Biology subjects

Pannekoek, Y.

Publications and source records attributed to Pannekoek, Y..

9 recordsLinked to original sources

bioRxiv2026

Monoclonal antibody-based serotyping of Listeria monocytogenes provides new insights in epidemiology and virulence

ObjectivesListeria monocytogenes is an opportunistic pathogen, associated with foodborne infections that disproportionately affect newborns, elderly and immunocompromised patients. L. monocytogenes can be classified on the antigenic and related structural variation of cell-associated wall teichoic acid (WTA) molecules through conventional serotyping techniques. The WTA structure of serovars (SV) 1/2, 1/2*, 3 and 7 consists of a linear poly-ribitolphosphate (RboP) polymer either with or without decoration with rhamnose (Rha) and/or N-acetylglucosamine (GlcNAc). Of these four SVs, SV1/2 (WTA with GlcNAc and Rha) causes [~] 99% of all listeriosis cases. However, conventional serotyping cannot accurately discriminate between these four SVs, particularly SVs1/2* (WTA with Rha). MethodsHere we applied two identified monoclonal antibodies (mAb), with specificity for the RboP backbone or GlcNAc modification to develop a discriminatory serotyping scheme for SV1/2, 1/2*, 3 and 7. Isogenic mutants for the different SVs were created in L. monocytogenes SV1/2 strain EGD-e. The typing scheme was then adapted to an immnoblot assay and applied to a collection of 317 previously classified listeriosis isolates from the Netherlands Reference Laboratory for Bacterial Meningitis. ResultsBinding of the RboP-specific mAb was limited to EGD-e wild type (SV1/2), but increased significantly for isogenic EGD-e mutants representing SV1/2*, 3 and 7. In contrast, the GlcNAc-specific mAb only recognized EGD-e mutants representing SVs 1/2 and 3. The combined staining profiles of the two mAbs allowed accurate discrimination of the four SVs as verified on clinical isolates. Applying this typing scheme to 317 listeriosis isolates previously typed as SV1/2, we confirmed SV designation in >90% of isolates, but also identified SV1/2* (5.4%), SV3 (0.6%) and SV7 (0.3%) isolates. SV1/2* isolates were also identified among meningitis patients. ConclusionThe increased discriminatory capacity of L. monocytogenes serotyping provides a more detailed insight of the epidemiological landscape and the critical factors for L. monocytogenes infections.

microbiology↗

bioRxiv2025

CLR-Seq: a pipeline to identify bacterial microbiota species with immunomodulatory potential through human C-type lectin receptor interaction

Bacterial glycans are key components in immune interactions. We lack insight into the diverse glycan landscape present in complex microbial communities since currentomics techniques do not capture this post-translational information. Here we employed C-type lectin receptors (CLRs), which are dedicated innate glycan-sensing receptors, as probes for bacterial cell sorting in combination with 16S rRNA gene sequencing to identify microbiota species with a specific CLR-reactive glycan profile. We established our experimental CLR-sequencing (CLR-seq) pipeline using soluble fluorescently-labeled human macrophage galactose C-type lectin (MGL, CD301) and langerin (CD207). Both receptors identified known langerin- or MGL-interacting Staphylococcus aureus strains in a synthetic microbial community even when present at low abundance. Subsequent application of CLR-seq on fecal microbiota samples from healthy donors identified specific langerin- and MGL-interacting bacterial species that were subsequently validated as monocultures. In summary, CLR-seq is a modular platform that allows identification of human microbiota species based on CLR-interacting glycans with easy expansion to other CLRs or microbiota samples from patients. Given that CLRs are densely expressed on dendrites of antigen-presenting cells, this pathway may play a role in cross-barrier recognition and sampling of the environment during homeostasis.

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

The two-component system ArlRS is essential for wall teichoic acid glycoswitching in Staphylococcus aureus

Staphylococcus aureus is among the leading causes of hospital-acquired infections. Critical to S. aureus biology and pathogenesis are the cell wall-anchored glycopolymers wall teichoic acids (WTA). Approximately one-third of S. aureus isolates decorates WTA with a mixture of 1,4- and {beta}1,4-N-acetylglucosamine (GlcNAc), which requires the dedicated glycosyltransferases TarM and TarS, respectively. Environmental conditions, such as high salt concentrations, affect the abundance and ratio of 1,4- and {beta}1,4-GlcNAc WTA decorations, thereby impacting biological properties such as antibody binding and phage infection. To identify regulatory mechanisms underlying WTA glycoswitching, we screened 1,920 S. aureus mutants (Nebraska Transposon Mutant Library) by immunoblotting for differential expression of WTA-linked 1,4- or {beta}1,4-GlcNAc using specific monoclonal antibody Fab fragments. Three two-component systems (TCS), GraRS, ArlRS, and AgrCA, were among the 230 potential hits. Using isogenic TCS mutants, we demonstrated that ArlRS is essential for WTA {beta}1,4-GlcNAc decoration through regulation of tarM but not tarS. ArlRS regulated tarM expression through the transcriptional regulator MgrA, which was responsive to Mg2+, but not Na+. Importantly, ArlRS-mediated regulation of WTA glycosylation affected S. aureus interaction with the innate receptor langerin and lysis by {beta}1,4-GlcNAc-dependent phages. Since WTA represents a promising target for future immune-based treatments and vaccines, our findings provide important insight to align targeting strategies with the S. aureus WTA glycosylation patterns during infection. ImportanceStaphylococcus aureus is a common colonizer of mucosal surfaces, but is also a frequent cause of severe infections in humans. Development of antibiotic resistance complicates treatment of S. aureus infections, increasing the need for antibiotic alternatives such as vaccines and therapies with bacterial viruses also known as phages. Wall teichoic acids (WTA) are abundantly-expressed glycosylated structures of the S. aureus cell wall that have gained attention as a promising target for new treatments. Importantly, WTA glycosylation patterns show variation depending on environmental conditions, thereby impacting phage binding and interaction with host factors, such as antibodies and innate pattern-recognition receptors. Here, we show that the two-component system ArlRS through its effector MgrA is involved in regulation of WTA glycosylation by responding to environmental changes in Mg2+ concentration. These findings may support the design of new treatment strategies that target WTA glycosylation patterns of S. aureus during infection.

