comparative exoproteome analyses at different temperatures
Tese apresentada para obtenção do grau de doutor em Engenharia Alimentar
Paula Cristina Branco Cabrita Cunha
Orientadora: Doutora Maria Luísa Lopes de Castro e Brito
Co-orientador: Doutor Ricardo Manuel de Seixas Boavida Ferreira Co-orientadora: Doutora Maria João Almeida Pessoa Trigo
JÚRI
Presidente: Reitor da Universidade de Lisboa
Vogais: Doutor António Carlos Matias Correia, Professor Catedrático Universidade de Aveiro
Doutor Timothy Alun Hogg, Professor Associado
Escola Superior de Biotecnologia da Universidade Católica Portuguesa
Doutora Maria Luísa Lopes de Castro e Brito, Professora Auxiliar com Agregação Instituto Superior de Agronomia da Universidade de Lisboa
Doutor Miguel Nobre Parreira Cacho Teixeira, Professor Auxiliar Instituto Superior Técnico da Universidade de Lisboa
Doutor Mário Emanuel Campos de Sousa Diniz, Investigador Auxiliar Faculdade de Ciências e Tecnologia da Universidade Nova de Lisboa
Lisboa 2014
À minha querida avó Maria para sempre profundamente inspiradora como ser humano. Aos meus doces rebentos Madalena e Francisco, por serem as minhas âncoras e, ao mesmo tempo, por nos permitirem voar nas asas dos vossos sonhos
"Quanto mais permitimos que cada voz cante com seu próprio tom, mais rica é a diversidade do canto em uníssono" Johannes Scheffler
generosidade. Tanto que seria necessário uma enciclopédia para testemunhar a cada um o que eu sinto, por serem todos tão especiais e particularmente marcantes. Por tudo o que foi possível fazer ao longo destes anos todos, um enorme e infinito OBRIGADA, mas que nunca será suficiente.
À Professora Luisa Brito (orientadora, Instituto Superior de Agronomia-ISA) pelo seu incansável apoio e por acreditar em mim e neste trabalho desde o início. O entusiasmo permanente por fazer ciência e a partilha de ideias, aos quais nunca fiquei indiferente e estiveram na génese desta caminhada. Hoje, sei que todos os ensinamentos e enorme amizade que ficaram irão ser o chão para percorrer as novas etapas que se avizinham.
Ao Professor Ricardo Boavida Ferreira (co-orientador, ISA/Instituto de Tecnologia Química e Biológica-ITQB) pela partilha dos seus largos conhecimentos e vasta experiência que foram determinantes na realização deste trabalho. Das inúmeras qualidades como pessoa, ressalto a contínua forma positiva de encarar a vida, tão sua característica, que foi importante para conseguir ultrapassar os normais, mas por vezes difíceis, obstáculos que fui encontrando ao longo deste percurso.
À Doutora Maria João Trigo (co-orientadora, Instituto Nacional de Investigação Agrária e Veterinária-INIAV, IP), pelo apoio fundamental que deu desde o início, sem o qual nunca teria sido possível a realização deste projeto. Vale sempre a pena acreditar…
Professor António Mexia, Engenheiro Manuel Candeias pelo acolhimento que me deram no Instituto Nacional de Recursos Biológicos, IP (actualmente INIAV, IP) e pelos incentivos e apoio para o início e prossecução deste trabalho.
À Fundação Marquês de Pombal, cujo financiamento deste doutoramento foi decisivo para o rumo que os trabalhos tomaram e para a qualidade dos seus resultados. Em particular, ao Sr. Presidente Dr. José Eugénio Salgado e ao seu sucessor Arquitecto Alfredo Romano de Castro pela simpatia e compreensão sempre demonstradas. À Alda e ao Pedro pela sua eficiência, enorme boa disposição e amizade.
À Regina Freitas e Sara Monteiro (ISA) e Cláudia Santos (ITQB) que me ajudaram, no início deste trabalho, a dar os meus primeiros passos na proteómica e me acolheram nos seus grupos de trabalho.
À Catarina Fonseca, Sandra Batista e Henrique Machado (ISA) pelas vossas motivação e energia contagiantes e pela partilha do gosto pela microbiologia. Por tudo o que aprendi com vocês pois “ensinar é aprender duas vezes”. Que daqui tenham levado mais um pedacinho de sonho para a construção dos vossos futuros…
A todos os que desenvolveram colaborações ao longo deste trabalho, com os quais foi um prazer trabalhar, não só pelo grande nível de profissionalismo de todos mas também pelas pessoas extraordinárias que foram.
Ao Professor José Luis Capelo (BIOSCOPE Group, Faculdade de Ciências, Universidade de Vigo, Espanha) e ao Ricardo Carreira (Faculdade de Ciência e Tecnologia/REQUIMTE, Universidade Nova de Lisboa) pelo enorme entusiasmo com que logo abraçaram a nossa colaboração na área da espectrometria de massa e pela dedicação com que sempre me surpreenderam.
Ao Rui Fernandes (Instituto de Biologia Molecular e Celular, Universidade do Porto) pela sua grande amabilidade e profissionalismo que permitiram a obtenção das imagens lindíssimas da “nossa” Listeria monocytogenes que apresento no meu trabalho e que pretendo partilhar com todos ao colocar aquele que considero o melhor exemplar na capa desta tese.
Ao Bruno Manadas (Centro de Neurociências e Biologia Celular, Universidade de Coimbra) pela simpatia, pela disponibilidade, pela partilha de conhecimentos no âmbito da espectrometria de massa.
