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Exploring the role of Campylobacter. coli GT-42 enzymes on LOS biosynthesis
- 1. Eläinlääketieteellinen tiedekunta Associate Professor Mirko Rossi CHRO 2017, Nantes ”Pathogenicity and Virulence factors” Exploring the role of C. coli GT-42 enzymes on LOS biosynthesis 12/09/2017CHRO2017/ Mirko Rossi 1
- 2. Eläinlääketieteellinen tiedekunta • Are sialyltransferases involved in the biosythesis of several LOS structures in C. jejuni related to ganglioside mimicry • Associated with the development of GBS • In C. jejuni: Cst-I, Cst-II and Cst-III have been characterized as mono/bi alfa- 2,3/alfa-2,8 sialyltransferases • GT-42 genes were detected also in C. coli (Skap-De Haan et al., 2014) but the function is still unknown 12/09/2017CHRO2017/ Mirko Rossi 2 Glycosyltransferases family 42 in Campylobacter LOS locus class A of C. jejuni Cst-II Gangliosides
- 3. Eläinlääketieteellinen tiedekunta • BLAST C. jejuni sialyltransferases vs NCBI nr • 7 phylogenetically distinct glycosyltransferases GT-42 in C. coli • Clustered into two monophyletic clusters: • C. jejuni and C. coli GT-42 • C. coli specific GT-42 12/09/2017CHRO2017/ Mirko Rossi 3 Campylobacter coli GT-42 NJ phylogeny of nt sequences of GT-42 homologues found in C. coli Skap-De Haan et al., 2014 Culebro et al., 2016 Richard et al., 2013
- 4. Eläinlääketieteellinen tiedekunta • 2574 genomes from ENA mapped against the 7 C. coli GT-42 groups and neuB using ReMatCh • 818 genomes possess one or multiple GT- 42 genes • C. coli-specific GT-42 are dominant; present in Clade 1a and Clade 3 • neuB is frequently not detected in GT-42 positive strains 12/09/2017CHRO2017/ Mirko Rossi 3 Frequency of GT-42 in C. coli Phylogeny based on atpA genes hierBAPS clustering (inner ring) Clade 3 Clade 2 Clade1bc Clade1a
- 5. Eläinlääketieteellinen tiedekunta LOS locus classes in GT-42 positive C. coli • 818 GT-42 + C. coli genomes mapped vs genes of C. coli LOS locus classes I to XII using ReMatCh (~100 genes) • LOS locus class I, II, III and IX; in addition to unknown classes • Strains having C. coli-specific GT-42 II, III and IX 12/09/2017CHRO2017/ Mirko Rossi 5 LOS locus class IX LOS locus class III LOS locus class II Cst-V Cst-IV Cst-VI Culebro et al., 2016
- 6. Eläinlääketieteellinen tiedekunta Role of GT-42 in C. coli LOS biosynthesis 12/09/2017CHRO2017/ Mirko Rossi 6 cst-V neuA LOS locus class IX LOS locus class III LOS locus class II cst-IV cst-VI neuCneuB C. coli 76339 (Clade 3) • LOS locus class IX: Cst-V • Contains also Cst-I on CPS • NANA detected by Dionex C. coli 65 (Clade 1a) • LOS locus class II: Cst-IV • Does not contain neuB • NANA not detected on LOS by ms/ms Skap-De Haan et al., 2014 Culebro et al., 2016 pseudogene
- 7. Eläinlääketieteellinen tiedekunta Cst-V is involved in LOS biosynthesis but not Cst-I 12/09/2017CHRO2017/ Mirko Rossi 7 WT ΔcstV→ ΔcstV← GGT16SrRNA cst-V CAT GGT WT ΔcstVΔggt::cstV Deletion of cstV induce a change in the electrophoresis motility of LOS in SDSPAGE Phenotype is partially restored after insertion of cstV within ggt locus cst-I … CPS locuscst-V neuALOS locus class IX neuCneuB C. coli 76339 Deletion of cstI does not produce any changes in the LOS
- 8. Eläinlääketieteellinen tiedekunta NeuB is involved in LOS biosynthesis 12/09/2017CHRO2017/ Mirko Rossi 8 cst-I … CPS locus Deletion of neuB produce same change in the electrophoresis motility of LOS in SDS- PAGE as observed in ΔcstV cst-V neuALOS locus class IX neuCneuB C. coli 76339 C. coli 76339 C. jejuni 81-176 LOS of C. coli 76339 isn’t sensitive to the action of C. perfringens neuroaminidase, indicating that NANA (=Sialic Acid), if present, is not terminal
- 9. Eläinlääketieteellinen tiedekunta Cst-IV is involved in LOS biosynthesis 12/09/2017CHRO2017/ Mirko Rossi 9 LOS locus class II cst-IV C. coli 65 Deletion of cstIV induce a change in the electrophoresis motility of LOS in SDS- PAGE neuB2 … Flagella locus WT WTΔcstIV→ … neuB3 Missing of NANA biosynthesis genes possible role of Leg and Pse Deletion of neuB2 (Leg) does not change LOS phenotype. Deletion of neuB3 (Pse) was unsuccessful (lethal?)
