Comprehensive proteomic analysis of exoproteins expressed by ERIC I, II, III and IV Paenibacillus larvae genotypes reveals a wide range of virulence factors

Abstract

American foulbrood is a quarantine disease of the honeybee Apis mellifera L. in many countries and contributes greatly to colony losses. We performed a label-free proteomics study of exoprotein fractions produced in vitro by Paenibacillus larvae reference strains of the ERIC I-IV genotypes. A quantitative comparison was performed of previous studied protein-based virulence factors and many newly identified putative virulence factors. Among the multiple proteases identified, key virulence factors included the microbial collagenase ColA and immune inhibitor A (InhA, an analog of the Bacillus thuringiensis protein InhA). Both of these virulence factors were detected in ERICs II-IV but were absent from ERIC I. Furthermore, the different S-layer proteins and polysaccharide deacetylases prevailed in ERICs II-IV. Thus, the expression patterns of these virulence factors corresponded with the different speeds at which honeybee larvae are known to be killed by ERICs II-IV compared to ERIC I. In addition, putative novel toxin-like proteins were identified, including vegetative insecticidal protein Vip1, a mosquitocidal toxin, and epsilon-toxin type B, which exhibit similarity to homologs present in Bacillus thuringiensis or Lysinibacillus sphaericus. Furthermore, a putative bacteriocin similar to Lactococcin 972 was identified in all assayed genotypes. It appears that P. larvae shares virulence factors similar to those of the Bacillus cereus group. Overall, the results provide novel information regarding P. larvae virulence potential, and a comprehensive exoprotein comparison of all four ERICs was performed for the first time. The identification of novel virulence factors can explain differences in the virulence of isolates.

Keywords: ADP-ribosylating toxin; American foulbrood; Apis mellifera; Bacillus thuringiensis; S-layer protein; bacteriocin; immune inhibitor A; microbial collagenase ColA; mosquitocidal toxin; polysaccharide deacetylase.

Figure 1.

Two heatmaps that visualize the proteome differences in exoproteins of the four genotypes. a) Heatmap generated after the missing values were replaced by the constant “0.” The presentation demonstrates that the number of proteins was not detected in all 7 biological replicates (denoted with a–g) within the genotype. b) Heatmap was generated after the missing values were replaced from a normal distribution, and the data were subtracted using a Z-score. Both data evaluation methods provide similar results in which ERIC I clusters separately from ERICs II–IV, which likely corresponds to the difference in the efficiency at which these genotypes kill honeybee larva. Overall, the results indicate a similarity between ERIC III and IV and a substantial difference between ERIC I and II.

Figure 2.

Two PCA charts that illustrate the proteome differences in exoproteins of the four genotypes. Each of the symbols represents one biological replicate, and the analysis clearly demonstrates the homogeneity of the samples or analyses. The same numerals used to generate the heatmaps in Figure. 1(a) and Figure 1(b) were used to produce the charts in Figures 2(a,b), respectively. The similarity in ERIC III and IV and separate positions of ERIC II and ERIC I are clearly demonstrated.