Biology subjects

Schipper, K.

Publications and source records attributed to Schipper, K..

14 recordsLinked to original sources

bioRxiv2026

UstiGate: Next generation toolkit for advanced genetic engineering of the basidiomycete chassis Ustilago maydis

The corn smut fungus Ustilago maydis is an established model for fungal biology and an emerging host for biotechnology. However, its broader application in synthetic biology has been constrained by a limited repertoire of standardized genetic parts and tools for multigene engineering. Here, we introduce UstiGate, a modular toolkit that combines MoClo-compatible DNA assembly with targeted genomic integration in U. maydis. The toolkit comprises 92 parts and vectors, including more than 20 promoter regions as well as terminators, selectable markers, fluorescent reporters and modules for different genomic integration sites. Quantitative reporter analyses at both, population and single-cell level, revealed a broad range of promoter strengths and characterized their behavior across growth phases, media, genomic loci and reporter contexts. We further identified a maltose-inducible, glucose-repressed promoter that enables autoinduction and established carotenoid-biosynthesis genes as integration sites supporting visual pre-selection of transformants. Finally, UstiGate enabled the assembly and simultaneous genomic integration of four transcriptional units in a single transformation. UstiGate thus provides a standardized and quantitatively characterized platform for standard applications as well as multigene engineering, enabling advanced synthetic biology applications such as pathway transplantation in U. maydis.

synthetic biology↗

bioRxiv2026

Disentangling the sucrose metabolism of the corn smut Ustilago maydis reveals unexpected complexity

The race for carbohydrates shapes organismic interactions. In plant pathogenic fungi, sucrose is a key nutrient as it constitutes the major transport sugar in plants. Here, we investigate sucrose acquisition in the corn smut fungus Ustilago maydis, a biotrophic pathogen that transitions from yeast-like to hyphal growth for infection. We establish that the fungus encodes a secreted acidic invertase, Suc2, with a dimeric canonical glycoside hydrolase 32 architecture. Comparative biochemical analyses across fungal homologs indicate that this dimeric architecture represents the predominant state, whereas higher-order oligomers, as initially described for Saccharomyces cerevisiae Suc2, are restricted to a subset of lineages. Unexpectedly, elimination of Suc2 did not impair yeast-like growth on sucrose. Similarly, deletion of genes for sucrose transporter Srt1 and cytosolic hydrolase Suc1, typically associated with intracellular sucrose metabolism, did not abolish growth. Instead, sucrose utilization during yeast-like growth depended on a repurposed non-canonical module comprising maltose transporter Agt1 and intracellular (iso)maltases. In contrast, pathogenic development strongly relied on the canonical intracellular sucrose utilization pathway mediated by Srt1 and Agt1. Infection was strongly diminished in strains unable to metabolise sucrose, confirming its central nutritional role for the fungus. Together, our work defines the complete sucrose utilization repertoire of U. maydis and uncovers a lifestyle-dependent metabolic switch between alternative sucrose acquisition strategies. Flexible carbon acquisition might represent a widespread adaptive strategy in basidiomycete pathogenic fungi.

microbiology↗

bioRxiv2024

Natural variation of the streptococcal Group A carbohydrate biosynthesis genes impacts host-pathogen interaction

Streptococcus pyogenes (S. pyogenes) is a leading cause of infection-related mortality in humans globally. The characteristic cell wall-anchored Group A Carbohydrate (GAC) is expressed by all S. pyogenes strains and consists of a polyrhamnose backbone with alternating N-acetylglucosamine (GlcNAc) side chains, of which 25% are decorated with glycerol phosphate (GroP). The genes in the gacA-L cluster are critical for GAC biosynthesis with gacH-L being responsible for the characteristic GlcNAc-GroP decoration, which confers the agglutination in rapid test diagnostic assays and contributes to S. pyogenes pathogenicity. Historical research papers described S. pyogenes isolates, so-called A-variant strains, that lost the characteristic GlcNAc side chain following serial animal passage. Genomic analysis of a single viable historic parent/A-variant strain pair revealed a premature inactivating stop codon in gacI, explaining the described loss of the GlcNAc side chain. Subsequently, we analyzed the genetic variation of the 12 gacA-L genes in a collection of 2,021 S. pyogenes genome sequences. Although all gac genes (gacA-L) displayed genetic variation, we only identified 26 isolates (1.3%) with a premature stop codon in one of the gac genes. Twelve out of 26 (46%) isolates contained a premature stop codon in gacH, which encodes the enzyme responsible for the GroP modification. To study the functional consequences of the different premature stop codons for GacH function, we plasmid-expressed three gacH variants in a S. pyogenes gacH-deficient strain. Cell wall analysis confirmed GacH loss-of-function for the studied gacH variants through the significant reduction of GAC GroP, complete resistance to killing by the human bactericidal enzyme group IIA-secreted phospholipase, and susceptibility to zinc toxicity. Overall, our data provide a comprehensive overview of the genetic variation of the gacA-L cluster in a global population of S. pyogenes strains and the functional consequences of rare inactivating mutations in gacH for host interaction.

