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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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Degradation of insoluble chitin structures by P. larvae PlCBP49.Peritrophic matrices were isolated from Spodoptera frugiperda larvae and incubated in an Ussing-chamber (A) with mock-treated chitin beads as negative control and chitin-binding fractions of either wild type bacteria or the corresponding mutant bacteria. Methylene blue efflux was used as a measure for permeability (B). PM permeability was significantly higher than negative control after incubation with ATCC9545 wt and DSM25430 wt chitin binding fractions. After incubation with ATCC9545 Δcbp and DSM25430 Δcbp chitin-binding fractions PM permeability was significantly lower compared to the incubation with chitin-binding fractions of the wild-type strains (ATCC9545, DSM25430). Bars represent mean values + SEM of at least three independent experiments, analyzed by student's t-test; *p value<0.05 and **p value<0.01.
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ppat-1004284-g005: Degradation of insoluble chitin structures by P. larvae PlCBP49.Peritrophic matrices were isolated from Spodoptera frugiperda larvae and incubated in an Ussing-chamber (A) with mock-treated chitin beads as negative control and chitin-binding fractions of either wild type bacteria or the corresponding mutant bacteria. Methylene blue efflux was used as a measure for permeability (B). PM permeability was significantly higher than negative control after incubation with ATCC9545 wt and DSM25430 wt chitin binding fractions. After incubation with ATCC9545 Δcbp and DSM25430 Δcbp chitin-binding fractions PM permeability was significantly lower compared to the incubation with chitin-binding fractions of the wild-type strains (ATCC9545, DSM25430). Bars represent mean values + SEM of at least three independent experiments, analyzed by student's t-test; *p value<0.05 and **p value<0.01.

Mentions: We recently showed that P. larvae is able to metabolize insoluble, colloidal chitin [19]. However, the data related to the chitin-degrading activity of PlCBP49 obtained so far were based on using EGC as a soluble substrate in zymograms. To further verify that degradation of insoluble recalcitrant polysaccharides is mediated by PlCBP49, as it is described for members of the AA10 family of LPMOs, we tested whether or not PlCBP49 might be able to degrade chitin-containing structures like an insect PM. To this aim, we used an Ussing chamber (Fig. 5A) to perform permeability assays with PMs which were isolated from S. frugiperda last instar larvae and subjected to the chitin bound fractions of the knock-out P. larvae strains (ATCC9545 Δcbp, DSM25430 Δcbp) and of the corresponding parent wild-type strains (ATCC9545, DSM25430). This comparative approach allows differences in PM permeabilization between mutant and wild-type bacteria to be linked with differences in PlCBP49 expression. Permeability of the PMs was measured as methylene blue (MB) efflux and was significantly higher after incubation with the chitin-binding fractions of the wild-type bacteria than after incubation with the chitin-binding fractions of the corresponding mutants (Fig. 5B). For P. larvae ATCC9545 MB efflux significantly (student's t-test, p-value = 0.0125) decreased from 0.0429±0.006 µg/ml/mm2/h in the presence of PlCBP49 expression (ATCC9545, mean values ± SEM) to 0.021±0.0016 µg/ml/mm2/h in the absence of PlCBP49 expression (ATCC9545 Δcbp, mean values ± SEM). Similar results were obtained for P. larvae DSM25430 (0.02418±0.002 µg/ml/mm2/h, mean values ± SEM) compared to P. larvae DSM25430 Δcbp (0.0094±0.0013 µg/ml/mm2/h, mean values ± SEM) which were also significantly different (student's t-test, p-value = 0.004). Remarkably, exposure of PMs to chitin-bound fractions of DSM25430 Δcbp resulted in PM permeability not significantly different from the negative control (student's t-test, p-value = 0.983) indicating that in the absence of PlCBP49 expression no PM degrading activity was active in these fractions. In contrast, chitin-bound fractions of ATCC9545 Δcbp resulted in PM permeability that was significantly higher than the negative control (student's t-test, p-value = 0.006) although also significantly reduced when compared to the effect achieved with ATCC9545 wild-type, meaning in the presence of PlCBP49 expression. These results indicated that the chitinolytic activity of PlCBP49 can act on chitin in its native crystalline form suggesting a role for PlCBP49 also in PM degradation observed during P. larvae infection of honey bee larvae [19].