- 10. Eläinlääketieteellinen tiedekunta Testing in vitro activity of C. coli GT-42 • Enzymatic activity using: • DONOR CMP-NANA • ACCEPTORS BODIPY or FCHASE-labeled Lac, LacNAc, Sialyl-Lactose, alpha/beta-GalNAc, GM3, alpha-Gal, beta-GlcNAc, LewisX • Mobility on TLC • Enzyme: • Native using the cell extracts of WTs and mutants • Overexpression using pCW and pCWmalET 12/09/2017CHRO2017/ Mirko Rossi 10
- 11. Eläinlääketieteellinen tiedekunta Testing in vitro activity of C. coli GT-42 12/09/2017CHRO2017/ Mirko Rossi 11 C. coli 76339 WT C. coli 76339 ΔcstV C. coli 76339 ΔcstI C. coli 65 Cst-I is very active on Lac, LacNAc, Sialyl-Lactose and alpha/beta-GalNAc Cst-V and Cst-IV are not active unknown donors and acceptors
- 12. Eläinlääketieteellinen tiedekunta Structural prediction of Cst-V and Cst-IV • Three- dimensional structures of Cst-V and Cst-IV were built with Phyre2 server • Both Cst-IV and Cst-V protein models were built with 100% confidence • Several substitutions at amino acids important for the interaction with CMP- NANA in Cst-II were identified 12/09/2017CHRO2017/ Mirko Rossi 12 Change of Cst-II activity Chiu et al., 2004
- 13. Eläinlääketieteellinen tiedekunta CONCLUSION • C. coli possess at least 7 types of glycosyltransferases belonging to CAZy family 42 probably involved both in LOS and CPS biosynthesis, clustering into two clusters: • C. jejuni and C. coli GT-42 (rarely found in the population) • C. coli specific GT-42 (most frequent) • C. coli specific GT-42 Cst-V and Cst-IV are involved in LOS biosynthesis of C. coli, but are unable to transfer NANA • C. coli specific GT-42 have acquired a different unknown function compared to others GT- 42 enzymes, diverging from C. jejuni sialyltransferase 12/09/2017CHRO2017/ Mirko Rossi 13
- 14. Eläinlääketieteellinen tiedekunta ACKNOWLEDGMENTS 12/09/2017CHRO2017/ Mirko Rossi 14 PhD student Alejandra Culebro Prof. Warren Wakarchuk, Ryerson University, Canada Prof. Emerita Marja-Liisa Hänninen (University of Helsinki, Finland) for providing the strains Dr. Michel Gilbert (NRC, Canada) for helping in the in vitro activity on FCHASE acceptors Dr. João Carriço and Miguel Machado (University of Lisbon, Portugal) for giving us the opportunity to use ReMatCh Funding: University of Helsinki Research grant MBDP doctoral program Walter Ehrströmin säätiö
- 15. Eläinlääketieteellinen tiedekunta 12/09/2017CHRO2017/ Mirko Rossi 15 THANKS FOR THE ATTENTION Contact me on twitter @happygipsy Slides are available on Slideshare If you are interested in ReMatCh: https://github.com/B-UMMI/ReMatCh
- 16. Eläinlääketieteellinen tiedekunta • Chiu et al. 2004. Structural analysis of the sialyltransferase CstII from Campylobacter jejuni in complex with a substrate analog. Nat Struct Mol Biol. 11(2):163-70 • Richards et al. 2013. Comparative characterization of the virulence gene clusters (lipooligosaccharide [LOS] and capsular polysaccharide [CPS]) for Campylobacter coli, Campylobacter jejuni subsp. jejuni and related Campylobacter species. Infect Genet Evol 14:200–213 • Skarp-de Haan, et al. 2014. Comparative genomics of unintrogressed Campylobacter coli clades 2 and 3. BMC Genomics 15:129 • Culebro et al. 2016. Large sequence diversity within biosynthesis locus and common biochemical features of Campylobacter coli lipooligosaccharides. J Bacteriol 198(20):2829-40. doi: 10.1128/JB.00347-16 (preprint http://dx.doi.org/10.1101/050328) • . 12/09/2017CHRO2017/ Mirko Rossi 16 REFERENCES
