Biology subjects

Titz, A.

Publications and source records attributed to Titz, A..

6 recordsLinked to original sources

bioRxiv2026

The Bacterial Kinase AnmK Integrates into Tick Genome and Biology

Horizontal Gene Transfer (HGT) contributes to eukaryotic evolution, potentially bringing phenotypic novelties to the recipient organisms. Ticks pose a severe threat to human health as vectors of various pathogens, including viruses, bacteria and protozoa, while stably hosting endosymbiotic bacteria. As such, these obligate blood-feeding parasites have been and continue to be exposed to HGT from a broad range of donors. To determine whether bacterial-to-tick HGT has contributed to important tick traits, we surveyed Ixodes tick genomes for HGT events. We revealed duplications of the known bacteria-derived gene dae2 and discovered two novel cases of bacterial HGT, the most remarkable of which involves a bacterial peptidoglycan metabolic gene (anmK) acquired by the common ancestor of ticks. The acquisition of an intron demonstrates "eukaryotization" of anmK within tick genomes. Transcript profiling revealed that anmK expression is upregulated during blood feeding, peaking in female ovaries, a niche occupied by horizontally acquired endosymbionts. Biochemical analysis confirmed that, to some extent, recombinant AnmK retains kinase activity on its cognate substrate - the bacterial cell wall component 1,6-anhydro-N-acetylmuramic acid. Immunolocalization showed that the enzyme is predominantly localized towards outer layers of the vitellogenic oocytes. Silencing of anmK in different tick species compromised blood-feeding and reproduction, demonstrating that this domesticated bacterial enzyme underpins reproductive fitness across tick species. Our findings exemplify the ability of horizontally acquired genes to integrate into the host biology and thereby shape host life history.

evolutionary biology↗

bioRxiv2024

A fucose-binding superlectin from Enterobacter cloacae with high Lewis and ABO blood group antigen specificity

Bacteria frequently employ carbohydrate-binding proteins, so-called lectins, to colonize and persist in a host. Thus, bacterial lectins are attractive targets for the development of new antiinfectives. To find new potential targets for antiinfectives against pathogenic bacteria, we searched for homologs of Pseudomonas aeruginosa lectins and identified homologs of LecA in Enterobacter species. Here, we recombinantly produced and biophysically characterized a homolog that comprises one LecA domain and one additional novel protein domain. This protein was termed Enterobacter cloacae lectin A (EclA) and found to bind L-fucose. Glycan array analysis revealed a high specificity for the LewisA antigen and the type II H-antigen (blood group O) for EclA, while related antigens LewisX, Y, and B as well as blood group A or B were not bound. We developed a competitive binding assay to quantify blood group antigen binding specificity in solution. Finally, the crystal structure of EclA could be solved in complex with methyl -L-selenofucoside. It revealed the unexpected binding of the carbohydrate ligand to the second domain, which comprises a novel fold that dimerizes via strand-swapping resulting in an intertwined beta sheet.

microbiology↗

bioRxiv2022

The Glycan-Specificity of the Pineapple Lectin AcmJRL and its Carbohydrate-Dependent Binding of the SARS-CoV-2 Spike Protein

The current SARS-CoV-2 pandemic has become one of the most challenging global health threats, with over 530 million reported infections by May 2022. In addition to vaccines, research and development have also been directed towards novel drugs. Since the highly glycosylated spike protein of SARS-CoV-2 is essential for infection, it constitutes a prime target for antiviral agents. The pineapple-derived jacalin-related lectin (AcmJRL) is present in the medication bromelain in significant quantities and has previously been described to bind mannosides. Here, we elucidated its ligand specificity by glycan array analysis, quantified the interaction with carbohydrates and validated high-mannose glycans as preferred ligands. Because the SARS-CoV-2 spike protein was previously reported to carry a high proportion of high-mannose N-glycans, we tested the binding of AcmJRL to recombinantly produced spike protein. We could demonstrate that AcmJRL binds the spike protein with a low micromolar KD in a carbohydrate-dependent fashion, suggesting its use as a potential SARS-CoV-2 neutralising agent.

biophysics↗

bioRxiv2021

NamZ1 and NamZ2 from the oral pathogen Tannerella forsythia are peptidoglycan processing exo-β-N-acetylmuramidases with distinct substrate specificity