Ao Doutor Paul Jenö e à Suzette Moes (Biozentrum, University of Basel, Switzerland) que sempre manifestaram muita simpatia e interesse neste projecto, e um grande sentido de trabalho em equipa, os quais espero, um dia, poder conhecer pessoalmente.
Ao Alexandre, Luísa, João e à Mariana pela transmissão de conhecimentos fundamentais sobre qPCR, pela disponibilização dos equipamentos e respectivos softwares. Ao Alexandre, em especial, pela disponibilização do geNorm, que foi determinante para a análise dos dados do qPCR.
À Elsa Neves, ao André Barata e ao António Lourenço colegas de trabalho que, tal como eu, lutavam no seu doutoramento, pela sua amizade e pela permanente e salutar muito boa disposição. Pelos preciosos conselhos, pelas dicas sobre o decorrer dos ensaios, pela partilha do espaço do laboratório de microbiologia do ISA. Infelizmente, não será fácil encontrar outros assim… Ao António em especial, colega nesta etapa “finalíssíma” dos nossos trabalhos, vive ao máximo aquilo que tens pois é o melhor do mundo…
laboratório. À Dona Lena e à Dona Manuela, tão necessárias à realização deste trabalho e pela sua permanente prestabilidade para ajudar. A todas pelo seu trabalho em equipa. Pela forma desinteressada com que invariavelmente partilharam as suas amizades. Por serem as peças fundamentais do excelente ambiente que sempre se viveu no laboratório muito importante para o sucesso de todos os trabalhos aí realizados. Por todos nós podermos contar com vocês…
À minha querida mãe, por ter incutido em mim o gosto pelo aperfeiçoamento ao longo da vida. Mas também pela ajuda fundamental como avó dos seus netos, colmatando de forma surpreendente as minhas “falhas” como mãe e, ao mesmo tempo, alavancou decisivamente o meu desempenho ao longo deste trabalho. Aos meus irmãos Nuno e Pedro, por tudo o que vivemos ao longo destes anos, e que tudo o que iremos viver para sempre…
Aos meus muito queridos Madalena, Francisco e Pedro que me deram sempre o privilégio de viver sua alegria e genuinidade tão próprias, que salutarmente permitiram que os “gigantes” que surgiram ao longo deste trabalho passassem a ser simples “moinhos de vento”… por vocês tudo vale a pena! Ao Pedro que tanto admiro, pela infinita paciência e grande companheirismo, e sem o qual nada disto teria sido possível. Pelos momentos mais difíceis que superámos juntos e pelos sonhos que se multiplicaram… "o amor não consiste em olhar um ao outro, mas sim em olhar juntos para a mesma direção."
A toda a minha família pelo seu apoio incondicional a este meu projecto. São, e serão sempre, os meus pilares nos quais me inspiro para ser uma pessoa melhor e mais forte cada dia que passa.
A todos os que partilharam momentos de vida comigo, mesmo os que aqui não mencionei, cujas simples palavras de apoio e incentivo foram tão importantes para mim pois “palavras que vêm do coração entram no coração”.
Título: Análise comparativa de exoproteomas de Listeria monocytogenes em função da estirpe e da temperatura
RESUMO
O presente trabalho centralizou-se na análise dos exoproteomas e transcriptomas de quatro estirpes de Listeria monocytogenes, geneticamente diferentes, incluindo uma estirpe persistente. Os resultados mostraram diferenças nos exoproteomas das estirpes durante o crescimento em meio mínimo a 37 °C, 20 °C e 11 °C. A 37 °C, a virulência das estirpes pôde ser inferida a partir dos níveis de listeriolisina O e de internalina C. A 11 °C e a 20 °C, observou-se a ausência de flagelina e de permease de oligopeptidos A, na estirpe persistente. O estudo de nove genes associados ao stresse provocado pelo frio, em duas estirpes crescidas a 11 °C, mostrou na estirpe persistente níveis de expressão significativamente superiores de dtpT e sigB, e significativamente inferiores de flaA, oppA,
lmo1722 e lmo0866. O estudo de 23 estirpes persistentes e não persistentes mostrou que a
mobilidade, per se, não parece determinante para a persistência. Uma análise exoproteómica, transcriptómica e fenotípica integradas, num conjunto mais alargado de estirpes persistentes e não persistentes, poderá fornecer maior informação sobre a regulação da expressão de genes associados à resposta ao stresse, nesta espécie. Este conhecimento contribuirá para a prevenção e controlo desta bactéria patogénica, em ambientes relacionados com a produção alimentar.
Palavras-chave: ciências de saúde pública, L. monocytogenes, proteínas extracelulares, qPCR, temperatura.
ABSTRACT
The present work focused on exoproteomic and transcriptomic analyses of four genetic distinct Listeria monocytogenes strains including one persistent strain. The results showed major differences in the exoproteomes of the strains grown in minimal medium at 37 °C, 20_°C and 11 °C. When grown at 37 °C, the virulence of the strains could be correlated with predicted from the levels of listeriolysin O and internalin C. At 11 °C and 20 °C, the main finding was the lack of flagellin and of oligopeptide permease protein A in the persistent strain. Transcriptional levels of nine cold stress related genes in two of these strains, grown at 11 °C, indicated that the persistent strain presented higher transcript levels of dtpT and
sigB, and lower transcript levels of flaA, oppA, lmo1722 and lmo0866. The swarming motility
of a group of 23 persistent and non-persistent strains, at the same temperature, indicated that motility per se should not be considered determinant for persistence. An integrated exoproteomic, transcriptomic and phenotypic analysis on a broader set of persistent and non-persistent strains may improve the knowledge on L. monocytogenes regulatory networks towards prevention and control of this foodborne pathogen in food and food related environments.
