Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens

For successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variability introduced by differing wet-lab procedures on the quality of the sequence data we conducted an interlaboratory study, involving four bacterial pathogens, which account for the majority of food-related bacterial infections: Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The participants, ranging from German federal research institutes, federal state laboratories to universities and companies, were asked to follow their routine in-house protocols for short-read sequencing of 10 cultures and one isolated bacterial DNA per species. Sequence and assembly quality were then analyzed centrally. Variations within isolate samples were detected with SNP and cgMLST calling. Overall, we found that the quality of Illumina raw sequence data was high with little overall variability, with one exception, attributed to a specific library preparation kit. The variability of Ion Torrent data was higher, independent of the investigated species. For cgMLST and SNP analysis results, we found that technological sequencing artefacts could be reduced by the use of filters, and that SNP analysis was more suited than cgMLST to compare data of different contributors. Regarding the four species, a minority of Campylobacter isolate data showed the in comparison highest divergence with regard to sequence type and cgMLST analysis. We additionally compared the assembler SPAdes and SKESA for their performance on the Illumina data sets of the different species and library preparation methods and found overall similar assembly quality metrics and cgMLST statistics

Detektion and characterization of cell wall proteins in Candida albicans

Candida albicans ist ein weit verbreiteter human pathogener Organismus, der sowohl oberflächliche wie auch systemische Infektionen verursacht. Diese treten vor allem bei Personen mit geschwächtem Immunsystem auf. Bei der Infektion spielen die Zellwand und ihre Bestandteile eine besonders wichtige Rolle. Dieses komplexe Netzwerk aus Glucan, Chitin, Mannan und Proteinen stellt die Naht¬stelle der Interaktion zwischen Wirt und Pathogen dar. In vorangegangenen Arbeiten konnte gezeigt werden, dass mehrere der in dieses Netzwerk eingebundenen Proteine ausschlaggebend für die Adhäsion und die Interaktion mit dem Wirt und seinem Immunsystem sind. Hinweise auf Änderungen im Zell¬wand-Proteom während des Übergangs vom Wachstum als Blastosporen hin zu Hyphen wurden zuerst im Microarray Experimenten gefunden, und konnten später in Untersuchungen bestätigt werden, die sich auf die Zellwandproteine konzentrierten. In dieser Arbeit wurden verschiedene Ansätze der Proben¬vorbereitung verglichen, um die kovalent an die Zellwand gebundenen Proteine oder deren Peptide durch massen¬spektro¬metrische Methoden zu identifizieren. Trypsin, Endo¬proteinase Glu-C und Bromcyan zur Freisetzung von Proteinfragmenten wurden allein oder in Kombination verwendet und miteinander verglichen. Zusätzlich untersucht wurden die Auswirkungen einer Vor¬behandlung der Zell¬wand durch eine β-1,3-Glucanase. Die Identifikation der Peptide erfolgte mittels zweier verschiedener Massenspektrometer. Insgesamt konnten 33 Proteine in der Wachstumsform der Blastosporen identifiziert werden, sechs davon mit vorhergesagtem GPI-Anker. Im Gegensatz dazu wiesen 14 der 18 identifizierten Proteine aus Hyphen-Zellen diese spezifische Ankersequenz auf, 12 davon sind in dieser Wachstumsform transkriptionell induziert. Zwischen den verwendeten Methoden wurde eine hohe Varianz in der Zahl identifizierter Proteine, bzw. der Zahl zu einem Protein gehörender Peptide, gefunden. Zusätzlich wurden die von C. albicans in synthetische Flüssigmedien sekretierten Proteine identifiziert. Unter den sowohl von Blastosporen wie auch Hyphen sekretierten Proteinen befand sich Sun41p. In einer früheren Arbeit wurde gezeigt, dass die Transkription dieses Proteins in Hyphen induziert wird. Die SUN Gen Familie, zu der es gehört, wurde in S. cerevisiae definiert und beinhaltet eine für Pilze spezifische Familie von Proteinen die eine hohe Übereinstimmung in ihrer C-terminalen Domäne aufweisen. Gene dieser Familie sind in unterschiedliche zelluläre Prozesse wie DNS-Replikation, Alterung, mitochondriale Biogenese und Cytokinese involviert. In C. albicans gehören zwei Gene, SUN41 und SIM1, dieser Familie an. Da Sun41p als potentieller Virulenz¬faktor Ziel einer gegen Pilze gerichteten Therapie darstellen kann, wurde seine Funktion durch die Konstruktion einer SUN41 Deletionsmutante untersucht. Dadurch konnte gezeigt werden, dass C. albicans Mutanten ohne SUN41 ähnliche Defekte aufweisen wie sie auch bei entsprechenden S. cerevisiae Mutanten gefunden wurden. Dies beinhaltet unter anderem Defekte bei der Cyto¬kinese. Zusätzlich zeigten die SUN41 Mutanten eine gesteigerte Empfindlichkeit gegenüber der die Zellwand schädigende Substanz Kongorot, wohingegen bei der Anwesenheit von Calcofluor Weiß keine Veränderung beobachtbar war. Im Vergleich mit dem Wildtyp wies der SUN41 Deletions-Stamm Defekte