Enzymatic engineering of polysialic acid on cells in vitro and in vivo using a purified bacterial polysialyltransferase

In vertebrates, polysialic acid (PSA) is typically added to the neural cell adhesion molecule (NCAM) in the Golgi by PST or STX polysialyltransferase. PSA promotes plasticity, and its enhanced expression by viral delivery of the PST or STX gene has been shown to promote cellular processes that are useful for repair of the injured adult nervous system. Here we demonstrate a new strategy for PSA induction on cells involving addition of a purified polysialyltransferase from Neisseria meningitidis (PSTNm) to the extracellular environment. In the presence of its donor substrate (CMP-Neu5Ac), PSTNm synthesized PSA directly on surfaces of various cell types in culture, including Chinese hamster ovary cells, chicken DF1 fibroblasts, primary rat Schwann cells, and mouse embryonic stem cells. Similarly, injection of PSTNm and donor in vivo was able to produce PSA in different adult brain regions, including the cerebral cortex, striatum, and spinal cord. PSA synthesis by PSTNm requires the presence of the donor CMP-Neu5Ac, and the product could be degraded by the PSA-specific endoneuraminidase-N. Although PSTNm was able to add PSA to NCAM, most of its product was attached to other cell surface proteins. Nevertheless, the PSTNm-induced PSA displayed the ability to attenuate cell adhesion, promote neurite outgrowth, and enhance cell migration as has been reported for endogenous PSA-NCAM. Polysialylation by PSTNm occurred in vivo in less than 2.5 h, persisted in tissues, and then decreased within a few weeks. Together these characteristics suggest that a PSTNm-based approach may provide a valuable alternative to PST gene therapy.Peer reviewed: YesNRC publication: Ye

Genotypes of 14 reference isolates (_ref) and 21 geographical isolates of <i>Pl</i>. <i>halstedii</i> for 22 KASP markers based on effector gene SNPs.

<p>The DNA base involved in polymorphism is indicated by a specific colour. Heterozygous DNA bases are separated with a slash. (–) sign corresponds to the absence of the indel (versus T in PhRXLR58_1). *Genomic sequences of effectors available for these pathotypes (S1_File). Not determined results: nd.</p

Workflow of the study.

<p>In grey, the procedure used to select candidate effector genes. Transcriptomic data from sunflower leaves infected by <i>Pl</i>. <i>halstedii</i> or isolated zoospores were analyzed by PSI-tBLASTn using the annotated sequences that were available in NCBI in March 2010 as models for RXLR and CRN effectors. In white, the procedure used to build the <i>Pl</i>. <i>halstedii</i> pathotype determination key. Identification of effector polymorphism was done by comparisons between genomic sequences obtained in 7 representative pathogen pathotypes (100, 300, 304, 334, 700, 703 and 710). Among the 22 <u>K</u>Bioscience <u>C</u>ompetitive <u>A</u>llele <u>S</u>pecific <u>P</u>CR (KASP) markers, eight were used in a determination key to discriminate <i>Pl</i>. <i>halstedii</i> pathotypes.</p

Polymorphism analysis of <i>Pl</i>. <i>halstedii</i> effector and non-effector genes.

<p>(A) Mean nucleotide diversity (π) calculated on non-effector genes (black bar) and effector genes (grey bar). Π values were calculated on DnaSP v5 software. Error bars represent two SEM. (B, C) Comparisons of polymorphism distributions (represented by SNP sparseness, i.e. minimum average distance between two polymorphisms) in <i>Pl</i>. <i>halstedii</i> effector (grey) and non-effector genes (black). (B) Frequency of genes in each class of index. (C) Frequency of predicted peptides in each class of SNP sparseness, among genes with nucleotide polymorphism.</p

Identification key for <i>Pl</i>. <i>halstedii</i> pathotypes using KASP markers designed on effector gene SNPs.

<p>First level (Key 1) separated the 14 <i>Pl</i>. <i>halstedii</i> pathotypes in 5 groups with 3 markers. Second level (Key 2) used five other markers to distinguish 12 subgroups of pathotypes, and especially 6 subgroups of multi-isolate pathotypes (Red boxes). 100 and 304, 314 and 714 pathotypes could not be distinguished. ¹<i>Pl</i>. <i>halstedii</i> pathotypes with only one isolate available.</p

<i>Plasmopara halstedii</i> geographical isolates used in this study.

<p><i>Plasmopara halstedii</i> geographical isolates used in this study.</p