microbiology↗

bioRxiv2024

Learning-Induced Effects of Practice Schedule Variability on Stimuli Discrimination Efficiency: High-Density EEG Multi-scale Analyses of Contextual Interference Effect

Contextual interference (CI) enhances motor learning by practicing skill variations in a random rather than blocked order. It has been demonstrated that performing aiming distances in a random order increased electrophysiological (EEG) markers of perceptual, attentional, and working memory processes. However, only the effect of CI on these markers before training was assessed, without evaluating whether they would decrease with learning in participants trained under the random compared to the blocked condition, indicating enhanced neural efficiency. To address this, 35 participants practiced an aiming task involving three distances over nine sessions across three weeks. They were divided into two groups: one trained with distances in a random order (HCI group) and the other in a blocked order (LCI group). Electrophysiological activity was recorded for all participants in the random condition before and after the training program using a high-density EEG multiscale approach, including topographical, source estimation, and source connectivity analyses. EEG analyses revealed post-training neural dynamic differences between groups. The HCI group showed reduced and shorter P3a-like activity compared to the LCI group, while the LCI group exhibited greater occipito-temporal-frontal gamma-band synchronization. These findings suggest that random practice enhances the efficiency of perceptual and attentional processes, particularly of stimuli discrimination, compared to blocked practice.

neuroscience↗

bioRxiv2024

Enhancing Perceptual, Attentional, and Working Memory Demands through Variable Practice Schedules: Insights from High-Density EEG Multi-Scale Analyses

Contextual interference (CI) enhances learning by practicing motor tasks in a random order rather than a blocked order. One hypothesis suggests that the benefits arise from enhanced early perceptual/attentional processes, while another posits that better learning is due to highly activated mnemonic processes. We propose to harness high-density electroencephalography in a multi-scale analysis approach, including topographic analyses, source estimations, and functional connectivity, to examine the intertwined dynamics of attentional and mnemonic processes within short time windows. We recorded scalp activity from 35 participants as they performed an aiming task at three different distances, under both random and blocked conditions using a crossover design. Our results showed that topographies associated with processes related to perception/attention (N1, P3a) and working memory (P3b) were more pronounced in the random condition. Source estimation analyses supported these findings, revealing greater involvement of the perceptual ventral pathway and the anterior cingulate and parietal cortices, along with increased functional connectivity in ventral alpha and frontoparietal theta band networks during random practice. Our results suggest that CI is driven, in the random compared to the blocked condition, by enhanced specific processes such as perceptual, attentional, and mnemonic, as well as large-scale general processes.

neuroscience↗

bioRxiv2024

The fungal protein Jps1 facilitates unconventional protein secretion through a direct phosphoinositide interaction

Protein secretion is indispensable for essential cellular processes in eukaryotic cells, contributing significantly to nutrient acquisition, defense or communication. Alternative pathways bypassing the endomembrane system collectively referred to as unconventional secretion are gaining increasing attention. A number of important molecules such as cytokines, fibroblast growth factor or viral proteins are being exported through these mechanistically diverse pathways. In the fungal model Ustilago maydis, cytokinesis-dependent unconventional secretion mediates export of the chitinase Cts1 via the fragmentation zone. This membrane-rich compartment is formed during cytokinesis between mother and daughter cells. Recently, we identified Jps1, a previously uncharacterized protein, as a crucial factor for Cts1 localization and export. Combining biochemical experiments and in vivo studies, we here uncover two pivotal features of Jps1: dimerization and phosphoinositide (PIP) binding. Our findings reveal that a conserved structural core domain mediates homodimerization, while surrounding flexible variable regions suggest potential diversification in different basidiomycete species. Jps1 does not harbor a canonical PIP-binding domain but instead specificity of the interaction with the preferred PIP PI(4,5)P2 is determined by basic residues. Importantly, loss of PI(4,5)P2 binding specificity results in mis-localisation, morphological defects and reduced extracellular Cts1 activity, particularly at low cell densities. This discovery sheds light on previously unknown key features of Jps1, elucidating its role in supporting Cts1 secretion, and representing a crucial step towards understanding the broader implications of unconventional secretion in eukaryotic cells.

