Antibiotics and translation

195 Pages·2012·13.18 MB·English

by  Starosta Agata Lucyna

Dissertation zur Erlangung des Doktorgrades der Fakultät fur Chemie und Pharmazie
der Ludwig-Maximilians-Universität München

Antibiotics and translation
Overcoming emerging bacterial resistance
to old and new antimicrobials

Agata Lucyna Starosta
aus Rzeszow, Polen

2011
Erklärung
Diese Dissertation wurde im Sinne von § 13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 (in der Fassung der Sechsten Änderungssatzung von 16. August 2010) von Herrn
Prof. Dr. Roland Beckmann betreut.

Ehrenwörtliche Versicherung
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.

München, am 24.11.2011
Agata Lucyna Starosta

Dissertation eingereicht am 24.11.2011
1. Gutachter: Herr Prof. Dr. Roland Beckmann
2. Gutachter: Herr Prof. Dr. Klaus Förstemann
Mündliche Prüfung am 31.01.2012

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Agata L. Starosta    List of contents 

List of contents

Acknowledgements .................................................................................................................................. 5 
List of original publications .................................................................................................................... 6 
Contribution report ................................................................................................................................. 8 
Abbreviations ........................................................................................................................................ 10 
Summary ................................................................................................................................................ 11 
1 Introduction ........................................................................................................................................ 12 
1.1 Traditional antibiotics .................................................................................................................. 13 
1.1.1 Introduction to antibiotics .................................................................................................... 13 
1.1.2 Targets for antibiotic action ................................................................................................. 14 
1.1.2.1 Inhibition of cell wall synthesis ..................................................................................... 14 
1.1.2.2 Inhibition of DNA replication ........................................................................................ 16 
1.1.2.3 Inhibition of RNA synthesis ........................................................................................... 16 
1.1.2.4 Inhibition of protein synthesis ....................................................................................... 16 
1.1.3 Protein synthesis ................................................................................................................... 17 
1.1.3.1 Inhibitors of the small ribosomal subunit .................................................................. 22 
1.1.3.2 Inhibitors of the large ribosomal subunit ................................................................... 23 
1.1.3.2.1 Inhibitors of the peptidyl-transferase center ...................................................... 23 
1.1.3.2.2 Inhibitors of the progressing nascent polypeptide chain .................................... 24 
1.1.3.2.3 Inhibitors of the GTPase-associated center (GAC) ............................................ 24 
1.1.4 Mechanism of cell death induced by bactericidal antibiotics .............................................. 25 
1.2 Antibiotics used in these studies .................................................................................................. 27 
1.2.1 Hygromycin A ....................................................................................................................... 27 
1.2.2 Macrolides ............................................................................................................................ 29 
1.2.3 Thiopeptides ......................................................................................................................... 31 
1.2.4 Orthosomycins ...................................................................................................................... 34 
1.2.5 Fusidic acid .......................................................................................................................... 35 
1.3 Alternative antimicrobials targeting virulence ............................................................................ 36 
1.3.1 Definition of virulence .......................................................................................................... 36 
1.3.2 Colonization ......................................................................................................................... 36 
1.3.3 Biofilm .................................................................................................................................. 36 
1.3.4 Quorum sensing .................................................................................................................... 37 
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Agata L. Starosta    List of contents 
1.3.5 Motility ................................................................................................................................. 37 
1.3.6 Secretion systems .................................................................................................................. 37 
1.3.7 Elongation factor P .............................................................................................................. 38 
2 Objectives of these studies .................................................................................................................. 41 
3 Cumulative thesis ............................................................................................................................... 43 
3.1 Hygromycin A ............................................................................................................................. 43 
3.1.1 Paper 1 .................................................................................................................................. 43 
3.1.2 Paper 2 .................................................................................................................................. 43 
3.2 Macrolides ................................................................................................................................... 47 
3.2.1 Paper 3 .................................................................................................................................. 47 
3.2.2 Paper 4 .................................................................................................................................. 47 
3.3 Thiopeptides ................................................................................................................................ 50 
3.3.1 Paper 5 .................................................................................................................................. 50 
3.3.2 Paper 6 .................................................................................................................................. 50 
3.4 Orthosomycins ............................................................................................................................. 52 
3.5 Fusidic acid.................................................................................................................................. 53 
3.5.1 Paper 7 .................................................................................................................................. 53 
3.6 Elongation factor P ...................................................................................................................... 54 
3.6.1 Paper 8 .................................................................................................................................. 54 
4 Conclusions ........................................................................................................................................ 58 
5 References .......................................................................................................................................... 59 