Editor's Notes
- Glycosyltransferases belonging to the family 42 as classified in the Cazy database, are sialyltransferases involved in the synthesis of LOS structure of C. jejuni related to ganglioside mimicry and associated with the development of the Guillain-Barré syndrome. Up to date three different GT-42 enzymes named Cst-I, Cst-II and Cst-III have been characterized in C. jejuni as monofunctional or bifunctional alpha-2,3 or 2,8 sialyltransferases. On the right a graphical representation of the biosynthesis of ganglioside-like structure on the LOS of C. jejuni expressing LOS locus class A which contains Cst-II. Our group and other have already detected orthologues of GT-42 enzymes in C. coli, but their role in the LOS biosynthesis in this species is still obscure. My presentation will focus on GT-42 enzyme in C. coli. I will present an overview of the types of GT-42 found in this species, their frequency in the population and their possible role in the LOS biosynthesis.
- Blasting the C. jejuni Csts against NCBI nr database, we retrieved 45 C. coli sequences forming 7 phylogenetically distinct GT-42 groups. They group into two separate monophyletic clusters: cluster A including C. jejuni orthologues Cst-I, Cst-II and Cst-III sialyltransferases, and cluster B which contains proteins exclusively found in C. coli including group 5, 6 and 7. Group 5 includes sequences similar to Cst-V while group 7 consists of sequences similar to Cst-IV, both GT-42 previously described in C. coli by our group.
- To investigate the frequency of the presence of GT-42 in C. coli population, we mapped all C. coli genomes currently available in ENA against the 45 sequences corresponding to the 7 GT-42 groups. We used ReMatCh for mining ENA and performing the mapping. The tree on the right represents the phylogeny of the strains based on atpA gene which clearly showed the division of the C. coli population in its main three monophyletic clades: Clades 1abc (red and violet), 2 (yellow) and 3 (green). As results, 818 over 2574 genomes possess one or multiple GT-42 genes. C. coli specific GT-42 genes of cluster B are more frequently detected than genes of GT-42 cluster A. In addition, GT-42 are practically restricted to Clade 1a and Clade3 C. coli strains. It is also interesting to notice that although NANA synthesis gene neuB is frequently found in C. coli, it is often absent in those strains possessing GT-42 enzyme of cluster B.
- We further analyzed the GT-42 positive genomes for predicting which type of LOS locus classes were linked to the detected GT-42 genes. Using ReMatCh we mapped the reads of the 818 genomes against all the genes of the known 11 C. coli LOS locus classes. We then summarized the results by showing the percentage of positive map of genes for each class in the two heatmaps; here showed on the right. The upper heatmap displays the results for all the 818 genomes, showing that the majority of the strains have predicted LOS classes I, II, III and IX and few have possibly novel classes (click). Limiting the analysis to those genomes possessing the C. coli specific GT-42 of the cluster B (lower heatmap), strains are positive for LOS locus class II, III and IX. Specifically Cst-V is restricted to class IX, Cst-VI to class III and Cst-IV to class II.