biochemistry↗

bioRxiv2023

The streptococcal phase-variable Type I Restriction-Modification system SsuCC20p dictates the methylome of Streptococcus suis and impacts virulence

Phase-variable Type I Restriction Modification (RM) systems are epigenetic regulatory systems that have been identified in numerous human bacterial pathogens. We previously showed that an emerging zoonotic lineage of Streptococcus suis acquired a phase-variable Type I RM system named SsuCC20p. The SsuCC20p locus was present in the genome of disease-associated isolates from multiple streptococcal species. This indicates that it is not restricted to S. suis and can be acquired through horizontal gene transfer. We demonstrate that SsuCC20p phase-variability relies on a recombinase present within the locus. In vitro, only SsuCC20p is responsible for the genome methylation profiles that were detected in the representative zoonotic S. suis isolate 861160. In addition, we show that, contrary to previous observations, hsdS genes located downstream of the hsdM gene and the recombinase gene, can contribute to the SsuCC20p genome methylation profile. SsuCC20p locked mutants expressing a single hsdS each showed unique genome methylation profiles. The differential genome methylation of the distinct locked mutants caused phase dependent differences in global gene expression in a growth condition dependent manner. We observed significant differences in virulence between hsdS locked mutants in a zebrafish larvae infection model. These data indicate that the streptococcal phase-variable Type I RM system SsuCC20p can impact bacterial virulence via epigenetic regulation of gene expression and potentially contributes to the zoonotic potential of S. suis. ImportancePhase-variation contributes to the virulence of bacterial pathogens as it allows a single strain to produce phenotypic diverse subpopulations. Phase-variable Restriction Modification (RM) systems are systems that allow for such phase-variation via epigenetic regulation of gene expression levels. The phase-variable RM system SsuCC20p was found in multiple streptococcal species and was acquired by an emerging zoonotic lineage of Streptococcus suis. We show that the phase-variability of SsuCC20p is dependent on a recombinase encoded within the SsuCC20p locus. We characterized the genome methylation profiles of the different phases of SsuCC20p and showed that the differential genome methylation within the phases causes differences in gene expression levels and virulence. Altogether, we show that the acquisition of a phase-variable RM system impacts virulence and can potentially contribute to the zoonotic potential of S. suis. Bacterial pathogens can increase their virulence through acquisition of mobile elements containing epigenetic regulatory systems such as RM systems.

microbiology↗

bioRxiv2022

Efficient SARS-CoV-2 detection utilizing chitin-immobilized nanobodies synthesized in Ustilago maydis

The COVID-19 pandemic has greatly impacted the global economy and health care systems, illustrating the urgent need for timely and inexpensive responses to a pandemic threat in the form of vaccines and antigen tests. The causative agent of COVID-19 is SARS-CoV-2. The spike protein on the virus surface interacts with the human angiotensin-converting enzyme (ACE2) via the so-called receptor binding domain (RBD), facilitating virus entry. The RBD thus represents a prime target for vaccines, therapeutic antibodies, and antigen test systems. Currently, antigen testing is mostly conducted by qualitative flow chromatography or via quantitative ELISA-type assays. The latter mostly utilize materials like protein-adhesive polymers and gold or latex particles. Here we present an alternative ELISA approach using inexpensive materials and permitting quick detection based on components produced in the microbial model Ustilago maydis. In this fungus, heterologous proteins like biopharmaceuticals can be exported by fusion to unconventionally secreted chitinase Cts1. As a unique feature, the carrier chitinase binds to chitin allowing its additional use as a purification or immobilization tag. In this study, we produced different mono- and bivalent SARS-CoV-2 nanobodies directed against the viral RBD as Cts1 fusions and screened their RBD binding affinity in vitro and in vivo. Functional nanobody-Cts1 fusions were immobilized on chitin forming an RBD tethering surface. This provides a solid base for future development of an inexpensive antigen test utilizing unconventionally secreted nanobodies as RBD trap and a matching ubiquitous and biogenic surface for immobilization.

molecular biology↗

bioRxiv2022

Neisseria meningitidis sibling small regulatory RNAs connect metabolism with colonization by controlling propionate use.

