101 research outputs found
Genome-Wide RNA Sequencing Analysis of Quorum Sensing-Controlled Regulons in the Plant-Associated Burkholderia glumae PG1 Strain
No full textBurkholderia glumae PG1 is a soil-associated motile plant-pathogenic bacterium possessing a cell density-dependent regulation system called quorum sensing (QS). Its genome contains three genes, here designated bgaI1 to bgaI3, encoding distinct autoinducer-1 (AI-1) synthases, which are capable of synthesizing QS signaling molecules. Here, we report on the construction of B. glumae PG1 ΔbgaI1, ΔbgaI2, and ΔbgaI3 mutants, their phenotypic characterization, and genome-wide transcriptome analysis using RNA sequencing (RNA-seq) technology. Knockout of each of these bgaI genes resulted in strongly decreased motility, reduced extracellular lipase activity, a reduced ability to cause plant tissue maceration, and decreased pathogenicity. RNA-seq analysis of all three B. glumae PG1 AI-1 synthase mutants performed in the transition from exponential to stationary growth phase revealed differential expression of a significant number of predicted genes. In comparison with the levels of gene expression by wild-type strain B. glumae PG1, 481 genes were differentially expressed in the ΔbgaI1 mutant, 213 were differentially expressed in the ΔbgaI2 mutant, and 367 were differentially expressed in the ΔbgaI3 mutant. Interestingly, only a minor set of 78 genes was coregulated in all three mutants. The majority of the QS-regulated genes were linked to metabolic activities, and the most pronounced regulation was observed for genes involved in rhamnolipid and Flp pilus biosynthesis and the type VI secretion system and genes linked to a clustered regularly interspaced short palindromic repeat (CRISPR)-cas gene cluster
RNA-seq basierte Identifikation und biochemische Charakterisierung des Pseudomonas aeruginosa LasR und Quorum Sensing Antiaktivators PA2226
No full textMany pathogenic bacteria regulate their virulence factor expression by Quorum Sens-ing (QS), the cell density dependent release and perception of small diffusible mole-cules, which are called autoinducers (AI). In the opportunistic pathogenic microorgan-ism Pseudomonas aeruginosa PAO1, in which nearly 10 % of the annotated genes are involved in regulation, Quorum Sensing regulated gene expression contributes to the formation and maintenance of biofilms and their tolerance to conventional antimicrobi-als. This makes it a promising target for infection control. Earlier it was shown that the enzymatic degradation of AI molecules is an efficient approach to control virulence of Pseudomonas. Acylases, lactonases and oxidoreductases are now known to cleave or modify the AI and thereby prevent activation of QS and in succession the controlled phenotypes like elastase, pyocyanin, hydrogen cyanide and biofilm formation. Within this framework, we have shown that the expression of a metagenome-derived short-chain dehydrogenase/reductase (SDR, BpiB09) resulted in significantly reduced pyocy-anin production, decreased motility, poor biofilm formation and decreased paralysis of nematodes. In this study and with the aim to unravel the impact on the complicated reg-ulatory system upon the expression of bpiB09, RNA sequencing (RNA-seq) was em-ployed to get detailed insight into the genetic alterations caused by BpiB09 and their impact on the bacterial metabolism. By analyzing the global transcriptome of PAO1 pBBR::bpiB09 via RNA-seq it was observed that the upregulation of the hypo-thetical protein PA2226 was not only associated with AI modification, but also with a 19 kb deletion in the bacterial chromosome. Within this thesis I could show, that the up-regulation of PA2226 was most likely responsible for several strong phenotypes con-nected to QS, including pyocyanin, elastase and 3-oxo-C12-HSL formation. For further verification of these phenotypes, several mutant strains with deletions in genes essential for QS were constructed. These strains included newly constructed lasI, rhlI and a dou-ble deletion mutant thereof as well as a mutant with a deleted PA2226. These mutations were verified by genetic complementation and PCR. Surprisingly, none of these mu-tants showed an altered biofilm phenotype compared to the parent strain. However, ∆lasI, ∆rhlI