Untersuchung der Funktion und des Glykosylierunsgprozesses der LTA in Listeria monocytogenes
Stoffwechselphysiologie, Biochemie und Genetik der Mikroorganismen
Zusammenfassung der Projektergebnisse
Lipoteichoic acid (LTA) is a cell wall polymer of Gram-positive bacteria that is attached to the membrane via a glycolipid linker. The synthesis of LTA begins in the cytoplasm of the cell with the synthesis of the glycolipid linker, which is subsequently flipped across the membrane. In L. monocytogenes, the LTA primase LtaP attaches the first glycerol-phosphate unit (GroP) which is then extended by the LTA synthase LtaS. The LTA structure can then be modified with D-alanine and sugar residues. Enzymes encoded in the dlt operon are responsible for the D-alanylation of LTA, however, enzymes involved in the glycosylation of LTA are either not known or barely studied. Recently, GtlA has been identified in L. monocytogenes as the putative glycosyltransferase, that produces a C55-galactose-intermediate. This sugar-lipid intermediate is subsequently transported across the membrane by an unknown flippase. I could show that GtcA, a member of the GtrA protein family, is important for the glycosylation of LTA in L. monocytogenes and in the model organism Bacillus subtilis. It was previously speculated, that GtcA might act as a flippase in the glycosylation process of wall teichoic acid (WTA), a second cell wall polymer of Gram-positive bacteria which is covalently linked to the peptidoglycan. I hypothesize that GtcA is responsible for the flipping of the sugar-lipid intermediate for both, glycosylation of LTA and WTA. However, biochemical experiments are necessary to confirm this hypothesis. After the C55-P-sugar intermediate is flipped across the membrane, the sugar residue will be transferred to the LTA backbone by a glycosyltransferase with extracellular activity, which appears to be a member of the GT-C family of glycosyltransferases. In the course of the project, I screened the genome of L. monocytogenes for membrane proteins that possess 8-14 transmembrane helices, a large outside loop of at least 50 amino acids and a DxD motif in the extracellular loop, typical criteria for GT-C enzymes. This analysis led to the identification of the putative glycosyltransferase GtlB. Absence of GtlB in L. monocytogenes resulted in the lack of galactose modifications on LTA. Furthermore, we could identify a DxxD motif in the extracellular loop of GtlB, which is essential for its activity. However, biochemical evidence for the transfer of sugar residues onto LTA by GtlB remains to be elucidated. As part of the project, I also investigated the role of FtsW and RodA homologs on the glycosylation of LTA due to the observation that L. monocytogenes encodes up to six homologs of these enzymes. FtsW and RodA act as glycosyltransferases and polymerize the glycan strand during peptidoglycan biosynthesis. However, none of these FtsW/RodA homologs are needed for the glycosylation of LTA. Further analysis of the function of five FtsW/RodA homologs led to the identification of two FtsW and three RodA enzymes, namely FtsW1 (Lmo1071), FtsW2 (Lmo2688), RodA1 (Lmo2427), RodA2 (Lmo2428) and RodA3 (Lmo2687). FtsW1 is essential for cell survival of L. monocytogenes, unless FtsW2 is overexpressed. Absence of one of the three RodA enzymes, RodA1, RodA2 or RodA3, had no impact on growth or cell length of L. monocytogenes. However, simultaneous deletion of rodA1 and rodA3 resulted in the formation of shorter cells. I was unable to generate an L. monocytogenes strain that lacks all three rodA genes, suggesting that one of the RodA enzymes needs to be present for survival. Further experiments that were conducted suggested that the activity of the five FtsW/RodA enzymes needs to be tightly regulated to maintain cell growth, cell shape and antibiotic resistance. Due to their importance for both, cell survival and antibiotic resistance, FtsW and RodA enzymes can be potentially used as targets for the development of new antimicrobials.
Projektbezogene Publikationen (Auswahl)
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(2018) Discovery of genes required for lipoteichoic acid glycosylation predicts two distinct mechanism for wall teichoic acid glycosylation. J Biol Chem. 293(9):3293-3306
Rismondo J, Percy MG, and Gründling A.
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(2018) Investigation of the phosphorylation of Bacillus subtilis LTA synthases by the serine/threonine kinase PrkC. Sci Rep. 8(1):17344
Pompeo F, Rismondo J, Gründling A, and Galinier A
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(2019) Cell shape and antibiotic resistance is maintained by the activity of multiple FtsW and RodA enzymes in Listeria monocytogenes. mBio. 10(4)
Rismondo J, Halbedel S, and Gründling A.
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(2019) Phage resistance at the cost of virulence: Listeria monocytogenes serovar 4b requires galactosylated teichoic acids for InlB-mediated invasion. Plos Pathog. 15(10):e1008032
Sumrall ET, Shen Y, Keller AP, Rismondo J, Pavlou M, Eugster MR, Boulos S, Disson O, Thouvenot P, Kilcher S, Wollscheid B, Cabanes D, Lecuit M, Gründling A, and Loessner MJ