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Agata L. Starosta    Ackowledgements 
Acknowledgements
This would not be possible without the support of many people that I had a pleasure of meeting
during my PhD studies.
First of all I would like to thank Dr. Daniel Wilson for giving me an opportunity to work in his
group in the Gene Center . I am grateful for the trust and support I received from him during all those
years. I appreciate each and every advice, discussion, criticism and praise I ever received from him.
That was a lesson I will never forget.
I would like to thank Prof. Roland Beckmann for all the expertise, good advices and support as
well as for providing us with a great scientific environment without which this work could not happen.
I am grateful to all our collaborators for having fruitful time collecting all the data: to Prof. B.
Cooperman for great work on thiopeptides; Prof. A. Bogdanov for macrolide story; Prof. K. Reynolds
for hygromycin A studies; Prof. C. Spahn for first and hopefuly not the last Nature paper; Prof. G.
Dinos for hosting me in Greece and support in experiments; and last but not least, to Prof. J. Remme
for great work on EF-P.
I would also like to thank people, whom I had a pleasure to have conducted the experiments with:
Aleksandra Mikolajka, Alexandra Dönhöfer, Viktorija Karpenko, Gemma Atkinson and Vidya Dhote.
Special thanks to Lauri Peil for being my Mass Spectrometry master, for all the hours we spent
discussing the most crazy ideas and then making them come true.
I would like to thank to all the former and present members of the Wilson and the Beckmann labs
for the atmosphere, discussions, parties, trips and many more.
Lots of thanks to Ingegerd Walz for all the help in the emergency situations.
I would like to thank Prof. Klaus Förstemann, Prof. Mario Halic and Prof. Knud Nierhaus for
being in my thesis committee.
I am very grateful to Marta Danecka and Jean-Paul Armache for surviving corrections of my
thesis.

Wielki dzieki Marto i Jasiu za te wszystkie lata spedzone razem w Monachium, i w Krakowie
(Marta). Bez waszego wsparcia wszystko byloby trudniejsze.

Najbardziej chcialabym podziekowac moim kochanym rodzicom i kochanej siostrze, za
wychowanie i wsparcie przez te wszystkie lata, ktore spedzilam z daleka od domu, bez was to
wszysko nie byloby mozliwe.

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Agata L. Starosta    List of original publications 
List of original publications
This thesis is based upon the following original publications
Reprints were made with permission of the publisher.

Paper 1
Palaniappan, N., Dhote, V., Ayers, S., Starosta, A.L., Wilson, D.N., and Reynolds, K.A.
(2009). Biosynthesis of the aminocyclitol subunit of hygromycin A in Streptomyces
hygroscopicus NRRL 2388. Chem Biol 16, 1180-1189.

Paper 2
Dhote, V., Starosta, A.L., Wilson, D.N., and Reynolds, K.A. (2009). The final step of
hygromycin A biosynthesis, oxidation of C-5''-dihydrohygromycin A, is linked to a putative
proton gradient-dependent efflux. Antimicrob Agents Chemother 53, 5163-5172.

Paper 3
Petropoulos, A.D., Kouvela, E.C., Starosta, A.L., Wilson, D.N., Dinos, G.P., and Kalpaxis,
D.L. (2009). Time-resolved binding of azithromycin to Escherichia coli ribosomes. J Mol
Biol 385, 1179-1192.

Paper 4
Starosta, A.L., Karpenko, V.V., Shishkina, A.V., Mikolajka, A., Sumbatyan, N.V.,
Schluenzen, F., Korshunova, G.A., Bogdanov, A.A., and Wilson, D.N. (2010). Interplay
between the ribosomal tunnel, nascent chain, and macrolides influences drug inhibition. Chem
Biol 17, 504-514.

Paper 5
Starosta, A.L.*, Qin, H.*, Mikolajka, A.*, Leung, G.Y., Schwinghammer, K., Nicolaou,
K.C., Chen, D.Y., Cooperman, B.S., and Wilson, D.N. (2009). Identification of distinct
thiopeptide-antibiotic precursor lead compounds using translation machinery assays. Chem
Biol 16, 1087-1096.

