Novel antibiotics from symbiotic peptides

Transcription

1 HU-NO Research conference and Knowledge exchange Novel antibiotics from symbiotic peptides Eva Kondorosi Biological Research Centre Hungarian Academy of Sciences Medicago truncatula-sinorhizobium meliloti symbiosis

2 Endosymbionts Nitrogen fixing root nodules Zone I. Meristem Zone II. Cellular infection Symbiotic cell differentiation Interzone II-III The most spectacular differentiation events Zone III. Nitrogen Fixation Aging of nodule cells Zone IV. Senescence

3 Differentiation of endosymbionts is provoked by the host plant Sinorhizobium meliloti Free-living Plant factors ~ 800 symbiotic peptides Van de Velde et al. 2010, Science Nitrogen fixing bacteroids Polyploid, Elongated and branched cells Altered cell envelope Higher membrane permeability Non-cultivable Irreversible differentiation Mergaert et al. 2006, PNAS

4 The largest family of plant effectors : NCRs (>700 genes) Nodule-specific Cysteine-Rich peptides (4 Cys) SIGNAL PEPTIDE CLEAVAGE SITE MATURE PEPTIDE (30-55 aa) X 2-7 -C 1 -X 5-6 -C 2 -X C 3 -X 4 -C 4 -X n Nodule-specific Cysteine-Rich peptides (6 Cys) X C 1 -X 5 -C 2 -X 1-8 -C 3 -X 5-8 -C 4 -X C 5 -X 1 -C 6 -X n SIGNAL PEPTIDE CLEAVAGE SITE MATURE PEPTIDE (30-55 aa) Isoelectric point: NCR peptides are delivered to the endosymbionts by the secretory pathway and ~200 peptides were found in the cytosol of bacteria

5 Control Figure NCR M.W p. I. A No treatment B C D E H I J L N U X

6 NCR247: multiple bacterial targets FITC-NCR2477 Enters the bacterial cytosol Binding to FtsZ inhibits FtsZ polymerization and FtsZ ring formation: No cell division Impact on transcription and translation: - decreased expression of ribosomal protein genes - interaction with several ribosomal proteins - inhibition of protein synthesis In vitro: Strong antimicrobial activity Farkas et al. PNAS 2014

7 Efficient killing of Gram-positive and Gram-negative bacteria NCR247 NCR335

8 Antifungal activity of NCRs Candida albicans Aspergilus niger

9 Can NCR peptides kill the ESKAPE pathogen strains? ESKAPE pathogens Enterococcus faecalis ATCC29212 Gram-positive Staphylococcus aureus HNCMO Gram-positive Klebsiella pneumoniae Gram-negative Acinetobacter baumannii Gram-negative Pseudomanas aeruginosa ATCC27853 Gram-negative Enterobacter species Gram-negative Escherichia coli ATCC8739 Gram-negative Listeria monocytogenes ATCC19111 Gram-positive Salmonella enteritidis ATCC13076 Gram-negative Log-phase bacteria, OD 600 = 0.1, µm peptide concentration Treatment in KH 2 PO 4 buffer: 3h 37 C Minimal Inhibitory Concentration (MIC) Minimal Bactericid Concentration (MBC)

