Biology subjects

Hensbergen, P.

Publications and source records attributed to Hensbergen, P..

4 recordsLinked to original sources

bioRxiv2026

Identification of novel cellular intermediates unveils unique enzymes for flagellar glycan biosynthesis in Clostridioides difficile

Glycosylation of bacterial surface proteins, such as flagellin (FliC), is important for their function and is often involved in virulence of pathogens. Glycans can be further modified by so-called post-glycosylation modifications (PGMs) often resulting in exclusive molecular structures. In Clostridioides difficile a unique glycan structure (Type A) decorates FliC (which forms the flagellar filament) that consists of an O-linked N-acetyl-{beta}-D-glucosamine (GlcNAc) modified with an N-methyl-L-threonine via a phosphodiester linkage. This PGM is synthesized by a set of four enzymes encoded in one operon (ftaABCD), but the exact biosynthesis pathway and biosynthetic intermediates remain unknown. In this study, we chemically synthesized two hitherto undescribed biosynthetic intermediates that we predicted based on bioinformatic analyses, CDP-threonine and CDP-N-methylthreonine. We showed that they are involved in the Type A PGM biosynthesis, as evidenced by mass spectrometric analyses of extracts of a set of C. difficile mutant strains. Furthermore, we characterized FtaC to be a SAM-dependent CDP-threonine N-methyltransferase, that installs the methyl group on CDP-threonine prior to transfer of the PGM to GlcNAc-FliC, and we revealed FtaD as the CDP-N-methylthreonine:GlcNAc N-methylthreoninephosphotransferase. Finally, using recombinantly expressed FtaC and FtaD in combination with synthetic CDP-threonine, we reconstituted the biosynthesis pathway of the Type A PGM in vitro. Overall, our results open avenues to explore these unique biosynthesis enzymes in molecular detail to provide new points of entry for the development of biosynthesis inhibitors and tools to study the role of this PGM in virulence and flagellar assembly.

microbiology↗

bioRxiv2024

Non-prime- and Prime-side Profiling of Pro-Pro Endopeptidase Specificity Using Synthetic Combinatorial Peptide Libraries and Mass Spectrometry

A group of bacterial proteases, the Pro-Pro endopeptidases (PPEPs), possess the unique ability to hydrolyze proline-proline bonds in proteins. Since a proteases function is largely determined by its substrate specificity, methods that can extensively characterize substrate specificity are valuable tools for protease research. Previously, we achieved an in-depth characterization of PPEP prime-side specificity. However, PPEP specificity is also determined by the non-prime-side residues in the substrate. To gain a more complete insight into the determinants of PPEP specificity, we characterized the non-prime- and prime-side specificity of various PPEPs using a combination of synthetic combinatorial peptide libraries and mass spectrometry. With this approach, we deepened our understanding of the P3-P3 specificities of PPEP-1 and PPEP-2, while identifying PPEP-2s endogenous substrate as the most optimal substrate in our library data. Furthermore, by employing the library approach, we investigated the altered specificity of mutants of PPEP-1 and PPEP-2. Additionally, we characterized a novel PPEP from Anoxybacillus tepidamans, which we termed PPEP-4. Based on structural comparisons, we hypothesized that PPEP-4 displays a PPEP-1-like prime-side specificity, which was substantiated by the experimental data. Intriguingly, another putative PPEP from Clostridioides difficile, CD1597, did not display Pro-Pro endoproteolytic activity. Collectively, we characterized PPEP specificity in detail using our robust peptide library method and, together with additional structural information, provide more insight into the intricate mechanisms that govern protease specificity.

molecular biology↗

bioRxiv2024

Proteolytic activity of surface exposed HtrA determines its expression level and is needed to survive acidic conditions in Clostridioides difficile

To survive in the host, pathogenic bacteria need to be able to react to the unfavourable conditions that they encounter, like low pH, elevated temperatures, antimicrobial peptides and many more. These conditions may lead to unfolding of envelope proteins and this may be lethal. One of the mechanisms through which bacteria are able to survive these conditions is through the protease/foldase activity of the high temperature requirement A (HtrA) protein. The gut pathogen Clostridioides difficile encodes one HtrA homolog that is predicted to contain a membrane anchor and a single PDZ domain. The function of HtrA in C. difficile is hitherto unknown but previous work has shown that an insertional mutant of htrA displayed elevated toxin levels, less sporulation and decreased binding to target cells. Here, we show that HtrA is membrane associated and localized on the surface of C. difficile and characterize the requirements for proteolytic activity of recombinant soluble HtrA. In addition, we show that the level of HtrA in the bacteria heavily depends on its proteolytic activity. Finally, we show that proteolytic activity of HtrA is required for survival under acidic conditions.

microbiology↗

bioRxiv2017

Proteomic identification of Axc, a novel beta-lactamase with carbapenemase activity in a meropenem-resistant clinical isolate of Achromobacterxylosoxidans

The development of antibiotic resistance during treatment is a threat to patients and their environment. Insight in the mechanisms of resistance development is important for appropriate therapy and infection control. Here, we describe how through the application of mass spectrometry-based proteomics, a novel beta-lactamase Axc was identified as an indicator of acquired carbapenem resistance in a clinical isolate of Achromobacter xylosoxidans.\n\nComparative proteomic analysis of consecutively collected susceptible and a resistant isolates from the same patient revealed that high Axc protein levels were only observed in the resistant isolate. Heterologous expression of Axc in Escherichia coli significantly increased the resistance towards carbapenems. Importantly, direct Axc mediated hydrolysis of imipenem was demonstrated using pH shift assays and 1H-NMR, confirming Axc as a legitimate carbapenemase. Whole genome sequencing revealed that the susceptible and resistant isolates were remarkably similar.\n\nTogether these findings provide a molecular context for the fast development of meropenem resistance in A. xylosoxidans during treatment and demonstrate the use of mass spectrometric techniques in identifying novel resistance determinants.

microbiology↗