- A schematic representation of the three LOS locus classes containing C. coli specific GT-42 are represented here. LOS class IX is the only one including also the three genes involved in the biosynthesis of Sialic Acid; in yellow. (click) Since the Cst-VI of LOS class III is likely a pseudogene (at least in the genomes we did manually check) we focused on the other two classes (click) selecting two strains. Cst-V positive strain C. coli 76339 belonging to Clade 3 which possesses also a GT-42 of cluster A in the capsule locus and detectable NANA on phenol extracted LOS using Dionex. We also selected the Cst-IV positive C. coli 65 strain which is characterized by the absence of NANA biosynthesis locus and for which NANA was not detected using ms/ms. We then performed mutational studies to investigate the possible role of Cst-V and Cst-IV in the biosynthesis of LOS. We also performed in vitro activity test to investigate the functionalities of these enzymes on several acceptors.
- Deleting cstV (click) by inserting the cassette either in the direction of the gene or in opposite direction, we observed a clear effect on the electrophoresis motility of LOS in SDSPAGE. (click) On the contrary, the deletion of Cst-I does not produce any detectable changes in the LOS. (click) The phenotype was partially restored after inserting the cstV within ggt gene under the its promoter, confirming that cstV is involved in decorating the LOS of C. coli 76339.
- (click) Similar changes in the electrophoresis motility of LOS of C. coli 76339 in SDS-PAGE have been showed after deleting neuB (the NANA synthase), indicating that most likely the LOS have been decorating with NANA. (click) Since the LOS of C. coli 76339 isn’t sensitive to the action of Clostridium perfringens neuraminidase, sialic acid is probably not a terminal sugar residue on the oligosaccharide chain, as also shown for C. jejuni 11168, or alternatively CstV transfers a sialidase resistant unidentified nonulosonate
- As observed for LOS class IX, deleting cstIV (click) by inserting the cassette in the same direction of the gene, we observed a clear effect on the electrophoresis motility in SDS-PAGE of the LOS of the strain C. coli 65. However, (click) differently from C. coli 76339, C. coli 65 does not possess NANA biosynthesis genes. We therefore investigated the possible role of other synthases locate in the Flagella region involved in the production of other nonulosonic acids such as Legionaminic and Pseudaminic acid. (click) Deletion of neuB2 does not produce any changes in the LOS, while it was not possible to obtain neuB3 mutants. Remain therefore unknown the possible substrate of cstIV and the type of decoration of the LOS class II.
- To compare with well characterized GT-42 enzymes, we performed enzymatic activity of C. coli GT-42 Cst-I, Cst-V and Cst-IV using CMP-NANA as donor and a long list of BODIPY or FCHASE labeled acceptors found to be substrates for GT-42 enzymes. We tested using both native proteins obtained from cell extract of C. coli strains and after overexpressing the proteins using pCW and pCWmalET as vectors.
- Here is shown the TLC mobility assay on the three major acceptors for C. jejuni Csts (Lac, LacNac and 3’Sialyllactose) tested using native proteins from cell extracts. C. coli 76339 showed activity on these acceptors due to action of Cst-I which resulted also very active on alpha and beta-GalNAc. On the contrary, C. coli specific GT-42 Cst-V and Cst-IV does not show any activity in any acceptors using CMP-NANA as donor. This results clearly showed that C. coli GT-42 enzymes of cluster B have functionally diverged from other characterized GT-42 by being specific for unknown acceptors (Cst-V) or both unknown donor and acceptors (Cst-IV).
- Comparing the predicted structure of Cst-IV and Cst-V with C. jejuni Cst-II we identified several amino acid substitutions that have demonstrated to be important in the interaction between Cst-II and CMP-NANA, supporting the theory of different donors (especially for Cst-IV).
- In conclusion. At least 7 types of glycosyltransferases belonging to CAZy family 42 have been found in C. coli. These enzymes are probably involved both in LOS and CPS biosynthesis. They form two major clusters. Cluster A including both C. jejuni and C. coli sialyltransferases which are relative infrequent in the C. coli population; and Cluster B composed by glycosyltranferases involved exclusively in the LOS biosynthesis of C. coli which are quite common among the available strains. Our study showed that C. coli specific GT-42 Cst-V and Cst-IV have acquired different unknown function (in term of donor and/or acceptor specificity) compared to others GT-42 enzymes, diverging from C. jejuni sialyltransferases. The possible implications for the ecology of this species and virulence are objectives for ongoing and future studies.