role of wall teichoic acid l-rhamnosylation in listeria monocytogenes resistance to antimicrobial

236 Pages·2015·4.2 MB·English

by  Filipe Carvalho

FILIPE FREITAS COELHO HENRIQUES DE CARVALHO

ROLE OF WALL TEICHOIC ACID L-RHAMNOSYLATION IN
LISTERIA MONOCYTOGENES RESISTANCE TO ANTIMICROBIAL
PEPTIDES AND SURFACE PROTEIN ANCHORING

Tese de Candidatura ao grau de Doutor em
Ciências Biomédicas, submetida ao Instituto de
Ciências Biomédicas Abel Salazar da
Universidade do Porto.

Orientador – Doutor Didier Jacques Christian
Cabanes
Categoria – Investigador principal
Afiliação – Instituto de Biologia Molecular e
Celular

Coorientador – Professor Doutor Rui Appelberg
Gaio Lima
Categoria – Professor catedrático
Afiliação – Instituto de Ciências Biomédicas
Abel Salazar da Universidade do Porto

De acordo com o disposto no ponto n.º 2 do Art.º 31º do Decreto-Lei n.º 74/2006,
de 24 de Março, aditado pelo Decreto-Lei n.º 230/2009, de 14 de Setembro, o
autor esclarece que na elaboração desta tese foram incluídos dados das
publicações abaixo indicadas, e declara ter participado activamente na concepção
e execução das experiências que estiveram na origem dos mesmos, assim como
na sua interpretação, discussão e redacção.

According to the relevant national legislation, the author clarifies that this thesis
includes data from the publications listed below, and declares that he participated
actively in the conception and execution of the experiments that produced such
data, as well as in their interpretation, discussion and writing.

PUBLICAÇÕES / PUBLICATIONS

 Carvalho F, Atilano ML, Pombinho R, Covas G, Gallo R, Filipe SR, Sousa S,
Cabanes D (2015) L-rhamnosylation of Listeria monocytogenes wall teichoic
acids promotes resistance to antimicrobial peptides by delaying interaction with
the membrane. PLoS Pathog 11(5):e1004919

 Carvalho F, Sousa S, Cabanes D (2014) How Listeria monocytogenes
organizes its surface for virulence. Front Cell Infect Microbiol 4(48).

FUNDING

The author was supported by national funds through a grant
(SFRH/BD/61825/2009) from Fundação para a Ciência e a Tecnologia (FCT).

The work here presented was funded by the Fundo Europeu de Desenvolvimento
Regional (FEDER) through the Programa Operacional Factores de
Competitividade (COMPETE), and by national funds through FCT, under projects
PTDC/SAU-IMU/111806/2009, PTDC/SAU-MIC/111581/2009FCOMP-01-0124-
FEDER-015844, ERA-Net PathoGenoMics LISTRESS ERA-PTG/0003/2010,
Infect-ERA/0001/2013PROANTILIS; and by project “NORTE-07-0124-FEDER-
000002-Host-Pathogen Interactions”, co-funded by the Programa Operacional
Regional do Norte (ON.2 – O Novo Norte), under the Quadro de Referência
Estratégico Nacional (QREN), through FEDER and FCT.

TABLE OF CONTENTS

ABSTRACT ........................................................................................................... 11
RESUMO............................................................................................................... 13
LIST OF ABBREVIATIONS .................................................................................. 15

CHAPTER I – INTRODUCTION ........................................................................... 19
A. Listeria monocytogenes ................................................................................ 21
A.1. History ................................................................................................ 21
A.2. Taxonomy, phylogeny and classification ............................................ 21
A.3. General features ................................................................................. 23
A.4. Listeriosis ........................................................................................... 25
A.4.1. Epidemiology ........................................................................ 25
A.4.2. Pathophysiology .................................................................... 25
A.4.3. Clinical manifestations and treatment ................................... 26
A.5. Cellular infection cycle ........................................................................ 28
A.5.1. Major virulence factors .......................................................... 29
B. Gram-positive cell envelope ......................................................................... 35
B.1. Peptidoglycan ..................................................................................... 35
B.1.1. Peptidoglycan metabolism .................................................... 36
B.1.1.1. Peptidoglycan assembly .......................................... 37
B.1.1.2. Peptidoglycan turnover ............................................ 39
B.2. Surface proteins and anchoring mechanisms .................................... 41
B.2.1. Cell wall-associated proteins ................................................ 42
B.2.1.1. LPXTG and NXXTX proteins ................................... 42
B.2.1.2. LysM proteins .......................................................... 45
B.2.1.3. GW proteins ............................................................ 45
B.2.2. Membrane-associated proteins ............................................ 47
B.2.2.1. Lipoproteins ............................................................. 47
B.2.2.2. Hydrophobic tail proteins ......................................... 48
B.2.3. Proteins with unknown association mechanism .................... 49
B.3. Teichoic acids ..................................................................................... 49
B.3.1. Lipoteichoic acids (LTAs) ...................................................... 50
B.3.1.1. LTA structure and biogenesis ................................. 50
B.3.1.2. LTA modifications and functions ............................. 51
B.3.2. Wall teichoic acids (WTAs) .................................................. 53
B.3.2.1. WTA structure and biogenesis ................................ 53
B.3.2.2. WTA modifications and functions ........................... 55
B.3.2.3. WTA diversity in Listeria monocytogenes ............... 57
C. Antimicrobial peptides .................................................................................. 59
C.1. General features and properties ........................................................ 59
C.2. Classes ............................................................................................. 61
C.2.1. Bacteriocins ......................................................................... 62
C.2.1.1. Gram-negative bacteriocins .................................... 62
C.2.1.2. Gram-positive bacteriocins ..................................... 64
C.2.2. Defensins ............................................................................. 69
C.2.3. Cathelicidins ........................................................................ 73
C.3. Mechanisms of action ........................................................................ 77
C.3.1. Cytoplasmic membrane disruption ...................................... 77
C.3.2. Inhibition of intracellular targets ........................................... 79
C.4. Bacterial mechanisms of resistance .................................................. 81
C.4.1. Modification of cell envelope components ........................... 81