bei der Bildung von Biofilmen auf, zeigte eine reduzierte Adhäsion auf einem epithelialen Gewebemodell und konnte unter den getesteten Bedingungen auf Festmedien keine Hyphen bilden. Die Ergebnisse deuten auf eine Funktion von Sun41p als Glycosidase hin, die an der Zellwand-Biogenese beteiligt ist und dadurch die Cytokinese, die Adhäsion am Wirtsgewebe, die Bildung von Biofilmen und die Virulenz beeinflusst. Dies weist auf eine wichtige Rolle von Sun41p bei der Interaktion des Pathogen mit dem Wirt hin.Candida albicans is a common human pathogen which may cause superficial and systemic infections especially in persons with weakened immune system. During infection, the cell wall and its components plays an important role because this complex network built of glucan, chitin, mannan and proteins is the interface between host and pathogen. It was shown that several of the embedded proteins are crucial for adhesion and interaction with the host. During yeast to hyphal transition significant changes in the cell wall proteome were first suggested by microarray experiments and further confirmed by studies focusing on cell wall proteomics. In this work, the effects of different sample preparations for solubilisation of covalently bound proteins or peptides thereof from isolated cell walls for identification with MS/MS were compared. Trypsin, endoproteinase Glu-C and cyanogene bromide alone or in combination were used for protein fragmentation, and the effects of a pre-treatment of the cell wall with a β-1,3-glucanase tested and compared to each other. Two different types of mass spectrometers were used for identification of the peptides. Overall, 33 proteins were identified in yeast growth form, 6 with a predicted GPI-anchor. In contrast, 14 of the identified 18 proteins in hyphal cells carry this specific anchor sequence. Based on the level of transcripts, 12 of these proteins are up regulated under hyphal inducing conditions. A large variance in the identification of proteins and the number of peptides corresponding to one protein depending on the method used was found. Additionally, proteins secreted from C. albicans growing in liquid synthetic media were identified. Among these proteins, Sun41p was found to be secreted to the media of cells growing as blastospores as well as hyphae. The transcription of this protein was found to be up regulated in hyphae in previous work. The SUN gene family, it belongs to, has been defined in S. cerevisiae and comprises a fungal specific family of proteins which show high similarity in their C-terminal domain. Genes of this family are involved in different cellular processes like DNA replication, ageing, mitochondrial biogenesis and cytokinesis. In C. albicans the SUN-family comprises two genes, SUN41 and SIM1. Being of interest as potential virulence factor and therefore potential target in an anti-fungal therapy, the function of Sun41p was examined by construction of a SUN41 deletion mutant. Using this, it could be demonstrated that C. albicans mutants lacking SUN41 show similar defects as found for S. cerevisiae including defects in cytokinesis. In addition, the SUN41 mutant showed a higher sensitivity towards the cell wall disturbing agent Congo red, whereas no difference was observed in the presence of Calcofluor white. Compared to the wild type SUN41 deletion strains exhibited a defect in biofilm formation, a reduced adherence on epithelial tissue models and are unable to form hyphae on solid media under the conditions tested. The results support a function of Sun41p as glycosidase involved cell wall biogenesis affecting cytokinesis, adhesion to host tissue, biofilm formation and virulence indicating an important role in host-pathogen interaction

Candida albicans Sun41p, a Putative Glycosidase, Is Involved in Morphogenesis, Cell Wall Biogenesis, and Biofilm Formation▿

The SUN gene family has been defined in Saccharomyces cerevisiae and comprises a fungus-specific family of proteins which show high similarity in their C-terminal domains. Genes of this family are involved in different cellular processes, like DNA replication, aging, mitochondrial biogenesis, and cytokinesis. In Candida albicans the SUN family comprises two genes, SUN41 and SIM1. We demonstrate that C. albicans mutants lacking SUN41 show similar defects as found for S. cerevisiae, including defects in cytokinesis. In addition, the SUN41 mutant showed a higher sensitivity towards the cell wall-disturbing agent Congo red, whereas no difference was observed in the presence of calcofluor white. Compared to the wild type, SUN41 deletion strains exhibited a defect in biofilm formation, a reduced adherence on a Caco-2 cell monolayer, and were unable to form hyphae on solid medium under the conditions tested. Interestingly, Sun41p was found to be secreted in the medium of cells growing as blastospores as well as those forming hyphae. Our results support a function of SUN41p as a glycosidase involved in cytokinesis, cell wall biogenesis, adhesion to host tissue, and biofilm formation, indicating an important role in the host-pathogen interaction