Paper 6
Mikolajka, A., Liu, H., Chen, Y., Starosta, A.L., Marquez, V., Ivanova, M., Cooperman,
B.S., and Wilson, D.N. (2011). Differential effects of thiopeptide and orthosomycin
antibiotics on translational GTPases. Chem Biol 18, 589-600.

Paper 7
Ratje, A.H., Loerke, J., Mikolajka, A., Brunner, M., Hildebrand, P.W., Starosta, A.L.,
Donhofer, A., Connell, S.R., Fucini, P., Mielke, T., Whitford, P. C., Onuchic, J. N., Yu, Y.,
Sanbonmatsu, K. Y., Hartmann, R. K., Penczek, P. A., Wilson, D. N., and Spahn, C. M.
(2010). Head swivel on the ribosome facilitates translocation by means of intra-subunit tRNA
hybrid sites. Nature 468, 713-716.

Paper 8
Peil L.*, Starosta, A.L.*, Virumäe, K, Aktinson, G.C., Tenson, T., Remme, J., Wilson, D.N.
(2011). Formation of ε(R)-β-lysyl-hydroxylysine on translation elongation factor EF-P
requires YjeK, YjeA and YfcM. Under revision at Nature Chemical Biology
* Equally contributed
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Agata L. Starosta    List of original publications 
List of publications not included in the thesis:

Paper 9
Wilson, D.N., Schluenzen, F., Harms, J.M., Starosta, A.L., Connell, S.R., and Fucini, P.
(2008). The oxazolidinone antibiotics perturb the ribosomal peptidyl-transferase center and
effect tRNA positioning. Proc. Natl Acad. Sci USA 105, 13339-13344.

Paper 10
Bhushan, S., Meyer, H., Starosta, A.L., Becker, T., Mielke, T., Berninghausen, O., Sattler,
M., Wilson, D.N., and Beckmann, R. (2010). Structural basis for translational stalling by
human cytomegalovirus and fungal arginine attenuator peptide. Mol Cell 40, 138-146.