PREAMBLE
Human listeriosis is a fatal disease caused by Listeria monocytogenes, particularly prevalent in industrialized countries where the mortality rate is high. This versatile bacterium is ubiquitous in natural environments and is transmitted by food. It is generally assumed that all L. monocytogenes strains are strictly pathogenic, but some questions remain unanswered about the different degrees of virulence found within the species. On the other hand, some strains of L. monocytogenes may become established in particular food processing facilities for long periods of time suggesting the existence of bacteria particularly adapted to specific niches. Refrigeration is an increasingly used, widespread process utilized along the food chain to guarantee food safety. However, L. monocytogenes is able to grow at low temperatures becoming a hazard particularly important in ready-to-eat (RTE) foods.
The extracellular proteins, or exoproteins, of L. monocytogenes vary both qualitatively and quantitatively within the species, according to the environmental conditions to which the strains are exposed. This type of proteins is most important in providing protection against both host defenses and saprophytic lifestyle stresses. Such protection is ensured through the coordination of complex transcriptional regulatory networks.
Nonetheless, there are few studies on the comparison of extracellular proteomic expression and/or gene expression among different strains of L. monocytogenes. If we consider comparisons of the response of persistent and non-persistent L. monocytogenes strains grown at low temperatures, there is even less reports.
Based on these premises, the objectives of the present work were:
- To analyze the interstrain exoproteome diversity of L. monocytogenes grown at the host temperature (37 °C). Additionally, this analysis attempted to identify correlations between the exoproteome and particularly high virulence of some strains of this foodborne pathogen.
- To study the interstrain exoproteome diversity of L. monocytogenes grown at low temperatures (20 °C and 11 °C). This comparison aimed at contributing to the elucidation of the nature of persistence in the food environment associated with some strains.
- To examine the pattern of gene expression, here intended as gene transcript levels, of nine cold stress related genes, including transcriptional regulators, in a persistent and in a non-persistent L. monocytogenes strain grown at 11 °C. This study planned to find which of these genes could account for interstrain diversity and persistence.
- To understand the importance played by motility on the persistence of some
L._monocytogenes strains. This comparison was performed on a larger set of persistent
and non-persistent strains, belonging to different genetic lineages and including food strains and clinical strains, with different levels of virulence.
TABLE OF CONTENTS
AGRADECIMENTOS ... vii
RESUMO ... xi
ABSTRACT ... xiii
PREAMBLE ... xv
LIST OF ABBREVIATIONS ... xxi
CHAPTER I – GENERAL INTRODUCTION - Is the exoproteome important for bacterial pathogenesis? Lessons learned from interstrain exoprotein diversity in L. monocytogenes grown at different temperatures ...1
Abstract ...3
I.1 Introduction ...5
I.2 The exoproteome ...8
I.2.1 Main challenges for exoproteomic analysis ... 9
I.2.2 Strategies to overcome the drawbacks of the exoproteome analysis ...13
I.2.3 Comparison of the exoproteins of distinct Listeria strains ...15
I.2.3.1 The importance of cell wall proteins in strain discrimination ...15
I.2.3.2. Studies on low-virulence strains ...16
I.2.4. Key exoproteins produced at 37 °C ...17
I.2.4.1 Lmo0443, a protein with a putative role in cell-wall structural maintenance ...17
I.2.4.2 Pleiotropic effects of listeriolysin O ...18
I.2.4.3 InlC, a multifunctional virulence factor ...22
I.2.5 Key exoproteins produced at low temperatures ...25
I.2.5.1. Flagellin ...25
I.2.5.2. Oligopeptide permease A ...28
I.3 Conclusions ... 30
I.4 Acknowledgements ... 30
I.5 References ... 31
CHAPTER II - A secretome-based methodology may provide a better characterization of the virulence of L. monocytogenes: preliminary results ... 47
Abstract ... 49
II.1 Introduction ... 51
II.2 Materials and methods ... 53
II.2.1 Strains used ...53
II.2.2 Bacterial cultures ...54
II.2.3 Protein precipitation ...54
II.2.4 SDS-PAGE ...54
II.2.5 Image scanning and data analysis ...55
II.2.6 MS protein digestion ...56
II.2.7 MALDI-TOF-MS analysis ...57
II.3. Results and discussion ... 58
II.3.1 SDS-PAGE analysis of secreted proteins ...58
II.3.2 Clustering analysis based on secretome profiles ...60
II.3.3 Invasion-associated protein p60 ...60
II.3.5 Internalin C ...63
II.4 Conclusions ... 64
II.5 Acknowledgements ... 66
II.6 References ... 67
II.7 Supplemental material... 73
CHAPTER III - Comparative analysis of the exoproteomes of L. monocytogenes strains grown at low temperatures ... 79
Abstract ... 81
III.1 Introduction ... 83
III.2 Materials and Methods ... 84
III.2.1 Bacterial strains ...84
III.2.2 Bacterial culture supernatants and protein precipitation ...84
III.2.3 SDS-PAGE and glycoprotein detection ...85
III.2.4 Protein sample preparation for LC-MS/MS ...85
III.2.5 Transmission electron microscopy (TEM)...86
III.2.6 Image scanning, protein quantification and data analysis ...86
III.3 Results ... 87
III.3.1 SDS-PAGE and clustering analysis of L. monocytogenes exoproteins ...87
III.3.2 L. monocytogenes exoproteome protein identification and TEM analysis ...89
III.4 Discussion ... 92
III.5 Conclusions ... 94
III.6 Acknowledgements ... 94
III.7 References ... 95
III.8 Supplemental material ... 99