Animal Species Identification of Meat Using MALDI-TOF Mass Spectrometry

We describe the animal species identification of meat using MALDI-TOF mass spectroscopy including the development and validation of a reliable method, qualified for use in the accredited official food-control laboratory. Previous publications had shown the potential of MALDI-TOF MS for animal species differentiation of several kind of food, including meat. Our aim was to establish a rapid and reliable method by means of a simplified sample preparation without prior tryptic digest, an existing popular MALDI system, an independent extensive reference database, and an adequate validation concept. In contrast to the previous works, we consequently use the MALDI user platform “MALDI-UP” to give other food control laboratories the possibility of exchanging reference and validation spectra.</p

Single-stranded DNA catalyzes hybridization of PCR-products to microarray capture probes.

Since its development, microarray technology has evolved to a standard method in the biotechnological and medical field with a broad range of applications. Nevertheless, the underlying mechanism of the hybridization process of PCR-products to microarray capture probes is still not completely understood, and several observed phenomena cannot be explained with current models. We investigated the influence of several parameters on the hybridization reaction and identified ssDNA to play a major role in the process. An increase of the ssDNA content in a hybridization reaction strongly enhanced resulting signal intensities. A strong influence could also be observed when unlabeled ssDNA was added to the hybridization reaction. A reduction of the ssDNA content resulted in a massive decrease of the hybridization efficiency. According to these data, we developed a novel model for the hybridization mechanism. This model is based on the assumption that single stranded DNA is necessary as catalyst to induce the hybridization of dsDNA. The developed hybridization model is capable of giving explanations for several yet unresolved questions regarding the functionality of microarrays. Our findings not only deepen the understanding of the hybridization process, but also have immediate practical use in data interpretation and the development of new microarrays

Regulation of Candida glabrata oxidative stress resistance is adapted to host environment

The human fungal pathogen Candida glabrata is related to Saccharomyces cerevisiae but has developed high resistance against reactive oxygen species. We find that induction of conserved genes encoding antioxidant functions is dependent on the transcription factors CgYap1 and CgSkn7 which cooperate for promoter recognition. Superoxide stress resistance of C. glabrata is provided by superoxide dismutase CgSod1, which is not dependent on CgYap1/Skn7. Only double mutants lacking both CgSod1 and CgYap1 were efficiently killed by primary mouse macrophages. Our results suggest that in C. glabrata the regulation of key genes providing stress protection is adopted to meet a host–pathogen situation

The E. coli S30 lysate proteome: A prototype for cell-free protein production

Protein production using processed cell lysates is a core technology in synthetic biology and these systems are excellent to produce difficult toxins or membrane proteins. However, the composition of the central lysate of cell-free systems is still a black box. Escherichia coli lysates are most productive for cell-free expression, yielding several mgs of protein per ml of reaction. Their preparation implies proteome fractionation, resulting in strongly biased and yet unknown lysate compositions. Many metabolic pathways are expected to be truncated or completely removed. The lack of knowledge of basic cell-free lysate proteomes is a major bottleneck for directed lysate engineering approaches as well as for assay design using non-purified reaction mixtures. This study is starting to close this gap by providing a blueprint of the S30 lysate proteome derived from the commonly used E. coli strain A19. S30 lysates are frequently used for cell-free protein production and represent the basis of most commercial E. coli cell-free expression systems. A fraction of 821 proteins was identified as the core proteome in S30 lysates, representing approximately a quarter of the known E. coli proteome. Its classification into functional groups relevant for transcription/translation, folding, stability and metabolic processes will build the framework for tailored cell-free reactions. As an example, we show that SOS response induction during cultivation results in tuned S30 lysate with better folding capacity, and improved solubility and activity of synthesized proteins. The presented data and protocols can serve as platform for the generation of customized cell-free systems and product analysis.Andalusian Government P11-CVI-7216 BIO198Spanish Ministry of Science and Innovation BFU2015-71017-

Hybridization of products of four asymmetric PCRs to seven probe pairs.

<p>Signal intensities of probe pairs 1–7 after hybridization of the target <i>dfrA1</i> which was amplified in four asymmetric PCRs containing different primer concentrations.</p

Schematic formation of a double-stranded full-length PCR-product and abortion products of different lengths.

<p>The abortion products of the sense strand lead to an excess of sense ssDNA in the vicinity of the forward primer binding site and those of the antisense strand in the vicinity of the reverse primer binding site, respectively. When both strands are formed to the same extend the equilibrium of sense and antisense strands lies in the middle of the product. The formation of one strand in excess leads to a shift of the equilibrium to one side.</p