and ∆lasI/rhlI showed strong phenotypes for motility, elastase, pyocyanin and 3-oxo-C12-HSL production. With a coordinated approach of RNA-seq of a PA2226 overproducer, QS reporter gene assays in E. coli and further phenotypical testing, PA2226 was established as responsible agent and QS core mechanisms were suspect-ed as its target. The obtained RNA-seq data were in good concordance with the recent-ly defined QS core regulon of P. aeruginosa PAO1. Among the differentially expressed genes the elastase lasB, the QS regulator rsaL, the AI synthase rhlI, the genes for hy-drogen cyanide formation hcnABC and ambBCDE, which are involved in IQS for-mation, were observed. Furthermore, it became evident that the overexpression of PA2226 had a major impact on genes belonging to the functional classes of secreted factors and protein secretion/export. Additional overexpression of PA2226 in E. coli showed no activation of the included QS reporter, exposing QS active promotors and LasR as essential parts of the mode of action. After excluding the potential interference of recombinant PA2226 with QS active promotors via electrophoretic mobility shift as-says (EMSA), LasR emerged to be the most likely target. It was possible to demon-strate that PA2226’s regulatory function is closely linked to this QS response regulator by coprecipitation of recombinant PA2226 and LasR from crude cell extracts, indicating that both proteins strongly interact in P. aeruginosa. This correlates with the finding that QS phenotypes were partially rescued, when LasR and PA2226 were co-expressed, but likewise opens the possibility for at least one other interaction partner. Taking ad-vantage of next generation sequencing to analyze a PAO1 strain with strong Quorum Quenching phenotypes enabled the identification and characterization of a novel major QS regulator, extending the knowledge of the complex QS network of P. aeruginosa.Viele pathogene Bakterien regulieren ihre Virulenz durch Quorum Sensing (QS), der zelldichteabhängigen Ausschüttung und Detektion kleiner diffusionsfähiger Moleküle, genannt Autoinducer (AI). In dem opportunistisch pathogenen Mikroorganismus Pseu-domonas aeruginosa, in welchem nahezu 10 % aller annotierter Gene in Regulations-prozessen involviert sind, trägt die Quorum Sensing regulierte Genexpression auch zur Bildung und Erhaltung von Biofilmen und der Toleranz entgegen Antibiotika bei. Dies macht es zu einem vielversprechenden Ziel zur Infektionskontrolle. Es wurde bisher gezeigt, dass der enzymatische Abbau der Autoinducermoleküle ein effizienter Ansatz zur Kontrolle der Virulenz in Pseudomonas ist. Acylasen, Laktonasen und Oxidoreduk-tasen degradieren oder modifizieren Autoinducermoleküle und verhindern damit die Aktivierung von QS und davon kontrollierten Phänotypen wie Elastase-, Pyocyanin-, Hydrogencyanid- und Biofilmbildung. In diesem Zusammenhang haben wir gezeigt, dass die Expression einer metagenomisch gewonnenen Dehydrogenase/Reduktase (BpiB09) zu signifikant reduzierter Produktion von Pyocyanin, geringer Biofilmbildung und reduzierter Paralyse von Nematoden führte. In dieser Studie wurde RNA-Sequenzierung (RNA-Seq) verwendet, um den Einfluss der Expression von bpiB09 auf das komplexe regulatorische Netzwerk zu enträtseln, sowie detaillierte Einsicht in durch BpiB09 hervorgerufene genetische Modifikationen und ihren Einfluss auf den bakteriel-len Metabolismus zu bekommen. Bei der Analyse des globalen Transkriptoms von PAO1 pBBR::bpiB09 mittels RNA-Seq wurde beobachtet, dass die Hochregulierung des hypothetischen Proteins PA2226 nicht nur in Verbindung mit der Modifikation des Autoinducers steht, sondern ebenso mit einer 19 kb Deletion im bakteriellen Chromo-som. In dieser Studie konnte ich zeigen, dass die Hochregulierung von PA2226 für eini-ge starke QS Phänotypen verantwortlich ist, darunter Pyocyanin-, Elastase- und 3-oxo-C12-HSl Bildung. Zur Verifizierung dieser Phänotypen wurden eine Reihe von mutierten Stämmen generiert, welche Deletionsmutationen für essentielle QS Gene beinhalten. Diese generierten Stämme beinhalten Einzeldeletionsmutanten für lasI und rhlI, eine Doppelmutante aus diesen, sowie eine Mutante mit deletiertem PA2226. Die Mutationen wurden mittels Komplementation und PCR überprüft. Überraschenderweise zeigte kei-ne