CHAPTER IV- Differential dtpT, sigB, flaA, oppA, lmo1722 and lmo0866 expression at low temperature and flagellar motility in persistent and non-persistent L. monocytogenes strains ... 107
Abstract ... 109
IV.1 Introduction ... 111
IV.2 Materials and methods ... 113
IV.2.1 Bacterial strains ...113
IV.2.2 Motility assays ...114
IV.2.3 Bacterial cell cultures and RNA isolation ...115
IV.2.4 cDNA synthesis ...115
IV.2.5 Real-time quantitative PCR ...116
IV.2.6 Analysis of the relative expression levels of the genes ...118
IV.2.7 Statistical analysis ...118
IV.2.8 Primer nucleotide sequences accession numbers ...119
IV.3 Results ... 119
IV.3.1 Motility analysis ...119
IV.3.2 Optimization of qPCR ...120
IV.3.3 Relative transcript levels of nine genes related with cold stress ...121
IV.4 Discussion ... 123
IV.4.1 Bacterial mobility varies widely among L. monocytogenes persistent strains ...123 IV.4.2 The correlation between flaA transcript levels and those of other low
IV.4.3 The regulation of dtpT expression levels and its correlation with fri. ...127
IV.4.4 Transcripts from three RNA helicase genes present in differing amounts in the two L. monocytogenes strains. ...128
IV.5 Conclusions ... 130
IV.6 Acknowledgements ... 131
IV.7 References ... 132
CHAPTER V – General discussion and conclusion ... 141
V.1 General discussion and conclusion ... 143
LIST OF ABBREVIATIONS
Alternative sigma factor
2D Two-dimensional
ABC ATP-binding cassette
ActA Actin A
AJ Adherens junction
ATCC The American Type Culture Collection bEBP Bacterial enhancer-binding protein
BSA Bovine serum albumin
CAN Acetonitrile
CBAA Centro de Botânica Aplicada à Agricultura CBB Coomassie Brilliant Blue
CDC Cholesterol-dependent cytolysin CIP Collection de l’Institute Pasteur
Cq Quantification cycle
CtsR Class three stress gene Repressor protein
DOC Sodium deoxycholate
DRAT Departamento dos Recursos Naturais, Ambiente e Território
DTT Dithiothreitol
Ecad Epithelial cadherin
EFSA European Food Safety Authorithy
EU European Union
FA Formic acid
FCT Fundação para a Ciência e Tecnologia
FlaA Flagellin
FMP Fundação Marquês de Pombal
Gap Glyceraldehyde 3-phosphate dehydrogenase
HrcA Heat-inducible transcription repressor HSD Honest significant difference
INIAV Instituto Nacional de Investigação Agrária e Veterinária
InlA Internalin A
InlB Internalin B
InlC Internalin C
InlJ Internalin J
INRA Institute Nationale de la Recherche Agronomique INRB Instituto Nacional dos Recursos Biológicos
IR Immunoglobulin-like
ISA Instituto Superior de Agronomia
LC Liquid chromatography
LC-MS/MS liquid chromatography- tandem mass spectrometry
LLO Listeriolysin O
LRR Leucine-rich repeat
MALDI-TOF-MS Matrix-assisted laser-desorption ionization - time of flight - mass spectrometry Met Mesenchymal epithelial transition factor
MSCAL2 ProteoMass Peptide MALDI-MS calibration kit MSDB Mass spectrometry protein sequence database MWB Modified Welshimer Broth
NCBInr National Center for Biotechnology Information non-redundant nLC-MS/MS nano-liquid chromatography- tandem mass spectrometry NoRT No reverse transcriptase
OppA Oligopeptide permease A PCR Polymerase chain reaction PdPs PrfA-dependent proteins PFA Plaque forming assay PMF Peptide mass fingerprint
PMSF Phenylmethylsulphonyl fluoride PrfA Positive regulatory factor A
qPCR Real-time quantitative PCR
RF Reference gene
RT Reverse transcriptase
RTE Ready-to-eat
SD Standard deviation
SDS Sodium dodecyl sulfate
SDS-PAGE Sodium dodecyl sulfate - polyacrylamide gel electrophoresis TCA Trichloroacetic acid
TEM Transmission electron microscopy TFA Trifluoroacetic acid
TN Threonine-asparagine
TSA-YE Tryptone soy agar – yeast extract
TSB Tryptone soy broth
UniProtKB UniProt knowledgebase
UPGMA Unweighted-pair group matching algorithm α-CHCA α-cyano-4-hydrocycinnamic acid
CHAPTER
I
GENERAL INTRODUCTION
Is the exoproteome important for bacterial pathogenesis?
Lessons learned from interstrain exoprotein diversity in
L.
_
monocytogenes grown at different temperatures
CHAPTER I – GENERAL INTRODUCTION - Is the exoproteome important for bacterial pathogenesis? Lessons learned from interstrain exoprotein diversity inL. monocytogenes grown at different temperatures
Cabrita, P, Trigo, MJ, Ferreira, RB, Brito, L (2014). Is the exoproteome important for bacterial pathogenesis? Lessons learned from interstrain exoprotein diversity in
Listeria monocytogenes grown at different temperatures. OMICS: A Journal of Integrative Biology. (Review article, in press)
Abstract
Bacterial exoproteomes vary in composition and quantity among species and within each species depending on the environmental conditions to which the cells are exposed. This article critically reviews the literature available on exoproteins synthesized by the foodborne pathogenic bacterium Listeria monocytogenes grown at different temperatures. The main challenges posed for exoproteome analyses and the strategies which are being used to overcome these constraints are discussed. Over thirty exoproteins from L. monocytogenes are considered and the multifunctionality of some of them is discussed. Thus, at the host temperature of 37 °C, good examples are provided by Lmo0443, a potential marker for low virulence, and by the virulence factors internalin C (InlC) and listeriolysin O (LLO). Based on the reported LLO-induced mucin exocytosis, a model is proposed for the involvement of extracellular LLO in optimizing the conditions for InlC intervention in the invasion of intestinal epithelial cells. At lower growth temperatures, exoproteins like flagellin (FlaA) and oligopeptide permease (OppA) may explain the persistence of particular strains in the food industry environment, eventually allowing the development of new tools to eradicate