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)

Degradation of insoluble chitin structures by P. larvae PlCBP49.Peritrophic matrices were isolated from Spodoptera frugiperda larvae and incubated in an Ussing-chamber (A) with mock-treated chitin beads as negative control and chitin-binding fractions of either wild type bacteria or the corresponding mutant bacteria. Methylene blue efflux was used as a measure for permeability (B). PM permeability was significantly higher than negative control after incubation with ATCC9545 wt and DSM25430 wt chitin binding fractions. After incubation with ATCC9545 Δcbp and DSM25430 Δcbp chitin-binding fractions PM permeability was significantly lower compared to the incubation with chitin-binding fractions of the wild-type strains (ATCC9545, DSM25430). Bars represent mean values + SEM of at least three independent experiments, analyzed by student's t-test; *p value<0.05 and **p value<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1004284-g005: Degradation of insoluble chitin structures by P. larvae PlCBP49.Peritrophic matrices were isolated from Spodoptera frugiperda larvae and incubated in an Ussing-chamber (A) with mock-treated chitin beads as negative control and chitin-binding fractions of either wild type bacteria or the corresponding mutant bacteria. Methylene blue efflux was used as a measure for permeability (B). PM permeability was significantly higher than negative control after incubation with ATCC9545 wt and DSM25430 wt chitin binding fractions. After incubation with ATCC9545 Δcbp and DSM25430 Δcbp chitin-binding fractions PM permeability was significantly lower compared to the incubation with chitin-binding fractions of the wild-type strains (ATCC9545, DSM25430). Bars represent mean values + SEM of at least three independent experiments, analyzed by student's t-test; *p value<0.05 and **p value<0.01.
Mentions: We recently showed that P. larvae is able to metabolize insoluble, colloidal chitin [19]. However, the data related to the chitin-degrading activity of PlCBP49 obtained so far were based on using EGC as a soluble substrate in zymograms. To further verify that degradation of insoluble recalcitrant polysaccharides is mediated by PlCBP49, as it is described for members of the AA10 family of LPMOs, we tested whether or not PlCBP49 might be able to degrade chitin-containing structures like an insect PM. To this aim, we used an Ussing chamber (Fig. 5A) to perform permeability assays with PMs which were isolated from S. frugiperda last instar larvae and subjected to the chitin bound fractions of the knock-out P. larvae strains (ATCC9545 Δcbp, DSM25430 Δcbp) and of the corresponding parent wild-type strains (ATCC9545, DSM25430). This comparative approach allows differences in PM permeabilization between mutant and wild-type bacteria to be linked with differences in PlCBP49 expression. Permeability of the PMs was measured as methylene blue (MB) efflux and was significantly higher after incubation with the chitin-binding fractions of the wild-type bacteria than after incubation with the chitin-binding fractions of the corresponding mutants (Fig. 5B). For P. larvae ATCC9545 MB efflux significantly (student's t-test, p-value = 0.0125) decreased from 0.0429±0.006 µg/ml/mm2/h in the presence of PlCBP49 expression (ATCC9545, mean values ± SEM) to 0.021±0.0016 µg/ml/mm2/h in the absence of PlCBP49 expression (ATCC9545 Δcbp, mean values ± SEM). Similar results were obtained for P. larvae DSM25430 (0.02418±0.002 µg/ml/mm2/h, mean values ± SEM) compared to P. larvae DSM25430 Δcbp (0.0094±0.0013 µg/ml/mm2/h, mean values ± SEM) which were also significantly different (student's t-test, p-value = 0.004). Remarkably, exposure of PMs to chitin-bound fractions of DSM25430 Δcbp resulted in PM permeability not significantly different from the negative control (student's t-test, p-value = 0.983) indicating that in the absence of PlCBP49 expression no PM degrading activity was active in these fractions. In contrast, chitin-bound fractions of ATCC9545 Δcbp resulted in PM permeability that was significantly higher than the negative control (student's t-test, p-value = 0.006) although also significantly reduced when compared to the effect achieved with ATCC9545 wild-type, meaning in the presence of PlCBP49 expression. These results indicated that the chitinolytic activity of PlCBP49 can act on chitin in its native crystalline form suggesting a role for PlCBP49 also in PM degradation observed during P. larvae infection of honey bee larvae [19].

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