Leonhard, T. | bioRxiv Science

bioRxiv2024

Discovery of a pentose as a cytosine nucleobase modification in Shewanella phage Thanatos-1 genomic DNA mediating enhanced resistance towards host restriction systems

Co-evolution of bacterial defense systems and phage counter defense mechanisms has resulted in an intricate biological interplay between bacteriophages and their prey. To evade nuclease-based mechanisms targeting the DNA, various bacteriophages modify their nucleobases, which impedes or even inhibits recognition by endonucleases. We found that Shewanella phage Thanatos-1 DNA is insensitive to multiple restriction enzymes and, partially, also to Cas I-Fv and Cas9 cleavage. Furthermore, the phage genome shows strongly impaired basecalling with nanopore sequencing. We characterised the phage adenine methyltransferase TH1_126 in methylase-free E. coli ER3413 and derived and confirmed its recognition motif 5-ATC-3. Moreover, the data pointed to an additional, much more substantial nucleobase modification. Using LC-MS, we identified a deoxypentose of unknown configuration attached to cytosine as a yet undiscovered phage DNA modification, which is present in Thanatos-1 genomic DNA, likely mediates the observed resistance to restriction endonucleases, as well as a strong reduction in Cas nuclease activity. To elucidate the underlying enzyme functions, we determined structural homologs of Thanatos-1 proteins among known glycosyltransferase folds and experimentally proved a UDP-xylose pyrophosphorylase function of phage protein TH1_063 by in vitro enzyme assays.

molecular biology↗

bioRxiv2023

Ciliopathy-associated missense mutations in IFT140 are hypomorphic and have edgetic effects on protein interaction networks

The mechanisms underlying recessive Mendelian diseases and the interplay between genotype and phenotype still need to be better understood. It is therefore necessary to characterise the functional effects of missense mutations at the protein level. Here we focus on missense mutations in the intraflagellar transport protein IFT140, which forms part of the IFT complex A (IFT-A), a crucial component of the ciliary machinery. Mutations in IFT140 can cause a vast spectrum of diseases belonging to the group of ciliopathies, reaching from isolated retinal dystrophy to severe skeletal abnormalities and multi-organ diseases such as Mainzer-Saldino and Jeune syndrome. We hypothesise that missense mutations in IFT140 are hypomorphic leading to quantitative effects on a subset of protein-protein interactions. This may affect complex stability as well as perturbations of protein interaction networks. In this work we assessed how 24 missense mutations in IFT140 affect interactions with other IFT and effector proteins using affinity purification coupled to mass spectrometry. Our data reveals that several mutations in IFT140 are hypomorphic and disrupt the stability of the IFT-A complex to varying degrees in a quantitative way. Allelic combination and the degree of IFT-A complex disruption in analysed missense mutations correlates with the severity of the observed phenotype in a subset of patients. In addition, we show that a distinct subset of mutations in IFT140 shows edgetic effects by disrupting specific PPIs rather than causing a total loss of IFT-A binding. This is the case e.g. with the disease-associated protein TULP3 which is involved in cilia-dependent sonic hedgehog signalling.

cell biology↗