L. monocytogenes, a major concern for public health.
I.1 Introduction
The foodborne pathogen L. monocytogenes, a gram-positive bacillus, is one of the most important causes of food-related infections in developed countries. These ubiquitous bacteria can grow and survive in diverse environments because of their tolerance and adaptability to different types of stresses, enabling colonization and persistence in multiple ecological niches (Hamon et al., 2006). This pathogen is responsible for the disease listeriosis that often results in self-limiting febrile gastroenteritis but can also be a potential threat to humans when it evolves to septicemia, central nervous system infections or maternofetal infections leading to stillbirths and abortions (Posfay-Barbe and Wald, 2009; Stavru et al., 2011; Disson and Lecuit, 2012). The incidence of listeriosis is lower than other common foodborne illnesses. However, it represents one of the most deadly bacterial infections, especially among elderly people (Gerner-Smidt et al., 2005; Schuppler and Loessner, 2010). In 2011, 1,476 confirmed human listeriosis cases were reported in the European Union (EU), with a high fatality rate of 12.7% (EFSA, 2013). In the United States, listeriosis is responsible for approximately 19% of the annual food-related deaths (Scallan et al., 2011).
L. monocytogenes consists of at least four evolutionary lineages (Ward et al., 2008; Orsi et al., 2011; Datta et al., 2013). Lineage I (flagellar antigen types b and d) contains strains that
are more likely to cause human disease than the isolates classified in lineages II (antigen type a or c), III and IV (some strains of 4b and the rarely detected serovars 4a and 4c). Although the 13 known serovars can cause human infection (Seeliger and Jones, 1986), epidemiological data show that the majority of human cases are associated with serovars 1/2a, 1/2b and 4b (Goulet et al., 2008; Schuppler and Loessner, 2010; Chenal-Francisque et
al., 2011). The strains within lineage II are usually present in food and food environments
more frequently than lineage I isolates (Nucera et al., 2010; Orsi et al., 2011; Wang et al., 2012; Parisi et al., 2013). However, some reports indicate the prevalence of serovar 4b in food and food-related isolates (Leite et al., 2006; Pagadala et al., 2012).
L. monocytogenes is a well-adapted saprophytic microorganism. The survival of this
bacterium under diverse environmental stresses strongly contributes to the niche persistence of some strains. The persistence of L. monocytogenes in food-processing facilities has been described in great detail (Leite et al., 2006; Keto-Timonen et al., 2007; D'Amico and Donnelly, 2008; Orsi et al., 2008; Ferreira et al., 2011; Fox et al., 2011). These strains become established for months or years as part of the resident microbiota, suggesting the niche-adaptation of the bacteria, which appear capable of withstanding and overcoming the significant challenges imposed, such as cold, unfavorable pH and salt stress. The increased diversity in consumer preferences for food products has led to the widespread implementation of cold chains to meet food safety demands (Rediers et al., 2009; Kuo and Chen, 2010). L. monocytogenes is able to grow at low temperatures (Tasara and Stephan, 2006; Chan and Wiedmann, 2009) for extended periods. The combination of these Listeria survival skills with periods of food storage at inappropriate temperatures may lead to significant growth of the pathogen within food matrices (Rosset et al., 2004). Notably, several studies have shown that different strains of L. monocytogenes usually respond differentially to low temperatures (Moorhead and Dykes, 2004; Pal et al., 2008; Kagkli et al., 2009).
The survival of a bacterial pathogen depends on its ability to maneuver and react actively with host cells. This host-pathogen interaction during listeriosis has been extensively studied (Vazquez-Boland et al., 2001; Cossart et al., 2003; Posfay-Barbe and Wald, 2009; Stavru et
al., 2011; Pizarro-Cerdá et al., 2012). Over several years, these studies have established that L. monocytogenes promotes its internalization into the host epithelial cells via an interaction
between the bacterial surface molecules internalin A (InlA) and internalin B (InlB), and the host cellular receptors epithelial cadherin (Ecad) and mesenchymal epithelial transition factor (Met), triggering several complex host cell pathways that lead to bacterial engulfment and escape from recognition by the innate immune system. In the subsequent stages of infection, the escape from the phagocytic vacuole is LLO-dependent, and the intracellular actin-based motility and the cell-to-cell spread are actin A (ActA)-dependent (Fig. I.1A). Recent cellular infection studies performed in vivo revealed that transcytosis could be used
for the early, rapid and efficient crossing of the intestinal barrier by L. monocytogenes. In fact, L. monocytogenes enters intestinal epithelial cells after InlA-Ecad interactions and, in a LLO and ActA-independent manner, crosses the epithelial cells by a furtive intra-vacuolar path (Fig. I.1B) (Nikitas et al., 2011).
Figure I.1 L. monocytogenes host-cell invasion.
A – Successive steps of L. monocytogenes infection of in vitro cultured cells (adapted from
http://alicelebreton.free.fr/research.html, last access 14th of January 2014);
B – Schematic representation of in vivo L. monocytogenes transcytosis through goblet cells (GC)
(adapted from Nikitas et al., 2011).