The Gram-negative periodontal pathogen Tannerella forsythia is inherently auxotrophic for N-acetylmuramic acid (MurNAc), which is an essential carbohydrate constituent of the peptidoglycan (PGN) of the bacterial cell wall. Thus, to build up its cell wall, T. forsythia strictly depends on the salvage of exogenous MurNAc or sources of MurNAc, such as polymeric or fragmentary PGN, derived from cohabiting bacteria within the oral microbiome. In our effort to elucidate how T. forsythia satisfies its demand for MurNAc, we recognized that the organism possesses three putative orthologs of the exo-{beta}-N-acetylmuramidase BsNamZ from Bacillus subtilis, which cleaves non-reducing end, terminal MurNAc entities from the artificial substrate pNP-MurNAc and the naturally-occurring disaccharide substrate MurNAc-{beta}-1,4-N-acetylglucosamine (GlcNAc). TfNamZ1 and TfNamZ2 were successfully purified as soluble, pure recombinant His6-fusions and characterized as exo-lytic {beta}-N-acetylmuramidases with distinct substrate specificities. The activity of TfNamZ1 was considerably lower compared to TfNamZ2 and BsNamZ, in the cleavage of pNP-MurNAc and MurNAc-GlcNAc. When peptide-free PGN glycans were used as substrates, we revealed striking differences in the specificity and mode of action of these enzymes, as analyzed by mass spectrometry. TfNamZ1, but not TfNamZ2 or BsNamZ, released GlcNAc-MurNAc disaccharides from these glycans. In addition, glucosamine (GlcN)-MurNAc disaccharides were generated when partially N-deacetylated PGN glycans from B. subtilis 168 were applied. This characterizes TfNamZ1 as a unique disaccharide-forming exo-lytic {beta}-N-acetylmuramidase (exo-disaccharidase), and, TfNamZ2 and BsNamZ as sole MurNAc monosaccharide-lytic exo-{beta}-N-acetylmuramidases. IMPORTANCETwo exo-{beta}-N-acetylmuramidases from T. forsythia belonging to glycosidase family GH171 (www.cazy.org) were shown to differ in their activities, thus revealing a functional diversity within this family: NamZ1 releases disaccharides (GlcNAc-MurNAc/GlcN-MurNAc) from the non-reducing ends of PGN glycans, whereas NamZ2 releases terminal MurNAc monosaccharides. This work provides a better understanding of how T. forsythia may acquire the essential growth factor MurNAc by the salvage of PGN from cohabiting bacteria in the oral microbiome, which may pave avenues for the development of anti-periodontal drugs. On a broad scale, our study indicates that the utilization of PGN as a nutrient source, involving exo-lytic N-acetylmuramidases with different modes of action, appears to be a general feature of bacteria, particularly among the phylum Bacteroidetes.

microbiology↗

bioRxiv2021

Exo-β-N-acetylmuramidase NamZ of Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidase

Endo-{beta}-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that potentially lyse bacterial cells. They catalyze an endo-lytic cleavage of the peptidoglycan, the structural component of the bacterial cell wall; i.e. they hydrolyze glycosidic N-acetylmuramic acid (MurNAc)-{beta}-1,4-N-acetylglucosamine (GlcNAc)-bonds within the heteroglycan backbone of peptidoglycan. In contrast, little is known about exo-{beta}-N-acetylmuramidases, catalyzing an exo-lytic cleavage of {beta}-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We identified ybbC of B. subtilis, renamed namZ, as encoding the reported exo-{beta}-N-acetylmuramidase. A {Delta}namZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl {beta}-MurNAc and the peptidoglycan-derived disaccharide MurNAc-{beta}-1,4-GlcNAc. Together with the exo-{beta}-N-acetylglucosaminidase NagZ and the exo-muramoyl-L-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. NamZ is a member of the DUF1343 protein family of unknown function and shows no significant sequence identity with known glycosidases. A structural model of NamZ revealed a putative active site located in a cleft within the interface of two subdomains, one of which constituting a Rossmann-fold-like domain, unusual for glycosidases. On this basis, we propose that NamZ represents the founding member of a novel family of peptidoglycan hexosaminidases, which is mainly present in the phylum Bacteroidetes and, less frequently, within Firmicutes (Bacilli, Clostridia), Actinobacteria and Gammaproteobacteria.

microbiology↗

bioRxiv2019

Induction of Rare Conformation of Oligosaccharide by Binding to Calcium-dependent Bacterial Lectin: X-ray Crystallography and Modelling Study

Pathogenic micro-organisms utilize protein receptors in adhesion to host tissues, a process that in some cases relies on the interaction between lectin and human glycoconjugates. Oligosaccharide epitopes are recognized through their three-dimensional structure and their flexibility is a key issue in specificity. In this paper, we analyse by X-ray crystallography the structures of the lectin LecB from two strains of Pseudomonas aeruginosa in complex with Lewis x oligosaccharide present on cell surfaces of human tissues. An unusual conformation of the glycan was observed in all binding sites with a non-canonical syn orientation of the N-acetyl group of N-acetyl-glucosamine. A PDB-wide search revealed that such an orientation occurs only in 2% of protein/carbohydrate complexes. Theoretical chemistry calculations showed that the observed conformation is unstable in solution but stabilised by the lectin. A reliable description of LecB/Lewis x complex by force field-based methods had proven as especially challenging due to the special feature of the binding site, two closely apposed Ca2+ ions which induce strong charge delocalisation. By comparing various force-field parametrisations, we design general protocols which will be useful in near future for designing carbohydrate-based ligands (glycodrugs) against other calcium-dependent protein receptors.

biophysics↗