37 research outputs found
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
Involvement of secondary metabolites in the pathogenesis of the American foulbrood of honey bees caused by Paenibacillus larvae
Covering: 2011 to end of 2014 The Gram-positive, spore-forming bacterium Paenibacillus larvae (P. larvae) is the causative agent of the epizootic American Foulbrood (AFB), a fatal brood disease of the western honey bee (Apis mellifera). AFB is one of the most destructive honey bee diseases since it is not only lethal for infected larvae but also for the diseased colonies. Due to the high impact of honey bees on ecology and economy this epizootic is a severe and pressing problem. Knowledge about virulence mechanisms and the underlying molecular mechanisms remain largely elusive. Recent genome sequencing of P. larvae revealed its potential to produce unknown secondary metabolites, like nonribosomal peptides and peptide-polyketide hybrids. This article highlights recent findings on secondary metabolites synthesized by P. larvae and discusses their role in virulence and pathogenicity towards the bee larvae
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
Characterization of plant growth-promoting rhizobacteria from perennial ryegrass and genome mining of novel antimicrobial gene clusters
Background Plant growth-promoting rhizobacteria (PGPR) are good alternatives for chemical fertilizers and pesticides, which cause severe environmental problems worldwide. Even though many studies focus on PGPR, most of them are limited in plant-microbe interaction studies and neglect the pathogens affecting ruminants that consume plants. In this study, we expand the view to the food chain of grass-ruminant-human. We aimed to find biocontrol strains that can antagonize grass pathogens and mammalian pathogens originated from grass, thus protecting this food chain. Furthermore, we deeply mined into bacterial genomes for novel biosynthetic gene clusters (BGCs) that can contribute to biocontrol. Results We screened 90 bacterial strains from the rhizosphere of healthy Dutch perennial ryegrass and characterized seven strains (B. subtilis subsp. subtilis MG27, B. velezensis MG33 and MG43, B. pumilus MG52 and MG84, B. altitudinis MG75, and B. laterosporus MG64) that showed a stimulatory effect on grass growth and pathogen antagonism on both phytopathogens and mammalian pathogens. Genome-mining of the seven strains discovered abundant BGCs, with some known, but also several potential novel ones. Further analysis revealed potential intact and novel BGCs, including two NRPSs, four NRPS-PKS hybrids, and five bacteriocins. Conclusion Abundant potential novel BGCs were discovered in functional protective isolates, especially in B. pumilus, B. altitudinis and Brevibacillus strains, indicating their great potential for the production of novel secondary metabolites. Our report serves as a basis to further identify and characterize these compounds and study their antagonistic effects against plant and mammalian pathogens
Genomic insights into the biosynthesis and physiology of the cyanobacterial neurotoxin 3-N-methyl-2,3-diaminopropanoic acid (BMAA)
Cyanobacteria are an ancient clade of photosynthetic prokaryotes, present in many habitats throughout the world, including water resources. They can present health hazards to humans and animals due to the production of a wide range of toxins (cyanotoxins), including the diaminoacid neurotoxin, β-N-methylaminoalanine (BMAA). Knowledge of the biosynthetic pathway for BMAA, and its role in cyanobacteria, is lacking. Present evidence suggests that BMAA is derived by 3-N methylation of 2,3-diaminopropanoic acid (2,3-DAP) and, although the latter has never been reported in cyanobacteria, there are multiple pathways to its biosynthesis known in other bacteria and in plants. Here, we used bioinformatics analyses to investigate hypotheses concerning 2,3-DAP and BMAA biosynthesis in cyanobacteria. We assessed the potential presence or absence of each enzyme in candidate biosynthetic routes known in Albizia julibrissin, Lathyrus sativus seedlings, Streptomyces, Clostridium, Staphylococcus aureus, Pantoea agglomerans, and Paenibacillus larvae, in 130 cyanobacterial genomes using sequence alignment, profile hidden Markov models, substrate specificity/active site identification and the reconstruction of gene phylogenies. Most enzymes involved in pathways leading to 2,3-DAP in other species were not found in the cyanobacteria analysed. Nevertheless, two species appear to have the genes sbnA and sbnB, responsible for forming the 2,3-DAP constituent in staphyloferrin B, a siderophore from Staphylococcus aureus. It is currently undetermined whether these species are also capable of biosynthesising BMAA. It is possible that, in some cyanobacteria, the formation of 2,3-DAP and/or BMAA is associated with environmental iron-scavenging. The pam gene cluster, responsible for the biosynthesis of the BMAA-containing peptide, paenilamicin, so far appears to be restricted to Paenibacillus larvae. It was not detected in any of the cyanobacterial genomes analysed, nor was it found in 93 other Paenibacillus genomes or in the genomes of two BMAA-producing diatom species. We hypothesise that the presence, in some cyanobacterial species, of the enzymes 2,3-diaminopropionate ammonia-lyase (DAPAL) and reactive intermediate deaminase A (RidA) may explain the failure to detect 2,3-DAP in analytical studies. Overall, the taxonomic distribution of 2,3-DAP and BMAA in cyanobacteria is unclear; there may be multiple and additional routes, and roles, for the biosynthesis of 2,3-DAP and BMAA in these organisms.Output Status: Forthcoming/Available Onlin
