larvae genotypes ERIC I or ERIC II, results in a change of expression of immune-relevant genes three days after infection. The increased expression of antimicrobial peptides (AMPs) was outstanding. Depending on the infecting P. larvae genotype different combinations of AMPs were expressed. Furthermore a stronger immune response was observed in ERIC IIinfected larvae and the assumed masking of P. larvae ERIC II through the S-layer protein could be rejected. These results point to a genotype-specific immune response of larvae to a P. larvae infection. Although insects are devoid of an adaptive immune system, in the present study it could be demonstrated that not solely the species of the pathogen, but also the infecting genotype is a crucial factor and has profound influence on the host immune response. Further investigations of the host-pathogen interaction of honey bee larvae and P. larvae need to consider the infecting genotype of the pathogen. In an additional experiment the expression of selected immune-relevant genes was analysed in individual larvae and the results of the comparative transcriptome analysis could be verified. In addition to the expression of different AMPs, depending on the infecting genotype, a stronger immune response in ERIC II-infected larvae could be approved. Since ERIC IIinfected larvae die within a very short time and mount a stronger immune response, it seems likely that the costly immune response contributes to the premature death of the larva. Taken together in this thesis the general virulence factor PlCBP49 was further biochemically characterized. In addition the investigation of the pathogenetic strategy of P. larvae ERIC II was advanced through the heterologous expression of SplA in P. larvae ERIC I. Furthermore the immune response of the larva was analysed and it could be demonstrated that not only the pathogen species, but also the genotype is crucial for the host immune response. Thesen results broaden our understanding of host-pathogen interaction of honey bee larvae and P. larvae.