dieser Mutanten einen veränderten Biofilm Phänotyp im Vergleich zum Wildtyp. An-dererseits zeigten die Mutanten ∆lasI, ∆rhlI und ∆lasI/rhlI starke Phänotypen für Motilität, sowie Elastase, Pyocyanin und 3-oxo-C12-HSL Produktion. In einem abgestimmten Ansatz aus RNA-Seq von einem PA2226 Überproduzierer, QS Reporter Gen Assays in E. coli und weiteren phänotypischen Tests, konnte PA2226 als verantwortliche Ursache identifiziert und zentrale QS Mechanismen als potenzielles Ziel ausgemacht werden. Die erhaltenen RNA-Seq Daten waren in großer Übereinstimmung mit dem kürzlich beschriebenen zentralem QS Regulon von P. aeruginosa PAO1. Unter den differenziell exprimierten Genen konnten die Elastase lasB, der QS Regulator rsaL, die AI Synthase rhlI, die Gene für Hydrogencyanidbildung hcnABC und ambBCDE, welche in der Syn-these von IQS involviert sind, beobachtet werden. Darüber hinaus wurde deutlich, dass die Überexpression von PA2226 großen Einfluss auf die Genexpression von Genen hat, welche zu den funktionellen Klassen der „Sekretierten Faktoren“ und der „Protein Sekretion/Export“ gehören. Weitere Überexpression von PA2226 in E. coli zeigte, dass der hier beinhaltende QS Reporter nicht aktiviert wird, was QS aktive Promotoren und LasR als essentielle Bestandteile des Mechanismus identifiziert. Nachdem eine Interak-tion von rekombinantem PA2226 mit QS aktiven Promotoren mittels Electrophoretic Mobility Shift Assay (EMSA) ausgeschlossen werden konnte, erschien LasR als wahr-scheinlichstes Ziel. Im Nachfolgenden konnte durch Kopräzipitation von rekombinantem PA2226 und LasR aus Zellrohextrakten gezeigt werden, dass die regulatorische Funkti-on von PA2226 eng mit diesem QS Regulator verbunden ist. Dies korreliert mit der Be-obachtung, dass QS Phänotypen partiell wiederhergestellt werden konnten wenn LasR und PA2226 gleichzeitig exprimiert wurden, öffnet aber die Möglichkeit mindestens ei-nes weiteren Interaktionspartners. Indem sich Next Generation Sequencing bei der Analyse eines PAO1 Stammes mit starken Quorum Quenching Phänotypen zu Nutze gemacht wurde, konnte ein neuer QS Regulator identifiziert und charakterisiert werden, womit das Wissen um das komplexe QS Netzwerk von P. aeruginosa erweitert wurde
A novel metagenomic short-chain dehydrogenase/reductase attenuates Pseudomonas aeruginosa biofilm formation and virulence on Caenorhabditis elegans.
No full textIn Pseudomonas aeruginosa, the expression of a number of virulence factors, as well as biofilm formation, are controlled by quorum sensing (QS). N-Acylhomoserine lactones (AHLs) are an important class of signaling molecules involved in bacterial QS and in many pathogenic bacteria infection and host colonization are AHL-dependent. The AHL signaling molecules are subject to inactivation mainly by hydrolases (Enzyme Commission class number EC 3) (i.e. N-acyl-homoserine lactonases and N-acyl-homoserine-lactone acylases). Only little is known on quorum quenching mechanisms of oxidoreductases (EC 1). Here we report on the identification and structural characterization of the first NADP-dependent short-chain dehydrogenase/reductase (SDR) involved in inactivation of N-(3-oxo-dodecanoyl)-L-homoserine lactone (3-oxo-C(12)-HSL) and derived from a metagenome library. The corresponding gene was isolated from a soil metagenome and designated bpiB09. Heterologous expression and crystallographic studies established BpiB09 as an NADP-dependent reductase. Although AHLs are probably not the native substrate of this metagenome-derived enzyme, its expression in P. aeruginosa PAO1 resulted in significantly reduced pyocyanin production, decreased motility, poor biofilm formation and absent paralysis of Caenorhabditis elegans. Furthermore, a genome-wide transcriptome study suggested that the level of lasI and rhlI transcription together with 36 well known QS regulated genes was significantly (≥10-fold) affected in P. aeruginosa strains expressing the bpiB09 gene in pBBR1MCS-5. Thus AHL oxidoreductases could be considered as potent tools for the development of quorum quenching strategies
Mycobacterium bovis Persistence in two Different Captive Wild Animal Populations in Germany: A Longitudinal Molecular Epidemiological Study Revealing Pathogen Transmission by Whole Genome Sequencing