Mining and characterization of antimicrobials from plant growth-promoting rhizobacteria isolated from perennial ryegrass
Plant growth-promoting rhizobacteria (PGPR) form a good alternative to chemical fertilizers and pesticides. Moreover, PGPR can produce antimicrobials that have the potential to be antibiotics, which are in great demand because of the rapidly emerging antibiotic resistance. This thesis describes the isolation and screening of seven PGPR strains for use on perennial ryegrass, a crucial pasture plant distributed worldwide. Genome mining of newly isolated PGPR strains reveals abundant biosynthetic gene clusters (BGCs) of secondary metabolites, some of which remain unknown. One of the most interesting strains, Brevibacillus laterosporus MG64, displays outstanding biocontrol activity but 80% of its BGCs have not been assigned to known products. Further characterization of its secondary metabolites led us to the discovery of three families of compounds: relacidines, bogorols, and succilins. Relacidines are a class of cationic circular lipopeptides that selectively combat Gram-negative bacteria by affecting the oxidative phosphorylation process. Bogorols are a class of cationic linear lipopeptides that are active against both Gram-positive bacteria and Gram-negative bacteria. Succilins are modified from bogorols by succinylation at Orn3. The lipoinitiation of bogorols and succilins is proven to be mediated by an adenylation domain, while the valinol at the C-terminus is formed by a two-step reduction. A comparative transcriptomics study reveals that the expression of sporulation genes and secondary metabolite encoding BGCs are regulated during the interaction with pathogens. This thesis provides a comprehensive understanding of the PGPR strain B. laterosporus MG64 and its antimicrobials and extends our knowledge on the biosynthetic machinery of lipopeptides
Genomic analysis of Paenibacillus larvae and its relation to virulence
Paenibacillus larvae is a Gram-positive sporulating bacterium that causes American foulbrood (AFB). It is one of the most dangerous bacterial pathogens of the honeybee (Apis mellifera). P. larvae spores are highly infectious to bee larvae and resist physicochemical influences. P. larvae is subtyped using repPCR with ERIC primers (Enterobacterial Repetitive Integrance Consensus) into five genotypes (ERIC I-V), which possess different colony morphology, metabolism and especially virulence. There is a significant genetic variability among isolates of P. larvae, which may contribute to differences in virulence. P. larvae isolates used in this work were obtained from clinical cases of American foulbrood as well as from a debris collected from bee hives with no American foulbrood symptoms from all over the Czech Republic in cooperation with the Beekeeping Research Institute, s.r.o., Dol. The isolates were obtained from larvae and hive debris. Both virulet and avirulet strains were sequenced using the SMRT (single molecule real time) method on the Sequel platform (PacBio). This method is suitable for Whole Genome Sequencing (WGS), because it allows sequencing of long reads with high accuracy, eliminating the effect of a large number of repetitive sequences during the genome assembly. Furthermore,...Paenibacillus larvae je grampozitivní sporulující bakterie, která je původcem moru včelího plodu. Jedná se o jednoho z nejvýznamnějších bakteriálních patogenů včely medonosné (Apis mellifera). Spory P. larvae jsou vysoce infekční pro včelí larvu a odolávají fyzikálně-chemickým vlivům. P. larvae se subtypizuje za využití repPCR s ERIC primery (Enterobacterial Repetitive Integrance Consensus). Dosud bylo popsáno 5 genotypů ERIC I-V, které se liší morfologií kolonií, metabolismem a především virulencí. Rovněž je mezi jednotlivými izoláty P. larvae významná genetická variabilita, která se může podílet na rozdílech ve virulenci. Izoláty P. larvae použité v této práci byly získány z klinických případů moru včelího plodu z celé České republiky ve spolupráci s Výzkumným ústavem včelařským, s.r.o., Dol. Jednalo se o bakteriální kultury získané kultivací infikovaných larev a měli. Také byly použity izoláty z měli získané ze včelstev bez klinických příznaků moru včelího plodu. Metodou SMRT (Single Molecule Real Time) na platformě Sequel (PacBio) byly sekvenovány virulentní i pravděpodobně avirulentní izoláty. Použitá metoda je vhodná pro celogenomové sekvenování bakteriálních genomů, protože umožňuje sekvenování dlouhých úseků DNA s vysokou přesností. Tím je eliminován efekt velkého množství repetitivních...Department of Genetics and MicrobiologyKatedra genetiky a mikrobiologiePřírodovědecká fakultaFaculty of Scienc
Characterization of the Paenibacillus larvae genotypes ERIC I-V and alternative therapeutic methods to fight American Foulbrood
In dieser Arbeit wurde der Erreger der Amerikanischen Faulbrut (AFB), Paenibacillus larvae charakterisiert. Dieses Bakterium wird in vier Genotypen mit unterschiedlicher Virulenz eingeteilt. Eine Therapie der AFB ist nicht möglich.
