42 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
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
Genomic Potential and Virulence Mechanisms of Paenibacillus larvae
Paenibacillus larvae, a Gram positive bacterial pathogen, causes American
Foulbrood (AFB), which is the most serious infectious disease of honey bees.
In order to investigate the genomic potential of P. larvae, two strains
belonging to two different genotypes were sequenced and used for comparative
genome analysis. The complete genome sequence of P. larvae strain DSM 25430
(genotype ERIC II) consisted of 4,056,006 bp and harbored 3,928 predicted
protein-encoding genes. The draft genome sequence of P. larvae strain DSM
25719 (genotype ERIC I) comprised 4,579,589 bp and contained 4,868 protein-
encoding genes. Both strains harbored a 9.7 kb plasmid and encoded a large
number of virulence-associated proteins such as toxins and collagenases. In
addition, genes encoding large multimodular enzymes producing nonribosomally
peptides or polyketides were identified. In the genome of strain DSM 25719
seven toxin associated loci were identified and analyzed. Five of them encoded
putatively functional toxins. The genome of strain DSM 25430 harbored several
toxin loci that showed similarity to corresponding loci in the genome of
strain DSM 25719, but were non-functional due to point mutations or disruption
by transposases. Although both strains cause AFB, significant differences
between the genomes were observed including genome size, number and
composition of transposases, insertion elements, predicted phage regions, and
strain-specific island-like regions. Transposases, integrases and recombinases
are important drivers for genome plasticity. A total of 390 and 273 mobile
elements were found in strain DSM 25430 and strain DSM 25719, respectively.
Comparative genomics of both strains revealed acquisition of virulence factors
by horizontal gene transfer and provided insights into evolution and
pathogenicity
Métodos para la investigación de la loque americana
American foulbrood is one of the most devastating diseases of the honey bee. It is caused by the spore-forming, Gram-positive rod-shaped bacterium Paenibacillus larvae. The recent updated genome assembly and annotation for this pathogen now permits in-depth molecular studies. In this paper, selected techniques and protocols for American foulbrood research are provided, mostly in a recipe-like format that permits easy implementation in the laboratory. Topics covered include: working with Paenibacillus larvae, basic microbiological techniques, experimental infection, and “’omics” and other sophisticated techniques. Further, this chapter covers other technical information including biosafety measures to guarantee the safe handling of this pathogen.La loque americana es una de las enfermedades más devastadoras de la abeja melífera, causada por el bacilo, formador de esporas Grampositivo Paenibacillus larvae. El reciente ensamblaje y anotación del genoma de este patógeno permite actualmente la realización de profundos estudios moleculares. En este trabajo, se proporcionan técnicas y protocolos seleccionados para la investigación de la loque americana, principalmente bajo la forma de protocolos de trabajo con una estructura similar al de las recetas, para facilitar su implementación en el laboratorio. Los temas desarrollados incluyen: el trabajo con Paenibacillus larvae, técnicas básicas microbiológicas, la infección experimental, y "'ómicas" y otras técnicas sofisticadas. Además, este capítulo abarca otro tipo de información técnica, incluyendo medidas de bioseguridad para garantizar la seguridad en el manejo de este patógeno.Trabajo publicado en Dietemann, V.; Ellis, J. D.; Neumann, P. (eds.) The Coloss Beebook, Volume II: standard methods for Apis mellifera pest and pathogen research. Journal of Apicultural Research, 52(1).Facultad de Ciencias Agrarias y Forestale
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
Liquid chromatography (LC) ESI-negative mass spectrometry (MS) analytics of ethyl acetate extracts of <i>P. larvae</i> secretomes.
<p>(A) Extracted ion chromatogram (<i>m/z</i> 881) of ethyl acetate extract <i>P. larvae</i> ATCC9545 wildtype (ERIC I; black line) and <i>P. larvae</i> ATCC9545 Δ<i>dhb</i>F (red line). (B) Extracted ion chromatogram (<i>m/z</i> 881) of ethyl acetate extract of <i>P. larvae</i> DMS25430 wildtype (ERIC II; black line) and <i>P. larvae</i> DMS25430 Δ<i>dhb</i>F (red line).</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