45 research outputs found
Production of the Catechol Type Siderophore Bacillibactin by the Honey Bee Pathogen Paenibacillus larvae
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
Biological effects of paenilamicin, a secondary metabolite antibiotic produced by the honey bee pathogenic bacterium Paenibacillus larvae
Paenibacillus larvae is the etiological agent of American Foulbrood (AFB) a world-wide distributed devastating disease of the honey bee brood. Previous comparative genome analysis and more recently, the elucidation of the bacterial genome, provided evidence that this bacterium harbors putative functional nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs) and therefore, might produce nonribosomal peptides (NRPs) and polyketides (PKs). Such biosynthesis products have been shown to display a wide-range of biological activities such as antibacterial, antifungal or cytotoxic activity. Herein we present an in silico analysis of the first NRPS/PKS hybrid of P. larvae and we show the involvement of this cluster in the production of a compound named paenilamicin (Pam). For the characterization of its in vitro and in vivo bioactivity, a knock-out mutant strain lacking the production of Pam was constructed and subsequently compared to wild-type species. This led to the identification of Pam by mass spectrometry. Purified Pam-fractions showed not only antibacterial but also antifungal and cytotoxic activities. The latter suggested a direct effect of Pam on honey bee larval death which could, however, not be corroborated in laboratory infection assays. Bee larvae infected with the non-producing Pam strain showed no decrease in larval mortality, but a delay in the onset of larval death. We propose that Pam, although not essential for larval mortality, is a virulence factor of P. larvae influencing the time course of disease. These findings are not only of significance in elucidating and understanding host-pathogen interactions but also within the context of the quest for new compounds with antibiotic activity for drug development.DFG, GRK 1121, Genetische und immunologische Determinanten von Pathogen-Wirt-InteraktionenDFG, EXC 314, Unifying Concepts in Catalysi
Paenibacillus larvae Chitin-Degrading Protein PlCBP49 Is a Key Virulence Factor in American Foulbrood of Honey Bees
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
Genetic models of apoptosis-induced proliferation decipher activation of JNK and identify a requirement of EGFR signaling for tissue regenerative responses in Drosophila
Recent work in several model organisms has revealed that apoptotic cells are able to stimulate neighboring surviving cells to undergo additional proliferation, a phenomenon termed apoptosis-induced proliferation. This process depends critically on apoptotic caspases such as Dronc, the Caspase-9 ortholog in Drosophila, and may have important implications for tumorigenesis. While it is known that Dronc can induce the activity of Jun N-terminal kinase (JNK) for apoptosis-induced proliferation, the mechanistic details of this activation are largely unknown. It is also controversial if JNK activity occurs in dying or in surviving cells. Signaling molecules of the Wnt and BMP families have been implicated in apoptosis-induced proliferation, but it is unclear if they are the only ones. To address these questions, we have developed an efficient assay for screening and identification of genes that regulate or mediate apoptosis-induced proliferation. We have identified a subset of genes acting upstream of JNK activity including Rho1. We also demonstrate that JNK activation occurs both in apoptotic cells as well as in neighboring surviving cells. In a genetic screen, we identified signaling by the EGFR pathway as important for apoptosis-induced proliferation acting downstream of JNK signaling. These data underscore the importance of genetic screening and promise an improved understanding of the mechanisms of apoptosis-induced proliferation
Identification and functional analysis of the S-layer protein SplA of Paenibacillus larvae, the causative agent of American Foulbrood of honey bees.