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Agata L. Starosta    Contribution report 
Contribution report
Work presented in this dissertation comprises part of the results of my doctoral
research conducted from October 2007 to November 2011 in cooperation with scientists from
the laboratories of: Professor K. Reynolds (Portland, Oregon, US), Professor. B. Cooperman
(Pennsylvania, Philadelphia,US), Professor A. Bogdanov (Moscow, Russia), Professor G.
Dinos (Patras, Greece), Professor C. Spahn (Berlin, Germany) and Professor J. Remme
(Tartu, Estonia).
Paper 1 (Palaniappan et al., 2009) and Paper 2 (Dhote et al., 2009)
I performed all the biochemical analysis determining the antimicrobial activities of
Hygromycin A (HygA) and intermediates of HygA, using (i) Escherichia coli lysate based in
vitro coupled transcription-translation assay (TT) with green fluorescence protein (GFP) as a
reporter, and (ii) the AcPhe-Puromycin (AcPhe-Puro) synthesis assay. These results comprise
Figure 6 (Paper 1), and Figure 4 (Paper 2), which I prepared for the papers as well as
contributing to the interpretation and analysis of these results.
Paper 3 (Petropoulos et al., 2009)
I cultivated Deinococcus radiodurans cells and used sucrose gradient centrifugation
protocol to isolated highly active 70S ribosomes, which were then used subsequently to
evaluate the binding mode of azithromycin (AZI) to bacterial ribosomes in collaboration with
Prof. Dinos.
Paper 4 (Starosta et al., 2010)
I carried out all the biochemical experiments to determine the inhibitory activity of
tylosin (Tyl) and derivatives of Tyl using (i) TT assay using GFP and firefly luciferase (Fluc)
as templates, and (ii) competition binding assay with [14C]-erythromycin and D. radiodurans
70S ribosomes. The results are depicted in Figures 3-6 and in Supplementary Table 1 of the
paper. In addition, I participated in the interpretation of the results and made Figures 1, 3-6 as
well as contributing to writing of the manuscript.
Paper 5 (Starosta et al., 2009)
I measured the potency of the entire library of thiopeptide precursor compounds to
inhibit synthesis of GFP in TT assay. I also measured the ability of precursors to restore
translation in the presence of inhibitory concentration of parental compounds, which is
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Agata L. Starosta    Contribution report 
presented in Figures 4-5. I prepared Figures 1-5 and participated in the preparations of the
draft of the manuscript.
Paper 6 (Mikolajka et al., 2011)
I evaluated the antimicrobial activity of thiostrepton, micrococcin and evernimicin
(Evn) in TT assay using GFP as a reporter gene. These results are presented in Supplementary
Figure 3, which I prepared together with Figure 5.
Paper 7 (Ratje et al., 2010)
I grew Thermus thermophilus cells and prepared highly pure 70S ribosomal particles
using sucrose gradient centrifugation protocol. The 70S ribosomes were used for preparation
of the complex of EF-G stalled with GDP and fusidic acid on the 70S ribosome.
Paper 8 (Peil et al. 2011)
I cloned and purified elongation factor P (EF-P) which was used to raise rabbit
polyclonal antibodies. I established the immuno-precipitation protocol using protein A and
prepared all constructs used for the rescue experiments. I identified the YfcM protein using
STRING database and cloned yfcM gene. I purified recombinant YfcM, which I characterized
using differential scanning fluorimetry (DSF). I optimized protocol for expression of EF-P
modified by 128 Da and established the in vitro hydroxylation assay. I prepared all figures
and participated in preparation of the manuscript. Additionally, I started initial
characterization of phenotypes of the efp and modification enzymes deletion strains.
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Agata L. Starosta    Abbreviations 
Abbreviations
· 30SIC – 30S initiation complex · MiC – Micrococcin
· 70SIC – 70S initiation complex · MIC – minimal inhibitory concentration
· A/A position – both anticodon stem loop and · mRNA – messenger RNA
aminoacyl arm of tRNA in A-site · MurNAc – N-acetyl-muramic acid
· A/T position – anticodon stem loop in A site, · NLPH – National Laboratory of Public Health
aminoacyl arm of tRNA bound to EF-Tu of the Ministry of Public Health and Population
· aa-tRNA – aminoacyl-tRNA · OMT – 5-O-mycaminosyl-tylonolid
· AB – subunits A and B of hygromycin A · ORF – open reading frame
· AcPhe-Puro – AcPhe-Puromycin · P/E - anticodon stem loop in P site, aminoacyl
· AHLs – acylated homoserine lactones arm bound to E-site
· A-site – aminoacyl-tRNA binding site · PBS – penicillin-binding proteins
· Avi – avilamycin · P-HygA – phosphorylated hygromycin A
· AZI – azithromycin · Pi – Inorganic phosphate
· Cryo-EM – cryo-electron microscopy · POST – post-translocation state
· Des-HygA – desmethylenehygromycin A · PRE – pre-translocation state
· DGI – German Society for Infectious Diseases  · Pseudomonas aeruginosa
DH-HygA – 5”-dihydrohygromycin A · P-site – peptidyl-tRNA binding site
·
· DHS – deoxyhypusine synthase · PTC – peptidyl-transferase center
· DOHH – deoxyhypusine hydroxylase · RF – release factor
· E. coli – Escherichia coli · rRNA – ribosomal RNA
· ECDC – European Center for Disease · SAM – S-adenosylmethionine
Prevention and Control · SAXS – small-angle X-ray scattering
· EF – elongation factor · SD – Shine-Dalgarno
· EHEC – enterohemorrhagic E. coli · SRL – sarcin-ricin loop
· Ery –erythromycin · SS – secretion systems
· E-site – exit site · SSU – 30S small ribosomal subunits
· Evn – evernimicin · SubA – subunit A, dehydrofucofuranose
· FA – fusidic acid moiety of HygA
· fMet-tRNAfMet – initiator-tRNA · SubB – subunit B, α-methyl cinnamic acid
· GAC – GTPase-associated center moiety of HygA
· GFP – green fluorescent protein · SubC – subunit C, aminocyclitol moiety of
· GlcNAc – N-acetylglucosamine HygA
· HygA – hygromycin A · ThS – thiostrepton
IC – half-inhibitory concentration · tRNA – transfer tRNA
· 50
· IF – initiation factor · TT – E. coli lysate-based in vitro coupled
· K. pneumoniae – Klebsiella pneumoniae transcription-translation assay
· LSU – 50S large subunit · Tyl – tylosin
· MBL – metallo-β-lactamase · UTR – untranslated region
· Met-HygA – methoxyhygromycin A

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