Abstract
ABSTRACTAmerican 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.
Highlights
Paenibacillus is an important bacterial genus with members that can be isolated from diverse environments, and they play important roles in soil, animals and plants
We identified two candidate protease markers with excellent potential to constitute important virulence factors, the microbial collagenase ColA (ColA) and the immune inhibitor A (InhA), which were detected in ERICs II–IV and missing in ERIC I
We have shown that the label-free proteomic analysis of the in vitro-produced exoprotein fraction is a useful strategy for identifying the virulence factors of the dangerous honeybee pathogen P. larvae
Summary
Paenibacillus is an important bacterial genus with members that can be isolated from diverse environments, and they play important roles in soil, animals and plants. Many Paenibacillus species have been found to produce biomolecules of scientific interest with potential industrial use. The genus Paenibacillus has recently been the subject of scientific reviews [1–3]. In an attempt to generate additional data on the relatively poorly understood Paenibacillus taxon, detailed studies have been performed to identify and characterize Paenibacillus isolates to facilitate their utilization [4,5]. In addition to the diverse beneficial Paenibacillus species, important pathogens of beneficial insects are members of this genus [1,6]. The most important insect-pathogenic member is Paenibacillus larvae [7], which is the causative agent of American foulbrood (AFB), a brood disease that causes substantial honeybee colony losses worldwide[8]
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