L. monocytogenes can switch between life in soil and in the mammalian host through
complex regulatory pathways that modulate the expression of factors involved in pathogenicity and/or in the adaptation to different abiotic life styles (Gray et al., 2006; Freitag et al., 2009; Toledo-Arana et al., 2009). These complex networks of metabolic pathways include several regulators such as the positive regulatory factor A (PrfA), class three stress gene repressor protein (CtsR), heat-inducible transcription repressor (HrcA) and the alternative sigma factors B, C, H and L, which play determinant roles in the bacterial stress response and adaptation to the environment, including gastrointestinal survival and the systemic stages of infection (Chaturongakul et al., 2008; Chaturongakul et al., 2011).
In the context of host-pathogen interactions, specific secreted proteins play important roles in evading and availing host cell defenses. However, the bacterium must first translocate
N o n -p h a g o c y ti c c e ll s L. monocytogenes-containing vacuole ActA-dependent actin tails Vacuole lysis by LLO
L. monocytogenes-containing vacuole Microtubule-dependent translocation In te s ti n a l g o b le tc e ll E n te ro c y te L. monocytogenes-containing vacuole Microtubule-dependent translocation In te s ti n a l g o b le tc e ll E n te ro c y te L. monocytogenes-containing vacuole Microtubule-dependent translocation Int es tin al g ob let ce ll En ter oc yte L. monocytogenes-containing vacuole Microtubule-dependent translocation Int es tin al g ob let ce ll En ter oc yte
A
B
these proteins to the cell surface and ultimately to the external milieu. The bacterial extracellular proteome or exoproteome undergoes multiple adaptations according to the bacterial environmental conditions (e.g., degradative enzymes during saprophytic conditions, as opposed to virulence factors when inside the host) (Conte et al., 2000; Lee and Schneewind, 2001; Midelet-Bourdin et al., 2006). Furthermore, the expression patterns of the exoproteome can undergo more pronounced alterations than the cytoplasmic proteins (Dumas et al., 2008, 2009b). Schliep et al. (2012) proposed that it is unnecessary to analyze all the proteins in a proteome and that a smaller subset of outer membrane proteins is sufficient for the identification of environmental patterns of expression.
In this review, recent studies on the L. monocytogenes exoproteome are summarized and discussed. The bacteria are repeatedly exposed to the host temperature or the low temperatures used for food preservation. Therefore, the interstrain exoprotein diversity may explain the particularly high virulence of some strains and the persistence of some others in the food environment.
I.2 The exoproteome
Successful infection by L. monocytogenes requires diverse adaptation strategies to efficiently avoid (by abolishing, counterattacking and/or circumventing) the host defense mechanisms. This process requires specific virulence factors such as extracellular proteins, which play an important role in pathogenicity (Vazquez-Boland et al., 2001; Trost et al., 2005; Desvaux and Hebraud, 2006; Fuchs et al., 2012). The recent development of powerful analytical tools such as mass spectrometry allowed the analysis of the pathogen exoproteome, which can provide insight into pathogenesis and therapeutics (Windle et al., 2010). The prompt investigation of extracellular proteins may well constitute an essential starting point in the global effort to understand and eradicate listeriosis. An important feature of such studies is the comparison between virulent and less virulent strains, and the identification of new key virulence factors.
The existence of the closely related species L. monocytogenes and L. innocua, of which only the first is pathogenic, motivated several researchers to screen for new potential virulence factors. Based on the genome analysis of sequences that encode proteins, Glaser et al. (2001) predicted that 86 proteins are secreted into the external milieu by L. monocytogenes. Trost et al. (2005) identified 45 putative secretory proteins from L. monocytogenes grown at 37 °C. This study experimentally demonstrated that almost half of the extracellular proteins identified had no recognizable signal sequence for secretion, which means that their extracellular location could not be predicted from their genomic sequences. Furthermore, the discrepancy between the expected and observed proteins in the extracellular milieu led to the proposal that this set of proteins should be called the “exoproteome” or “extracellular proteome”, instead of the secretome, because they may or may not be actively secreted (Desvaux et al., 2009).