Für diese Arbeit wurden Honigproben auf die Abundanz von P. larvae untersucht. Hierbei konnte ein fünfter Genotyp isoliert und beschrieben werden. Durch die Genomsequenzierung der fünf Genotypen konnten Differenzierungen im Hinblick auf NRPS/PKS-Gencluster erfolgen. Die hierdurch kodierten Sekundärmetabolite stellen potentielle Virulenzfaktoren dar. Paenilarvin A-C wurden hinsichtlich ihrer biologischen Aktivität gegenüber Brut und Vertretern des Mikrobioms der Biene getestet. Eine Bestätigung als Virulenzfaktor konnte nicht erfolgen. Weiterführende Arbeiten zeigten, dass Paenilarvine eine biologische Aktivität gegenüber einer Vielzahl an Mikroorganismen aufweisen. Diese Ergebnisse lassen eine Funktion als Fitnessfaktoren bei der Schaffung der ökologischen Nische vermuten.
Ergänzend wurden Versuche zur biologischen Aktivität von Thyrotricinen gegenüber Brut und dem Mikrobiom der Biene durchgeführt. Die Schadschwelle der Konzentration für Bienenbrut konnte bestimmt werden. Diese lag deutlich über der minimalen Hemmkonzentrationen gegenüber P. larvae. Basierend auf diesen Ergebnissen wurden Tests auf die therapeutische Eignung gegen die AFB in vivo durchgeführt. Dieser Effekt konnte nicht gezeigt werden. Stattdessen wiesen die Testindividuen eine erhöhte Mortalität auf.
Weiterhin wurden P. larvae-spezifische Bakteriophagen (Phagen) hinsichtlich ihrer potentiellen therapeutischen Eignung analysiert. Zehn Phagen-Isolate konnten in dieser Arbeit charakterisiert werden. Ein Therapieerfolg konnte durch die singuläre Applikation des Phagen HB10c2 nicht erzielt werden, jedoch wurden Erkenntnisse für die sichere Anwendung und Voraussetzungen für eine Phagentherapie etabliert. Durch Modifizierungen der Therapieversuche wurde für die kontinuierliche Applikation des Phagen HB? ein Therapieerfolg gezeigt.
Durch die Sequenzierung des Phagen HB10c2 konnten Markergene identifiziert werden, die zur Charakterisierung der Phagen-Isolate herangezogen wurden. Diese ergab, dass alle isolierten Phagen über eine Integrase verfügen und somit zu den lysogenen Phagen zählen. Dieses Ergebnis deutet auf eine koevolutionäre Adaption der Bakteriophagen an ihren Wirt und dessen Lebensraum Bienenvolk vor dem Hintergrund der jahreszeitlichen Entwicklungsstadien hin.This work focusses on Paenibacillus larvae, the causative agent of American Foulbrood (AFB) in honeybees. This bacterium can be subdivided into four genotypes that differ in their virulence. A therapeutic approach does not exist.
During this work, honey samples were analyzed for the abundance of P. larvae. Thereby, a new fifth, highly virulent genotype was discovered and described. Sequencing of all five genotypes allowed differentiations between ERIC I-V. Therefore, genomes were analyzed for NRPS/PKS gene clusters. These encoded secondary metabolites are known as potential virulence factors. Paenilarvins were analyzed for their biological activity against bee brood and representative bacterial strains of the bees gut microbiota. Paenilarvins could not be verified as virulence factors. Further studies on this topic indicated an activity of paenilarvins against a number of microorganisms. According to these results, paenilarvins may act as fitness factor to create an ecological niche for P. larvae.
Additionally experiments were performed to determine the biological activity of thyrotricins (Trcs) against honeybee brood and microbiota of A. mellifera. The ecologically harmless concentration against bee larvae, which was much higher than the minimal inhibitory concentration against the P. larvae, was determined. Based on these results, Trcs were used for analysis of their therapeutic potential against AFB. Nevertheless, treated individual showed an increase of mortality.
As an alternative therapy, P. larvae-directed bacteriophages (phages) were analyzed for their potential therapeutic use. A number of ten phage isolates were characterized. Phage HB10c2 was tested for its application in a primary study. Decreasements of mortality in treated individuals could not be. But the essentials for a safe application of phages in honeybees could be derived from these results. By modifications of phage application with phage HB? a decrease of mortality in treated individuals could be shown.
Furthermore, sequencing of phage HB10c2 indicated some marker genes that were used for the characterization of phage isolates during this work. Thereby, all phage were identified to carry an integrase in their genomes, which indicate them as lysogenic phages. Taken together, these results indicate co-evolutionary adaption of the phages and their host P. larvae in their shared environment honeybee, respective to the developmental stages of the colony among the seasons