Neisseria meningitidis (the meningococcus) colonizes the human nasopharynx, primarily as a commensal, but sporadically causing septicemia and meningitis. During colonization and invasion, it encounters different niches with specific nutrient compositions. Small non-coding RNAs (sRNAs) are used to fine-tune expression of genes allowing adaptation to their physiological differences. We have previously characterized sRNAs (Neisseria metabolic switch regulators [NmsRs]) controlling switches between cataplerotic and anaplerotic metabolism. Here we extend the NmsRs regulon by studying methylcitrate lyase (PrpF) and propionate kinase (AckA-1) involved in the methylcitrate cycle, and serine hydroxymethyltransferase (GlyA) and 3-hydroxyacid dehydrogenase (MmsB) involved in protein degradation. These proteins were previously shown to be dysregulated in a {Delta}nmsRs strain. Levels of transcription of target genes and NmsRs were assessed by RT-qPCR. We also used a novel gene-reporter system, in which the 5UTR of the target gene is fused to mcherry to study NmsRs-target gene interaction in the meningococcus. Under nutrient-rich conditions, NmsRs downregulate expression of PrpF and AckA-1, by direct interaction with the 5 UTR of their mRNA. Overexpression of NmsRs impaired growth under nutrient-limiting growth conditions with pyruvate and propionic acid as the only carbon sources. Our data strongly suggest that NmsRs downregulate propionate metabolism by lowering methylcitrate enzyme activity under nutrient-rich conditions. Under nutrient-poor conditions, NmsRs are downregulated, increasing propionate metabolism, resulting in higher tricarboxylic acid (TCA) activities. This allows metabolism to support nasopharynx colonization with breakdown products of amino acids functioning as anaplerotic substrates, as highlighted by NmsRs regulation of GlyA and MmsB. SIGNIFICANCENeisseria meningitidis colonizes the human adult nasopharynx, forming a reservoir for the sporadic occurrence of epidemic invasive meningococcal disease like septicemia and meningitis. Propionic acid generated by other bacteria that co-inhabit the human adolescent/adult nasopharynx can be utilized by meningococci for replication in this environment. Here we showed that the sibling small RNAs designated NmsRs (Neisseria metabolic switch regulators) riboregulate propionic acid utilization by meningococci, and thus, colonization. Under conditions mimicking the nasopharyngeal environment, NmsRs upregulated expression of enzymes of the methylcitrate cycle. This leads to the conversion of propionic acid to pyruvate and succinate, resulting in higher tricarboxylic acid cycle activity, allowing colonization of the nasopharynx. NmsRs link metabolic state with colonization, which is a crucial step on the trajectory to invasive meningococcal disease.

microbiology↗

bioRxiv2022

Genetic diversity of Staphylococcus aureus wall teichoic acid glycosyltransferases affects immune recognition

Staphylococcus aureus (S. aureus) is a leading cause of skin and soft tissue infections and (hospital-acquired) systemic infections. Wall teichoic acids (WTAs) are cell wall-anchored glycopolymers that are important for S. aureus nasal colonization, endocarditis, and antibiotic resistance. WTAs consist of a polymerized ribitol phosphate (RboP) chain that can be glycosylated with N-acetylglucosamine (GlcNAc) by three glycosyltransferases: TarS, TarM, and TarP. TarS and TarP modify WTA with {beta}-linked GlcNAc on the C-4 ({beta}1,4-GlcNAc) and the C-3 position ({beta}1,3-GlcNAc) of the RboP subunit, respectively, whereas TarM modifies WTA with -linked GlcNAc at the C-4 position (1,4-GlcNAc). Importantly, these WTA glycosylation patterns impact immune recognition and clearance of S. aureus. Previous studies suggest that tarS is near-universally expressed within the S. aureus population, whereas a smaller proportion co-express either tarM or tarP. To gain more insight in the presence and genetic variation of tarS, tarM, and tarP in the S. aureus population, we analyzed a collection of 25,652 S. aureus genomes within the PubMLST database. Over 99% of isolates contained tarS. Co-expression of tarS/tarM or tarS/tarP occurred in 37% and 7% of isolates, respectively, and was associated to specific S. aureus clonal complexes. We also identified 26 isolates (0.1%) that contained all three glycosyltransferase genes. At sequence level, we identified tar alleles with amino acid substitutions in critical enzymatic residues or with premature stop codons. Several tar variants were expressed in a S. aureus tar-negative strain. Analysis using specific monoclonal antibodies and human langerin showed that WTA glycosylation was severely attenuated or absent. Overall, our data provide a broad overview of the genetic diversity of the three WTA glycosyltransferases in the S. aureus population and the functional consequences for immune recognition.

