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Paenibacillus larvae is the etiological agent of American foulbrood (AFB) in honeybees. Recently, different genotypes of P. larvae (ERIC I to ERIC IV) were defined, and it was shown that these genotypes differ inter alia in their virulence on the larval level. On the colony level, bees mitigate AFB through the hygienic behavior of nurse bees. Therefore, we investigated how the hygienic behavior shapes P. larvae virulence on the colony level. Our results indicate that P. larvae virulence on the larval level and that on the colony level are negatively correlated.

Paenibacillus larvae, the etiological agent of the globally occurring epizootic American Foulbrood (AFB) of honey bees, causes intestinal infections in honey bee larvae which develop into systemic infections inevitably leading to larval death. Massive brood mortality might eventually lead to collapse of the entire colony. Molecular mechanisms of host-microbe interactions in this system and of differences in virulence between P. larvae genotypes are poorly understood. Recently, it was demonstrated that the degradation of the peritrophic matrix lining the midgut epithelium is a key step in pathogenesis of P. larvae infections. Here, we present the isolation and identification of PlCBP49, a modular, chitin-degrading protein of P. larvae and demonstrate that this enzyme is crucial for the degradation of the larval peritrophic matrix during infection. PlCBP49 contains a module belonging to the auxiliary activity 10 (AA10, formerly CBM33) family of lytic polysaccharide monooxygenases (LPMOs) which are able to degrade recalcitrant polysaccharides. Using chitin-affinity purified PlCBP49, we provide evidence that PlCBP49 degrades chitin via a metal ion-dependent, oxidative mechanism, as already described for members of the AA10 family. Using P. larvae mutants lacking PlCBP49 expression, we analyzed in vivo biological functions of PlCBP49. In the absence of PlCBP49 expression, peritrophic matrix degradation was markedly reduced and P. larvae virulence was nearly abolished. This indicated that PlCBP49 is a key virulence factor for the species P. larvae. The identification of the functional role of PlCBP49 in AFB pathogenesis broadens our understanding of this important family of chitin-binding and -degrading proteins, especially in those bacteria that can also act as entomopathogens., Author Summary American Foulbrood and its etiological agent, Paenibacillus larvae, pose a serious threat to global honey bee health. So far, molecular mechanisms of host-microbe interactions are poorly understood in this system and no key virulence factor for the entire species has been identified. Here, we demonstrate that P. larvae expresses a chitin-binding and -degrading protein PlCBP49 harboring one module that belongs to the auxiliary activity 10 (AA10) family of lytic polysaccharide monooxygenases (LPMOs). We provide evidence that PlCBP49 degrades chitin via a metal ion-dependent, oxidative mechanism, as already described for other members of the AA10 enzyme family. Using P. larvae mutants lacking PlCBP49 expression, we demonstrate that PlCBP49 is crucial for the degradation of the chitin-rich peritrophic matrix, a key step in pathogenesis of P. larvae infections. In the absence of PlCBP49 expression the peritrophic matrix remained nearly intact and about 95% of the infected larvae survived infection. This clearly indicated that PlCBP49 is a key virulence factor of P. larvae. These results constitute important progress in our understanding of both P. larvae pathogenesis and the biological role of LPMOs in entomopathogens. Furthermore, knowing PlCBP49 and its role in pathogenesis opens new possibilities to develop curative measures for this disease.

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., Author Summary Paenibacillus larvae is the most devastating bacterial pathogen of honey bees. However, the molecular interactions between infected larvae and P. larvae are poorly understood and little more than speculation exist concerning virulence factors. Recently, a putative S-layer protein has been identified in P. larvae. We here demonstrate that only representatives of P. larvae genotype ERIC II harbor a functional splA-gene and that SplA is a true S-layer protein with self-assembly capability. The presence of a functional S-layer protein is novel for P. larvae. When elucidating the biological function of SplA we broke new ground by establishing primary cell culture for pupal gut cells and by developing a genetic system for disruption of gene expression in this important honey bee pathogen. By using these novel methods we were able to prove that SplA serves as a shape-determining factor, mediates adhesion to host cells, and is a key virulence factor of P. larvae ERIC II. These results present an immense advancement in our understanding of P. larvae pathogenesis. Furthermore, we propose P. larvae as a model system for the analysis of the in vivo functions of S-layer proteins because P. larvae SlpA knockout-mutants retain viability and are thus suitable for functional studies.

Paenibacillus larvaesubsp.larvae(P. l. larvae) is the aetiological agent of American foulbrood (AFB), the most virulent bacterial disease of honey bee brood worldwide. In many countries AFB is a notifiable disease since it is highly contagious, in most cases incurable and able to kill affected colonies. Genotyping of field isolates ofP. l. larvaerevealed at least four genotypes (AB,Ab,abandαB) present in Germany which are genotypically different from the reference strain DSM 7030. Therefore, based on these data, five different genotypes ofP. l. larvaeare now identified with genotypeABstanding out with a characteristic brown-orange and circled two-coloured colony morphology. Analysing the metabolic profiles of three German genotypes (AB,Abandab) as well as of the reference strain using the Biolog system, a characteristic biochemical fingerprint could be obtained for each strain. Cluster analysis showed that while genotypesAb,aband the reference strain DSM 7030 are rather similar, genotypeABis clearly different from the others. Analysis of all isolates for plasmid DNA revealed two different plasmids present only in isolates belonging to genotypeAB. Therefore, genotypeABis remarkable in all aspects analysed so far. Future analysis will show whether or not these differences will expand to differences in virulence.