Production of the Catechol Type Siderophore Bacillibactin by the Honey Bee Pathogen Paenibacillus larvae

Abstract

The Gram-positive bacterium Paenibacillus larvae is the etiological agent of American Foulbrood. This bacterial infection of honey bee brood is a notifiable epizootic posing a serious threat to global honey bee health because not only individual larvae but also entire colonies succumb to the disease. In the recent past considerable progress has been made in elucidating molecular aspects of host pathogen interactions during pathogenesis of P. larvae infections. Especially the sequencing and annotation of the complete genome of P. larvae was a major step forward and revealed the existence of several giant gene clusters coding for non-ribosomal peptide synthetases which might act as putative virulence factors. We here present the detailed analysis of one of these clusters which we demonstrated to be responsible for the biosynthesis of bacillibactin, a P. larvae siderophore. We first established culture conditions allowing the growth of P. larvae under iron-limited conditions and triggering siderophore production by P. larvae. Using a gene disruption strategy we linked siderophore production to the expression of an uninterrupted bacillibactin gene cluster. In silico analysis predicted the structure of a trimeric trithreonyl lactone (DHB-Gly-Thr)3 similar to the structure of bacillibactin produced by several Bacillus species. Mass spectrometric analysis unambiguously confirmed that the siderophore produced by P. larvae is identical to bacillibactin. Exposure bioassays demonstrated that P. larvae bacillibactin is not required for full virulence of P. larvae in laboratory exposure bioassays. This observation is consistent with results obtained for bacillibactin in other pathogenic bacteria.

Molecular pathogenesis of American Foulbrood: how Paenibacillus larvae kills honey bee larvae

“…It is about 11 kb in length, consists of five genes, and shows strong homology to the bacillibactin gene clusters of Bacillus subtilis and the B. cereus sensu lato group as well as to the paenibactin gene cluster of Paenibacillus elgii, [17 ,36 ]. Mass spectrometric fragmentation analyses confirmed that the NRPS machinery is responsible for the production of the catechol-type siderophore bacillibactin (Dhb) in P. larvae [36 ]. Production of Dhb by P. larvae could only be demonstrated in culture under iron limited conditions.…”

“…Production of Dhb by P. larvae could only be demonstrated in culture under iron limited conditions. Larval infection assays performed with gene inactivation mutants of P. larvae (DdhbF) and the corresponding wild-type strain did not give any hints for a role of Dhb during pathogenesis: neither total mortality nor disease progression were affected by the absence of Dhb production [36 ]. However, the experimental design did not allow to test whether the lack of Dhb production had any effect on the growth of other bacteria like P. alvei.…”

Current knowledge and perspectives of Paenibacillus: a review

“…These multienzyme complexes consist of various modules that each incorporate one or more specific amino acids into a peptide backbone (see also “Nonribosomal lipopeptides” section). The three types of siderophores are classified based on their functional groups, being catecholates, hydroxamates, and α-hydroxy carbolates [38]. However, few siderophores have thus far been characterized from Paenibacillus .…”

“…Paenibacillus larvae produces a catecholate -type siderophore called bacillibactin, which is also made by Bacillus subtilis and members of the Bacillus cereus sensu lato group [38]. Bacillibactin is a cyclic trimeric lactone of 2,3-dihydroxybenzoate (DHB)-glycine-threonine.…”

“…ERIC I and II are the types typically isolated from afflicted hives, as ERIC III and IV are less virulent [38, 140–145]. While ERIC II is the most virulent strain, ERIC I is more prevalent globally, likely because it can infect all honeybee subspecies, whereas ERIC II is restricted to certain subspecies [137].…”