microbiology↗

bioRxiv2021

Establishing regulated unconventional secretion for production of heterologous proteins

Heterologous protein production is a highly demanded biotechnological process. Secretion of the product to the culture broth is advantageous because it drastically reduces downstream pro-cessing costs. We exploit unconventional secretion for heterologous protein expression in the fungal model microorganism Ustilago maydis. Proteins of interest are fused to carrier chitinase Cts1 for export via the fragmentation zone of dividing yeast cells in a lock-type mechanism. Here, we are first to develop regulatory systems for unconventional protein secretion. This enables uncoupling the accumulation of biomass and protein synthesis of a product of choice from its export. Regulation was successfully established at two different levels using transcriptional and post-translational induction strategies. As a proof-of-principle, we applied autoinduction based on transcriptional regulation for the production and secretion of functional anti-Gfp nanobodies. The presented developments comprise tailored solutions for differentially prized products and thus constitute another important step towards a competitive protein production platform.

molecular biology↗

bioRxiv2020

Polycistronic gene expression in the model microorganism Ustilago maydis

AbtractEukaryotic microorganisms transcribe monocistronic mRNAs to encode proteins. For synthetic biological approaches like metabolic engineering, precise co-expression of several proteins in space and time is advantageous. A straightforward approach is the application of viral 2A peptides to design synthetic polycistronic mRNAs in eukaryotes. Here, we establish such a system in the well-studied model microorganism Ustilago maydis. Using two fluorescence reporter proteins, we compared the activity of five viral 2A peptides. Their activity was evaluated in vivo using fluorescence microscopy and validated using fluorescence resonance energy transfer (FRET). Activity ranged from 20 to 100% and the best performing 2A peptide was P2A from porcine teschovirus-1. As proof of principle, we followed regulated gene expression efficiently over time and synthesised a tri-cistronic mRNA encoding biosynthetic enzymes to produce mannosylerythritol lipids (MELs). In essence, we evaluated 2A peptides in vivo and demonstrated the applicability of 2A peptide technology for U. maydis in basic and applied science.

microbiology↗

bioRxiv2020

A novel factor essential for unconventional secretion of chitinase Cts1

Subcellular targeting of proteins is essential to orchestrate cytokinesis in eukaryotic cells. During cell division of Ustilago maydis, for example, chitinases must be specifically targeted to the fragmentation zone at the site of cell division to degrade remnant chitin and thus separate mother and daughter cells. Chitinase Cts1 is exported to this location via an unconventional secretion pathway putatively operating in a lock-type manner. The underlying mechanism is largely unexplored. Here, we applied a forward genetic screen based on UV mutagenesis to identify components essential for Cts1 export. The screen revealed a novel factor termed Jps1 lacking known protein domains. Deletion of the corresponding gene confirmed its essential role for Cts1 secretion. Localization studies demonstrated that Jps1 colocalizes with Cts1 in the fragmentation zone of dividing yeast cells. While loss of Jps1 leads to exclusion of Cts1 from the fragmentation zone and strongly reduced unconventional secretion, deletion of the chitinase does not disturb Jps1 localization. Yeast-two hybrid experiments suggest that the two proteins interact. In essence, we identified a novel component of unconventional secretion that functions in the fragmentation zone to enable export of Cts1. We hypothesize that Jps1 acts as an anchoring factor, supporting the proposed novel lock-type mechanism of unconventional secretion.

microbiology↗

bioRxiv2019

Retrieval of a well-established skill is resistant to distraction: evidence from an implicit probabilistic sequence learning task

The characteristics of acquiring new sequence information under dual-task situations have been extensively studied so far. Such a concurrent task has often been found to affect performance. In real life, however, we mostly perform a secondary task when the primary one is already well-acquired. The effect of a secondary task on the ability to retrieve well-established sequence representations remains elusive. The present study investigates whether accessing a well-acquired probabilistic sequence knowledge is affected by a concurrent task. Participants acquired non-adjacent regularities in a perceptual-motor implicit probabilistic sequence learning task. After a 24-hour offline period, participants were tested on the same sequence learning task under dual-task or single-task conditions. Here we show that although the secondary task significantly prolonged the overall reaction times in the primary task, the access to the previously learned probabilistic representations remained intact. Our results highlight the importance of studying the dual-task effect not only in the learning phase but also during memory access.

neuroscience↗