No full textBovine tuberculosis (bTB) caused by Mycobacterium bovis is a transmissible disease notifiable to the World Organization for Animal Health and to European Union, with ongoing efforts of surveillance and eradication in every EU member state. In Germany, a country which has been declared officially free from bovine tuberculosis since 1997 by the EU, M. bovis infections still occur sporadically in cattle and other mammals, including humans. Here, the transmission routes of a bovine TB outbreak in a wildlife park in Germany affecting different cervid species, bison, lynx, and pot-bellied pig were followed employing whole genome sequencing (WGS) combined with spoligotyping and MIRU-VNTR-typing. One single M. bovis strain persisted from 2002 – 2015, and transmission between the park and a distantly located captive cervid farm was verified. The spoligotyping pattern remained identical while MIRU-VNTR-typing of 24 loci of the standardized panel and locus 2163a as additional locus revealed one change at locus 2165 in a strain from a fallow deer, and one at locus 2461 in isolates from red deer over the whole time period. WGS analysis confirmed close relatedness of the isolates, with a maximum of 12 SNPs detected between any two sequenced isolates. In conclusion, our data confirm a longitudinal outbreak of M. bovis in a German wildlife park and provide first insights into the dynamics of different genotyping markers in M. bovis
A novel mechanism of unstable heteroresistance via reversible ompP2 mutations in Haemophilus influenzae
No full textObjectives: Heteroresistance, the presence of a resistant subpopulation within a predominantly susceptible bacterial isolate, is usually difficult to detect and can cause treatment failure. Mechanisms driving unstable heteroresistant phenotypes, which rapidly revert in the absence of antibiotics, remain poorly understood. Methods: To investigate the mechanisms underlying heteroresistance in Haemophilus influenzae, we isolated four heteroresistant clones and characterized them through experimental evolution, gradient diffusion assays, population analysis profiling (PAP), and whole genome sequencing. Results: All clones initially exhibited increased ceftriaxone minimum inhibitory concentrations (MICs) but rapidly reverted to wild-type susceptibility within 4 days in drug-free conditions. The main mechanism was reversible structural changes of the ompP2 gene. Three clones exhibiting a 168 bp genomic inversion affecting ompP2 reverted to wild-type sequence. A fourth clone with an ompP2 frameshift insertion reproducibly acquired compensatory deletions restoring the open reading frame. PAP analysis still detected minority subpopulations with 4x-8x increased MICs at day 4. We identified site-specific DNA breakpoints with a palindrome and a homologous sequence facilitating rapid structural genome changes and phenotypic switching. Conclusion: We discovered a novel mechanism of unstable heteroresistance in H. influenzae, mediated by reversible structural changes in the gene ompP2. Rapid phenotypic switching was induced by ceftriaxone exposure and likely reflects the mutational plasticity of ompP2
Implementation of targeted next-generation sequencing for the diagnosis of drug-resistant tuberculosis in low-resource settings: a programmatic model, challenges, and initial outcomes
No full textTargeted next-generation sequencing (tNGS) from clinical specimens has the potential to become a comprehensive tool for routine drug-resistance (DR) prediction of Mycobacterium tuberculosis complex strains (MTBC), the causative agent of tuberculosis (TB). However, TB mainly affects low- and middle-income countries, in which the implementation of new technologies have specific needs and challenges. We propose a model for programmatic implementation of tNGS in settings with no or low previous sequencing capacity/experience. We highlight the major challenges and considerations for a successful implementation. This model has been applied to build NGS capacity in Namibia, an upper middle-income country located in Southern Africa and suffering from a high-burden of TB and TB-HIV, and we describe herein the outcomes of this process
The Janthinobacterium sp. HH01 genome encodes a homologue of the V. cholerae CqsA and L. pneumophila LqsA autoinducer synthases.