I.2.1 Main challenges for exoproteomic analysis
Table I.1 presents some of the exoproteins of the serovar 1/2a strain EGD-e (and strain 10403S) according to the main functional categories. These EGD-e proteins were identified in the first post-genomic study of the exoproteome of L. monocytogenes by Trost et al. (2005). Subsequently, other groups have also identified these proteins. Trost et al. (2005) combined predictive bioinformatics and two proteomic approaches: two-dimensional (2D) gel electrophoresis, and matrix-assisted laser-desorption ionization - time of flight - mass spectrometry (MALDI-TOF-MS) peptide mass fingerprint (PMF) and post source decay fragmentation followed by nano-liquid chromatography - tandem mass spectrometry (nLC-MS/MS). Using the two approaches, these authors identified 58 and 47 exoproteins, respectively, in the culture supernatant of this pathogen, resulting in a total of 105 exoproteins. Dumas et al. (2009a) used 2D gel electrophoresis and MALDI-TOF-MS for the comparison of the exoproteomes of 12 L. monocytogenes strains including EGD-e, and found that 43 of the 60 exoproteins identified were present in the supernatants of all strains. Therefore, these exoproteins were considered to comprise the core exoproteome of the species (Table I.1). Besides this core exoproteome, 17 proteins (variante exoproteome) appeared
Ta b le I .1 Exo p ro tei n s id ent ifi ed, u si n g d ifferent ap p ro ac h es, in t h e su p erna tan ts o f 3 7 C -g ro wn cu lt u res o f L. m o n o cy to g en es 1 /2 a st rai n s (E GD -e an d 1 0 4 0 3 S) Lo cus na m e P ro te in acr o ny m P ro te in na m e & C ha racte ri sti cs / Func ti o na l des cr ipti o n Str ai n N o o rtho lo gu e in L. in n o cu a C o re exo pr o teo m e R ef er en ces Sp ec if ic v ir u le n ce f ac to rs lm o 0 2 0 1 P lc A a) P ho spha ti dy lino si to l-spe ci fi c pho spho lipa se C EG D -e Tr o st et a l., 2 0 0 5 ; D u m as et a l., 2009a lm o 0 2 0 2 LLO Li ster io lys in O EG D -e, 1 0 4 0 3 S Tr o st et a l., 2 0 0 5 ; P o rt an d Fr ei tag, 2 0 0 7 ; D um as et a l., 2 0 0 9a lm o 0 2 0 3 Mpl Zi nc m eta llo p ro tei n as e EG D -e Tr o st et a l., 2 0 0 5 lm o 0 2 0 4 A ctA A cti n as sem bl y-in duc ing pr o te in EG D -e, 1 0 4 0 3 S Tr o st et a l., 2 0 0 5 ; P o rt an d Fr ei tag, 2 0 0 7 ; D um as et a l., 2 0 0 8; D um as et a l., 2009a lm o 0 2 0 5 P lc B P ho spho lipa se C EG D -e, 1 0 4 0 3 S Tr o st et a l., 2 0 0 5 ; P o rt an d Fr ei tag, 2 0 0 7 ; D um as et a l., 2 0 0 9a lm o 0 4 3 3 Inl A Inter na lin A EG D -e Tr o st et a l., 2 0 0 5 ; C al vo et a l., 2005 lm o 0 4 3 4 Inl B Inter na lin B EG D -e Sc ha um bu rg et a l. , 2 0 0 4 ; T ro st et a l., 2 0 0 5 lm o 1 7 8 6 Inl C Inter na lin C EG D -e, 1 0 4 0 3 S Tr o st et a l., 2 0 0 5 ; P o rt an d Fr ei tag, 2 0 0 7 ; D um as et a l., 2 0 0 8; D um as et a l., 2009a C e ll su rf ac e p ro te in s an d m e ta b o lis m o f ce ll w al l lm o 0 2 6 3 Inl H Inter na lin H EG D -e Tr o st et a l., 2 0 0 5 ; C al vo et a l., 2005 lm o 0 5 8 2 Iap b) P ro ba b le endo pe pti da se p6 0 EG D -e Sc ha um bu rg et a l. , 2 0 0 4 ; T ro st et a l., 2 0 0 5 ; C al vo et a l., 2005; D um as et a l., 2 0 0 8 ; D u m as et a l., 2009a lm o 0 8 8 0 Lm o 0 8 8 0 Lys M do m ai n p ro te in EG D -e Tr o st et a l., 2 0 0 5 ; C al vo et a l., 2005 lm o 1 4 3 8 P bp P eni ci lli n -bi n di n g p ro tei n EG D -e Tr o st et a l., 2 0 0 5 ; C al vo et a l., 2 0 0 5 ; D u m as et a l., 2009a lm o 1 5 2 1 Lm o 1 5 2 1 N -ace ty lm ur am o yl -L -a lan ine am ida se EG D -e Tr o st et a l., 2 0 0 5 ; D u m as et a l., 2 0 0 8 ; D u m as et a l., 2009a lm o 1 6 6 6 Lm o 1 6 6 6 LP X TG do m ai n p ro te in EG D -e Tr o st et a l., 2 0 0 5 ; C al vo et a l., 2005 lm o 1 8 8 3 Lm o 1 8 8 3 C hi ti na se EG D -e Tr o st et a l., 2 0 0 5 ; D u m as et a l., 2009a lm o 2 5 0 4 Lm o 2 5 0 4 c) P uta ti ve p epti da se EG D -e Tr o st et a l., 2 0 0 5 ; D u m as et a l., 2008 lm o 2 5 0 5 P 4 5 P epti do gl yc an lyt ic pr o tei n P 4 5 EG D -e Sc ha um bu rg et a l. , 2 0 0 4 ; T ro st et a l., 2 0 0 5 ; C al vo et a l., 2005; D um as et a l., 2009a