10 Peptides pi vs. antimicrobial activity Peptide Isoelectric Point Sequence PGLa GWTLNSAGYLLGKINLKALAALAKKIL NCR FLPTSRNCITNKDCRQVRNYIARCRKGQCLQSPVR NCR AFIQLSKPCISDKECSIVKNYRARCRKGYCVRRRIR TP GWTLNSAGYLLGKINLKALAALAKKIL NCR ITISNSSFGRIVYWNCKTDKDCKQHRGFNFRCRSGNCIPIRR GoNCR IITNRRCKSHKDCPGFGKCLKLKCVYTPKR NCR RNGCIVDPRCPYQQCRRPLYCRRR NCR FGMTLRPCLTDKDCPRMPPHNIKCRKGHCVPIGKPFK NCR FPDKIFIGDCKTDKDCKPKRGVNFRCRKGKCYPR NCR335C 9.98 HFSLILSDCKTNKDCPKLRRANVRCRKSYCVPI PsNCR AVATKCKTDVDCPPSTSIRRYKCTKNKCRYTRLSYG NCR MKNGCKHTGHCPRKMCGAKTTKCRNNKCQCV NCR SFSQMINFRGCKRDKDCPQFRGVNIRCRSGFCTPIDS NCR YKNRCFRDSDCPKEMCNHPKIPKCVNNAYCKCVVAMYFPPK NCR EDIGHIKYCGIVDDCYKSKKPLFKIWKCVENVCVLWYK NCR RECHANSHCVGKITCVLPQKPECWNYACVCYDSNKYR NCR AVHKECKTDVDCRQIWFVTKCINHECQPIL NCR AYIECEVDDDCPKPMKNSHPDTYYKCVKHRCQWAWK NCR LFECNRDFVCGNDDECVYPYAVQCIHRYCKCLKSRN NCR KDLPFNICEKDEDCLEFCAHDKVAKCMLNICFCF NCR DDVVFQYVFDGCRIDADCPISGLQLLKWMCINNECEFNHVRPRYV NCR KSYGPCTTLQDCETHNWFEVCSCIDFECKCWSLL NCR MEVGRRANVECESDKDCQEHWSEFFIIQCIDNICVPSERPL NCR QTEPPGPLIPCEFDYDCPLIDCIRTSDSRCINGNCHCRE E.coli Salmonella Pseudomonas Listeria Staphylococcus Enterococcus ++ + bactericid bacteriostatic - no effect + charge alone is not sufficient for killing

11 Many NCRs and their shorter derivatives kill pathogen bacteria Név oldódás IE Enterococ Pseudom Salmonel Staphyloc E. coli Listeria Klebsiella cus onas la occus NCR169 MQ NCR247 MQ NCR247 oxidált MQ NCR247C tem MQ NCR044 MQ NCR035 MQ NCR055 MQ NCR147 MQ NCR183 MQ NCR183C KQRR (23-42) MQ NCR192 MQ 50 TP10 MQ TP10 KIAL (7-16) MQ TP10 KAIL (11-21) MQ transportan MQ Transportan analog MQ NCR280 MQ ,25 2,25 9 2,25 18 NCR335 pep C-terminális fele MQ 10, NCR335 N-term. darab MQ N RLLR (1-15) MQ N RPNR (7-21) MQ NCR335C KLPI (17-33) MQ 11, NCR335C KDPI (13-33) MQ 10, C VRPI (23-33) MQ 9, C VRPI (23-33)oxidált MQ 9,75 25 NCR458 ARRR(23-36) MQ NCR504C FDRL (35-60) MQ PsNCR351 MQ GoNCR308 MQ GoNCR308 oxidált MQ GoNCR308C FGKR (16-30)MQ GoNCR308C FGCV (16-25)MQ GoNCR308C FGLK (16-23)MQ GoNCR308C KCKR (23-30)MQ GoNCR313C HRIP (23-44) MQ AMP1/RPRR MQ AMP2/QQIL MQ NCR200 20%DMF

12 Haemolysis Konc. 335C 335C C C µm LDH (U/L) Index abs. 405 abs. 560 LDH (U/L) Index abs. 405 abs. 560 LDH (U/L) Index abs. 405 abs. 560 LDH (U/L) Index abs. 405 abs Melittin NA Konc. 335N 1-19 µm LDH (U/L) Index abs. 405 abs Melittin NA 1.4 No or very little haemolysis!

13 Haemolysis Konc. µm LDH (U/L) Index 247 abs. 405 abs Melittin NA 1.4 No toxicity!

14 Antimicrobial activity of NCR peptides Depends on the charge and amino acid sequence Fast killing (few minutes, faster than many classic antibiotics) Wide spectrum or specific No toxicity on blood cells Multiple bacterial/fungal targets (cell envelope: OM&IM and multiple targets in the cytosol) Low probability for resistance

15 NCRs IM OM Gram-

16 Bioinformatics analysis suggests anticancer activity for many antimicrobial NCRs Cationic NCR peptides inhibit the proliferation of immortalized human epithel (tumor) cells

17 Present and future goals Designing shorter, even more effective or more specific peptide derivatives Determine the antimicrobial activity of these synthetic peptides Determine their mode of action Screen for additional activities (collaborating partners are needed!) Development of novel drugs

18 Thank you for your attention

19 NCR055 NCR124 no treatment MUTANTS AFFECTED IN SURFACE POLYSACCHARIDE PRODUCTION ARE MORE SENSITIVE TOWARDS NCR ACTION wild-type exoa exob lpsb