microbiology↗

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

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↗

bioRxiv2022

Interference with lipoprotein maturation sensitizes methicillin-resistant Staphylococcus aureus to human group IIA secreted phospholipase A2 and daptomycin.

Methicillin-resistant Staphylococcus aureus (MRSA) has been classified as a high priority pathogen by the World Health Organization underlining the high demand for new therapeutics to treat infections. Human group IIA secreted phospholipase A2 (hGIIA) is among the most potent bactericidal proteins against Gram-positive bacteria, including S. aureus. To determine hGIIA-resistance mechanisms of MRSA we screened the Nebraska Transposon Mutant Library using a sublethal concentration of recombinant hGIIA. We identified and confirmed the role of lspA, encoding the lipoprotein signal peptidase LspA, as a new hGIIA resistance gene in both in vitro assays and an infection model in hGIIA-transgenic mice. Increased susceptibility of the lspA mutant was associated with faster and increased cell wall penetration of hGIIA. Moreover, lspA deletion also increased susceptibility to daptomycin, a last-resort antibiotic to treat MRSA infections. Exposure of MRSA wild-type to the LspA-specific inhibitors globomycin and myxovirescin A1 induced a lspA mutant phenotype with regard to hGIIA and daptomycin killing. Analysis of >26,000 S. aureus genomes showed that LspA is highly sequence-conserved, suggesting that LspA inhibition could be applied universally. The role of LspA in hGIIA resistance was not restricted to MRSA since Streptococcus mutans and Enterococcus faecalis were also more hGIIA-susceptible after lspA deletion or LspA inhibition, respectively. Overall, our data suggest that pharmacological blocking of LspA may disarm Gram-positive pathogens, including MRSA, to enhance clearance by innate host defense molecules and clinically-applied antibiotics.

microbiology↗

bioRxiv2020

Chlamydia gallinacea: genetically armed as a pathogen however a phenotypical commensal?

Chlamydia gallinacea is an obligate intracellular bacterium that has recently been added to the family of Chlamydiaceae. C. gallinacea is genetically diverse, widespread in poultry and a suspected cause of pneumonia in slaughterhouse workers. In poultry, C. gallinacea infections appear asymptomatic, but studies about the pathogenic potential are limited. In this study two novel sequence types of C. gallinacea were isolated from apparently healthy chickens. Both isolates (NL_G47 and NL_F725) were closely related to each other and showed 99.1% DNA sequence identity to C. gallinacea Type strain 08-1274/3. To gain further insight in the pathogenic potential, infection experiments in embryonated chicken eggs and comparative genomics with Chlamydia psittaci were performed. C. psittaci is an ubiquitous zoonotic pathogen of birds and mammals, and infection in poultry can result in severe systemic illness. In experiments with embryonated chicken eggs C. gallinacea induced mortality was observed, potentially strain dependent but lower compared to C. psittaci induced mortality. Comparative analyses confirmed all currently available C. gallinacea genomes possess the hallmark genes coding for known and potential virulence factors as found in C. psittaci albeit to a reduced number of orthologues or paralogs. The presence of (potential) virulence factors and the observed mortality in embryonated eggs indicates C. gallinacea should rather be considered as a (conditional) pathogen than an innocuous commensal. ImportanceChlamydiaceae are a family of bacteria comprising human and animal pathogens including the recently recognized Chlamydia gallinacea. C. gallinacea is widespread in poultry without causing clinical signs, which raises questions about its pathogenic potential. To assess this potential, two novel C. gallinacea strains were isolated, tested in infection experiments in embryonated chicken eggs and compared to C. psittaci. C. psittaci infection in poultry can result in severe systemic illness, depending on the conditions, and infections can be transmitted to humans. In the experiments C. gallinacea infection induced mortality of the embryo, but to a lower extent than infection with C. psittaci. Subsequent genome comparisons confirmed both C. gallinacea strains possess potential virulence genes typical for chlamydia, but fewer than C. psittaci. These results indicate C. gallinacea does have a pathogenic potential which warrants further research to elucidate its role as a poultry pathogen.

microbiology↗