The gram-positive, spore-forming bacterium Paenibacillus larvae is the etiological agent of American Foulbrood (AFB), a globally occurring, deathly epizootic of honey bee brood. AFB outbreaks are predominantly caused by two genotypes of P. larvae, ERIC I and ERIC II, with P. larvae ERIC II being the more virulent genotype on larval level. Recently, comparative proteome analyses have revealed that P. larvae ERIC II but not ERIC I might harbour a functional S-layer protein, named SplA. We here determine the genomic sequence of splA in both genotypes and demonstrate by in vitro self-assembly studies of recombinant and purified SplA protein in combination with electron-microscopy that SplA is a true S-layer protein self-assembling into a square 2D lattice. The existence of a functional S-layer protein is novel for this bacterial species. For elucidating the biological function of P. larvae SplA, a genetic system for disruption of gene expression in this important honey bee pathogen was developed. Subsequent analyses of in vivo biological functions of SplA were based on comparing a wild-type strain of P. larvae ERIC II with the newly constructed splA-knockout mutant of this strain. Differences in cell and colony morphology suggest that SplA is a shape-determining factor. Marked differences between P. larvae ERIC II wild-type and mutant cells with regard to (i) adhesion to primary pupal midgut cells and (ii) larval mortality as measured in exposure bioassays corroborate the assumption that the S-layer of P. larvae ERIC II is an important virulence factor. Since SplA is the first functionally proven virulence factor for this species, our data extend the knowledge of the molecular differences between these two genotypes of P. larvae and contribute to explaining the observed differences in virulence. These results present an immense advancement in our understanding of P. larvae pathogenesis
Biological Role of Paenilarvins, Iturin-Like Lipopeptide Secondary Metabolites Produced by the Honey Bee Pathogen Paenibacillus larvae.
The Gram-positive bacterium Paenibacillus larvae (P. larvae) is the causative agent of a deadly honey bee brood disease called American Foulbrood (AFB). AFB is a notifiable epizootic in most countries and, hence, P. larvae is of considerable relevance for veterinarians and apiculturists alike. Over the last decade, much progress has been made in the understanding of the (patho)biology of P. larvae. Recently, several non-ribosomally produced peptides (NRP) and peptide/polyketide (NRP/PK) hybrids produced by P. larvae were identified. Among these NRPs were iturin-like lipopeptides, the paenilarvins A-C. Iturins are known to exhibit strong anti-fungal activity; for some iturins, cytotoxic activity towards mammalian erythrocytes and human cancer cell lines are described. We here present our results on the analysis of the natural function of the paenilarvins during pathogenesis of P. larvae infections. We demonstrated production of paenilarvins in infected larvae. However, we could neither demonstrate cytotoxicity of paenilarvins towards cultured insect cells nor towards larvae in feeding assays. Accordingly, exposure bioassays performed with larvae infected by wild-type P. larvae and a knockout mutant of P. larvae lacking production of paenilarvins did not substantiate a role for the paenilarvins as virulence factor. Further experiments are necessary to analyze the relevance of the paenilarvins' anti-fungal activity for P. larvae infections in the presence of fungal competitors in the larval midgut or cadaver
The <i>dhb</i> gene cluster of <i>P. larvae</i>.
<p>(A) Gene arrangement of the <i>dhb</i> gene cluster of <i>P. larvae</i> in comparison to the <i>dhb</i> gene clusters of <i>B. subtilis</i> and <i>B. cereus</i> involved in the synthesis of bacillibactin and of the <i>pae</i> gene cluster of <i>P. elgii</i> involved in the synthesis of paenibactin. (B) Domain arrangement within the dimodular genes <i>pae</i>F (<i>P. elgii</i>) and <i>dhb</i>F (<i>B. subtilis</i>, <i>B. cereus</i>, <i>P. larvae</i>) and the predicted amino acids activated by the A-domains. A, adenylation domain; C condensation domain; T, thiolation domain; TE, thioesterase domain. Domain prediction was performed using SBSPKS <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0108272#pone.0108272-Anand1" target="_blank">[44]</a>.</p
Primers used for construction of gene knock-outs in <i>P. larvae</i> ATCC9545 (ERIC I) and DSM25430 (ERIC II).
<p>Primers used for construction of gene knock-outs in <i>P. larvae</i> ATCC9545 (ERIC I) and DSM25430 (ERIC II).</p
Liquid chromatography (LC) ESI-negative tandem mass spectrometry (MS/MS) analytics of bacillibactin.
<p>MS/MS analytics of ethyl acetate extract of <i>P. larvae</i> ATCC9545 (A; ERIC I), <i>P. larvae</i> DSM25430 (B; ERIC II), and of commercial bacillibactin (C) are shown. The single charged molecular ion of bacillibactin (m/z 881) was chosen for fragmentation with collision-induced dissociation (CID; 30 eV). Fragments are indicated in the spectrum; fragment <i>m/z</i> 249.1 is attributed to the decarboxylation of DHB-Gly-Thr.</p