No full textJanthinobacteria commonly form biofilms on eukaryotic hosts and are known to synthesize antibacterial and antifungal compounds. Janthinobacterium sp. HH01 was recently isolated from an aquatic environment and its genome sequence was established. The genome consists of a single chromosome and reveals a size of 7.10 Mb, being the largest janthinobacterial genome so far known. Approximately 80% of the 5,980 coding sequences (CDSs) present in the HH01 genome could be assigned putative functions. The genome encodes a wealth of secretory functions and several large clusters for polyketide biosynthesis. HH01 also encodes a remarkable number of proteins involved in resistance to drugs or heavy metals. Interestingly, the genome of HH01 apparently lacks the N-acylhomoserine lactone (AHL)-dependent signaling system and the AI-2-dependent quorum sensing regulatory circuit. Instead it encodes a homologue of the Legionella- and Vibrio-like autoinducer (lqsA/cqsA) synthase gene which we designated jqsA. The jqsA gene is linked to a cognate sensor kinase (jqsS) which is flanked by the response regulator jqsR. Here we show that a jqsA deletion has strong impact on the violacein biosynthesis in Janthinobacterium sp. HH01 and that a jqsA deletion mutant can be functionally complemented with the V. cholerae cqsA and the L. pneumophila lqsA genes
LEF1 supports metastatic brain colonization by regulating glutathione metabolism and increasing ROS resistance in breast cancer
No full textMore than half of all brain metastases show infiltrating rather than displacing growth at the macro-metastasis/organ parenchyma interface (MMPI), a finding associated with shorter survival. The lymphoid enhancer-binding factor-1 (LEF1) is an epithelial-mesenchymal transition (EMT) transcription factor that is commonly overexpressed in brain-colonizing cancer cells. Here, we overexpressed LEF1 in an in vivo breast cancer brain colonization model. It shortened survival, albeit without engaging EMT at the MMPI. By differential proteome analysis, we identified a novel function of LEF1 as a regulator of the glutathione (GSH) system, the principal cellular redox buffer. LEF1 overexpression also conferred resistance against therapeutic GSH depletion during brain colonization and improved management of intracellular ROS. We conclude that besides EMT, LEF1 facilitates metastasis by improving the antioxidative capacity of epithelial breast cancer cells, in particular during colonization of the brain parenchyma
MTBseq: a comprehensive pipeline for whole genome sequence analysis of Mycobacterium tuberculosis complex isolates
No full textAnalyzing whole-genome sequencing data of Mycobacterium tuberculosis complex (MTBC) isolates in a standardized workflow enables both comprehensive antibiotic resistance profiling and outbreak surveillance with highest resolution up to the identification of recent transmission chains. Here, we present MTBseq, a bioinformatics pipeline for next-generation genome sequence data analysis of MTBC isolates. Employing a reference mapping based workflow, MTBseq reports detected variant positions annotated with known association to antibiotic resistance and performs a lineage classification based on phylogenetic single nucleotide polymorphisms (SNPs). When comparing multiple datasets, MTBseq provides a joint list of variants and a FASTA alignment of SNP positions for use in phylogenomic analysis, and identifies groups of related isolates. The pipeline is customizable, expandable and can be used on a desktop computer or laptop without any internet connection, ensuring mobile usage and data security. MTBseq and accompanying documentation is available from https://github.com/ngs-fzb/MTBseq_source
Increased Levels of sCD30 Have No Impact on the Incidence of Early ABMR and Long-Term Outcome in Intermediate-Risk Renal Transplant Patients With Preformed DSA
No full textBackground: It is still incompletely understood why some patients with preformed donor-specific anti-HLA antibodies (DSA) have reduced kidney allograft survival secondary to antibody-mediated rejection (ABMR), whereas many DSA-positive patients have favorable long-term outcomes. Elevated levels of soluble CD30 (sCD30) have emerged as a promising biomarker indicating deleterious T-cell help in conjunction with DSA in immunologically high-risk patients. We hypothesized that this would also be true in intermediate-risk patients.Methods: We retrospectively analyzed pre-transplant sera from 287 CDC-crossmatch negative patients treated with basiliximab induction and tacrolimus-based maintenance therapy for the presence of DSA and sCD30. The incidence of ABMR according to the Banff 2019 classification and death-censored allograft survival were determined.Results: During a median follow-up of 7.4 years, allograft survival was significantly lower in DSA-positive as compared to DSA-negative patients (p < 0.001). In DSA-positive patients, most pronounced in those with strong DSA (MFI > 5,000), increased levels of sCD30 were associated with accelerated graft loss compared to patients with low sCD30 (3-year allograft survival 75 vs. 95%). Long-term survival, however, was comparable in DSA-positive patients irrespective of sCD30 status. Likewise, the incidence of early ABMR and lesion score characteristics were comparable between sCD30-positive and sCD30-negative patients with DSA. Finally, increased sCD30 levels were not predictive for early persistence of DSA.Conclusion: Preformed DSA are associated with an increased risk for ABMR and long-term graft loss independent of sCD30 levels in intermediate-risk kidney transplant patients
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