CHAPTER II – PROJECT PRESENTATION ...................................................... 85

CHAPTER III – RESULTS ................................................................................... 89
Part I – L-Rhamnosylation of Listeria monocytogenes wall teichoic acids
promotes resistance to antimicrobial peptides by delaying interaction with
the membrane .................................................................................................... 93
I.1. Abstract ............................................................................................... 97
I.2. Author Summary ................................................................................. 99
I.3. Introduction ........................................................................................ 101
I.4. Results .............................................................................................. 105
I.4.1. The rmlACBD locus is required for the presence of L-rhamnose
in Lm WTAs .................................................................................. 105
I.4.2. RmlT is required for the incorporation of L-rhamnose into Lm
WTAs ............................................................................................ 108
I.4.3. WTA L-rhamnosylation promotes Lm resistance to AMPs .... 110
I.4.4. WTA L-rhamnosylation interferes with Lm cell wall crossing by
AMPs ............................................................................................. 112
I.4.5. WTA L-rhamnosylation delays AMP interaction with the Lm
plasma membrane ......................................................................... 114
I.4.6. WTA L-rhamnosylation is crucial for AMP resistance in vivo and
Lm virulence .................................................................................. 117
I.5. Discussion ......................................................................................... 121
I.6. Materials and methods ....................................................................... 127
I.6.1. Bacterial strains and growth conditions ................................ 127
I.6.2. Construction and complementation of mutant strains ........... 127
I.6.3. Gene expression analyses ................................................... 126
I.6.4. WTA PAGE analysis ............................................................ 129
I.6.5. Purification of cell wall components ...................................... 129
I.6.6. Extraction of bacterial cytoplasmic content .......................... 130
I.6.7. HPLC analyses ..................................................................... 131
I.6.8. Intracellular multiplication ..................................................... 132
I.6.9. Resistance to salt stress and lysozyme ................................ 132
I.6.10. AMP susceptibility .............................................................. 132
I.6.11. Cytochrome c binding ......................................................... 133
I.6.12. Zeta potential measurements ............................................. 133
I.6.13. Flow cytometry analyses .................................................... 133
I.6.14. SYTOX Green uptake ........................................................ 134
I.6.15. Binding of AMP to purified cell walls ................................... 135
I.6.16. Immunoelectron microscopy................................................ 135
I.6.17. Animal infections ................................................................ 136
I.6.18. Ethics statement ................................................................. 136
I.6.19. Statistical analyses ............................................................. 137
I.7. Acknowledgements ............................................................................ 139
I.8. Tables ................................................................................................ 141
I.9. Supplementary information ................................................................ 143

Part II – L-Rhamnosylation of Listeria monocytogenes wall teichoic acids is
required for efficient surface anchoring of GW proteins .............................. 149
II.1. Introduction ....................................................................................... 151
II.2. Results ............................................................................................. 153
II.2.1. WTA L-rhamnosylation-deficient Lm is less autolytic due to
deficient surface anchoring of the autolysin Ami ........................... 153
II.2.2. Study of the WTA L-rhamnosylation-dependent surface
localization of Lm GW proteins ...................................................... 155
II.2.3. WTA L-rhamnosylation is required for host cell invasion ..... 158
II.3. Discussion ........................................................................................ 161
II.4. Materials and methods ..................................................................... 167
II.4.1. Bacterial strains and growth conditions .............................. 167
II.4.2. Construction of strains expressing FLAG-tagged cell wall-
binding domains of GW proteins .................................................. 167
II.4.3. Autolysis assay ................................................................... 168
II.4.4. Analysis of Lm surface and secreted protein extracts ......... 168
II.4.5. Cell line infection assays .................................................... 169
II.5. Tables .............................................................................................. 171

CHAPTER IV – GENERAL DISCUSSION ......................................................... 175

CHAPTER V – REFERENCES .......................................................................... 183

CHAPTER VI – APPENDICES ........................................................................... 233