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Paenibacillus larvae chitin-degrading protein PlCBP49 is a key virulence factor in American Foulbrood of honey bees.

Garcia-Gonzalez E, Poppinga L, Fünfhaus A, Hertlein G, Hedtke K, Jakubowska A, Genersch E - PLoS Pathog. (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bee Research, Department for Molecular Microbiology and Bee Diseases, Hohen Neuendorf, Germany; Humboldt Universität Berlin, Institut für Biologie, Berlin, Germany.

ABSTRACT
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.

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Identification of chitin-binding and –degrading proteins in the supernatants of cultured P. larvae.(A) Chitin-binding proteins isolated via chitin-beads from culture supernatants of ATCC9545 (P. larvae ERIC I) and DSM25430 (P. larvae ERIC II) were subjected to SDS-PAGE analysis. PlCBP49 is marked by an arrowhead. (B) Proteins isolated via chitin-beads from culture supernatants of ATCC9545 (P. larvae ERIC I) and DSM25430 (P. larvae ERIC II) were subjected to zymography using ethylene glycol chitin- (EGC-) impregnated gels to assess their chitinolytic activity. PlCBP49 is marked by an arrow head.
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ppat-1004284-g001: Identification of chitin-binding and –degrading proteins in the supernatants of cultured P. larvae.(A) Chitin-binding proteins isolated via chitin-beads from culture supernatants of ATCC9545 (P. larvae ERIC I) and DSM25430 (P. larvae ERIC II) were subjected to SDS-PAGE analysis. PlCBP49 is marked by an arrowhead. (B) Proteins isolated via chitin-beads from culture supernatants of ATCC9545 (P. larvae ERIC I) and DSM25430 (P. larvae ERIC II) were subjected to zymography using ethylene glycol chitin- (EGC-) impregnated gels to assess their chitinolytic activity. PlCBP49 is marked by an arrow head.

Mentions: Recently, degradation of the larval midgut PM by P. larvae was demonstrated to be a key step in the pathogenesis of P. larvae infections [19]. In order to identify proteins which might be involved in this process, we isolated and analyzed chitin-binding proteins from P. larvae culture supernatants using chitin-coated beads. SDS-PAGE analysis of the chitin-binding fractions revealed two chitin-binding proteins (CBP) migrating around 60 kD (CBP60) and 49 kD (PlCBP49I) secreted by ATCC9545 (P. larvae ERIC I), while only one band migrating around 49 kD (PlCBP49II) was visible in the supernatant of DSM25430 (P. larvae ERIC II) (Fig. 1A).


Paenibacillus larvae chitin-degrading protein PlCBP49 is a key virulence factor in American Foulbrood of honey bees.

Garcia-Gonzalez E, Poppinga L, Fünfhaus A, Hertlein G, Hedtke K, Jakubowska A, Genersch E - PLoS Pathog. (2014)

Identification of chitin-binding and –degrading proteins in the supernatants of cultured P. larvae.(A) Chitin-binding proteins isolated via chitin-beads from culture supernatants of ATCC9545 (P. larvae ERIC I) and DSM25430 (P. larvae ERIC II) were subjected to SDS-PAGE analysis. PlCBP49 is marked by an arrowhead. (B) Proteins isolated via chitin-beads from culture supernatants of ATCC9545 (P. larvae ERIC I) and DSM25430 (P. larvae ERIC II) were subjected to zymography using ethylene glycol chitin- (EGC-) impregnated gels to assess their chitinolytic activity. PlCBP49 is marked by an arrow head.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117609&req=5

ppat-1004284-g001: Identification of chitin-binding and –degrading proteins in the supernatants of cultured P. larvae.(A) Chitin-binding proteins isolated via chitin-beads from culture supernatants of ATCC9545 (P. larvae ERIC I) and DSM25430 (P. larvae ERIC II) were subjected to SDS-PAGE analysis. PlCBP49 is marked by an arrowhead. (B) Proteins isolated via chitin-beads from culture supernatants of ATCC9545 (P. larvae ERIC I) and DSM25430 (P. larvae ERIC II) were subjected to zymography using ethylene glycol chitin- (EGC-) impregnated gels to assess their chitinolytic activity. PlCBP49 is marked by an arrow head.
Mentions: Recently, degradation of the larval midgut PM by P. larvae was demonstrated to be a key step in the pathogenesis of P. larvae infections [19]. In order to identify proteins which might be involved in this process, we isolated and analyzed chitin-binding proteins from P. larvae culture supernatants using chitin-coated beads. SDS-PAGE analysis of the chitin-binding fractions revealed two chitin-binding proteins (CBP) migrating around 60 kD (CBP60) and 49 kD (PlCBP49I) secreted by ATCC9545 (P. larvae ERIC I), while only one band migrating around 49 kD (PlCBP49II) was visible in the supernatant of DSM25430 (P. larvae ERIC II) (Fig. 1A).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Institute for Bee Research, Department for Molecular Microbiology and Bee Diseases, Hohen Neuendorf, Germany; Humboldt Universität Berlin, Institut für Biologie, Berlin, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus