Role of Sialyl-O-Acetyltransferase CASD1 on GD2 Ganglioside O-Acetylation in Breast Cancer Cells.

The O-acetylated form of GD2, almost exclusively expressed in cancerous tissues, is considered to be a promising therapeutic target for neuroectoderm-derived tumors, especially for breast cancer. Our recent data have shown that 9-O-acetylated GD2 (9-OAcGD2) is the major O-acetylated ganglioside species in breast cancer cells. In 2015, Baumann et al. proposed that Cas 1 domain containing 1 (CASD1), which is the only known human sialyl-O-acetyltransferase, plays a role in GD3 O-acetylation. However, the mechanisms of ganglioside O-acetylation remain poorly understood. The aim of this study was to determine the involvement of CASD1 in GD2 O-acetylation in breast cancer. The role of CASD1 in OAcGD2 synthesis was first demonstrated using wild type CHO and CHOΔCasd1 cells as cellular models. Overexpression using plasmid transfection and siRNA strategies was used to modulate CASD1 expression in SUM159PT breast cancer cell line. Our results showed that OAcGD2 expression was reduced in SUM159PT that was transiently depleted for CASD1 expression. Additionally, OAcGD2 expression was increased in SUM159PT cells transiently overexpressing CASD1. The modulation of CASD1 expression using transient transfection strategies provided interesting insights into the role of CASD1 in OAcGD2 and OAcGD3 biosynthesis, and it highlights the importance of further studies on O-acetylation mechanisms.

The sialyl-O-acetylesterase NanS of Tannerella forsythia encompasses two catalytic modules with different regiospecificity for O7 and O9 of sialic acid.

The periodontal pathogen Tannerella forsythia utilizes host sialic acids as a nutrient source. To also make O-acetylated sialyl residues susceptible to the action of its sialidase and sialic acid uptake system, Tannerella produces NanS, an O-acetylesterase with two putative catalytic domains. Here, we analyzed NanS by homology modeling, predicted a catalytic serine-histidine-aspartate triad for each catalytic domain and performed individual domain inactivation by single alanine exchanges of the triad nucleophiles S32 and S311. Subsequent functional analyses revealed that both domains possess sialyl-O-acetylesterase activity, but differ in their regioselectivity with respect to position O9 and O7 of sialic acid. The 7-O-acetylesterase activity inherent to the C-terminal domain of NanS is unique among sialyl-O-acetylesterases and fills the current gap in tools targeting 7-O-acetylation. Application of the O7-specific variant NanS-S32A allowed us to evidence the presence of cellular 7,9-di-O-acetylated sialoglycans by monitoring the gain in 9-O-acetylation upon selective removal of acetyl groups from O7. Moreover, we established de-7,9-O-acetylation by wild-type NanS as an easy and efficient method to validate the specific binding of three viral lectins commonly used for the recognition of (7),9-O-acetylated sialoglycans. Their binding critically depends on an acetyl group in O9, yet de-7,9-O-acetylation proved advantageous over de-9-O-acetylation as the additional removal of the 7-O-acetyl group eliminated ligand formation by 7,9-ester migration. Together, our data show that NanS gained dual functionality through recruitment of two esterase modules with complementary activities. This enables Tannerella to scavenge 7,9-di-O-acetylated sialyl residues and provides a novel, O7-specific tool for studying sialic acid O-acetylation.

Phenotypic Diversification of Microbial Pathogens-Cooperating and Preparing for the Future.

Recent studies revealed an amazing phenotypic heterogeneity between genetically identical individual cells within populations of microbial pathogens. During the course of an infection, subpopulations occur, which differ in certain virulence-relevant factors, stress adaptation functions or physiological and metabolic abilities. The mechanisms driving this heterogeneity are divergent reactions of the pathogens to differences in host tissue microenvironments. In addition, certain genetic regulatory circuits with positive feedback loops and stochastic differences in gene expression can generate endogenous fluctuations in regulatory components leading to bistable expression of virulence-associated functions. Here, we focus on the occurrence of phenotypic heterogeneity in populations of well-studied examples of pathogens, which enables cooperative, social behavior where a subpopulation of producers shares fitness- and/or virulence-relevant goods and traits with non-producers. We further highlight that this strategy allows preadaptation of a subgroup of cells to recurrent and thus predictable changes of the environment that they encounter during the different stages of the infection. The diversity within bacterial communities has a significant influence on the survival of the pathogens within their hosts and the progression of the disease.