T a b le I .1 ( co n t) Lo cus N am e P ro te in acr o ny m P ro te in na m e & C ha racte ri sti cs / Func ti o na l des cr ipti o n Str ai n N o o rtho lo gu e in L. in n o cu a C o re exo pr o teo m e R ef er en ces lm o 2 5 2 2 Lm o 2 5 2 2 c) Lys M do m ai n p ro te in E G D -e, 1 0 4 0 3 S Tr o st et a l., 2 0 0 5 ; P o rt an d Fr ei tag, 2 0 0 7 ; D um as et a l., 2 0 0 8; D um as et a l., 2009a lm o 2 5 5 8 Lm o 2 5 5 8 A uto lys in, a m ida se E G D -e Sc ha um bu rg et a l. , 2 0 0 4 ; Tr o st et a l. , 2 0 0 5 lm o 2 5 9 1 Lm o 2 5 9 1 c) Sur face p ro te in (G W r epeat ) si m ila r to N -ace ty lm ur am ida se E G D -e Tr o st et a l., 2 0 0 5 ; D u m as et a l., 2 0 0 8 ; D u m as et a l., 2009a lm o 2 6 9 1 Lm o 2 6 9 1 N -ace ty lm ur am ida se Mu rA E G D -e, 1 0 4 0 3 S Sc ha um bu rg et a l. , 2 0 0 4 ; Tr o st et a l. , 2 0 0 5 ; C al vo et a l., 2 0 0 5 P o rt an d Fr ei ta g, 2 0 0 7 ; D um as et a l., 2009a lm o 2 7 1 4 Lm o 2 7 1 4 P uta ti ve D -al an yl -D -al ani ne car bo xyp epti da se E G D -e Tr o st et a l., 2 0 0 5 ; C al vo et a l., 2 0 0 5 ; D u m as et a l., 2009a lm o 2 7 5 4 Lm o 2 7 5 4 P eni ci lli n -bi n di n g p ro tei n E G D -e, 1 0 4 0 3 S Tr o st et a l., 2 0 0 5 ; P o rt an d Fr ei tag, 2 0 0 7 T ra n sp o rt /b in d in g p ro te in s, li p o p ro te in s a n d m e m b ra n e b io e n e rget ic s lm o 0 0 1 3 Q o xA d) AA3 -6 0 0 qui no l o xi da se subu n it II E G D -e Sc ha um bu rg et a l. , 2004; Tr o st et a l. , 2 0 0 5 ; D um as et a l., 2 00 D um as et a l., 2 0 0 9 a; P o rt an d Fr ei ta g, 2 0 0 7 lm o 0 1 3 5 Lm o 0 1 3 5 P uta ti ve o lig o pept ide A B C t ran spo rte r E G D -e, 1 0 4 0 3 S Sc ha um bu rg et a l. , 2 0 0 4 ; Tr o st et a l. , 2 0 0 5 ; P o rt an d Fr ei ta g, 2 0 0 7 ; D um as et a l., 2009a lm o 1 0 6 8 Lm o 1 0 6 8 b) Li po p ro tei n E G D -e Sc ha um bu rg et a l. , 2004; Tr o st et al ., 2 0 0 5 ; D um as et a l., 2 00 lm o 1 3 8 8 Tc sA C D 4 + T-ce ll-sti m ul at ing an ti gen E G D -e, 1 0 4 0 3 S Sc ha um bu rg et a l. , 2 0 0 4 ; Tr o st et a l. , 2 0 0 5 ; P o rt an d F re ita g, 2 0 0 7 ; D um as et a l., 2 0 0 8 ; D u m as et a l., 2009a lm o 1 6 7 1 Lm o 1 6 7 1 A B C tr an spo rter an d ad hes in p ro te in E G D -e Sc ha um bu rg et a l. , 2 0 0 4 ; Tr o st et a l. , 2 0 0 5 lm o 1 7 3 8 Lm o 1 7 3 8 A m ino ac id A B C tr an spo rte r (bi nd ing pr o tei n ) E G D -e Sc ha um bu rg et a l. , 2 0 0 4 ; Tr o st et a l. , 2 0 0 5 lm o 1 8 4 7 MntA Mang an es e-bi nd ing pr o tei n E G D -e Sc ha um bu rg et a l. , 2 0 0 4 ; Tr o st et a l. , 2 0 0 5 lm o 2 1 9 6 O ppA O lig o pept ide A B C t ran spo rt er O ppA E G D -e, 1 0 4 0 3 S Tr o st et a l., 2 0 0 5 ; P o rt an d Fr ei tag, 2 0 0 7 ; D um as et a l., 2 0 0 9a lm o 2 6 3 7 Lm o 2 6 3 7 P uta ti ve ph er o m o ne li po p ro tei n E G D -e, 1 0 4 0 3 S Sc ha um bu rg et a l. , 2 0 0 4 ; Tr o st et a l. , 2 0 0 5 ; P o rt an d Fr ei ta g, 2007
T a b le I .1 ( co n t) Lo cus N am e P ro te in acr o ny m P ro te in na m e & C ha racte ri sti cs / Func ti o na l des cr ipt io n Str ai n N o o rtho lo gu e in L. in n o cu a C o re exo pr o teo m e R ef er en ces R e gu la ti o n a n d s e n si n g lm o 0 4 4 3 Lm o 0 4 4 3 Tr an sc ri pt io na l r eg u lat o r, LytR/C ps A /P sr f am ily E G D -e Sc ha um bu rg et a l. , 2004; Tr o st et a l. , 2 0 0 5 ; D um as et a l. , 2 00 8 ; D um as et a l., 2009a lm o 2 5 1 8 Lm o 2 5 1 8 Tr an sc ri pt io na l r eg u lat o r, L yt R f am ily E G D -e Tr o st et a l., 2 0 0 5 ; D u m as et a l., 2 0 0 8 ; D u m as et a l., 2009a a) M em b er o f the var ian t exo pr o teo m e o f L. m o n o cy to g en es ( D u m as et a l., 2 0 0 9 a) . b) P ro te in und er exp res sed in th e str ai ns f ro m s er o va r 1 /2 a, r el at ive to s tr ai ns f ro m s er o va rs 1 /2 b an d 4 b ( D um as et a l. , 2 0 0 8 ). c) P ro tei n o ve rexp res sed in th e st rai ns f ro m s er o va r 1 /2 a, r el at ive to s tr ai ns f ro m s er o var s 1 /2 b an d 4 b (D u m as et a l. , 2 00 8 ). d) P ro te in ab sent o r do wnr eg u lat ed in a L. m o n o cy to g en es m uta nt wi th c o ns ti tut ive exp res si o n o f p rf A g ene ( P o rt an d F rei ta g, 2 0 0 7 ).
The total number of exoproteins identified by Dumas et al. (2009a) was lower than by Trost
et al. (2005). For example, the virulence factor Mpl was identified by Trost et al. (2005)
(Table I.1) and not by Dumas et al. (2009a). However, the virulence factor PlcA belonged to the variant exoproteome of L. monocytogenes identified by Dumas et al. (2009a) (Table I.1). Another study using the same 12 L. monocytogenes strains and the same experimental techniques as used by Dumas et al. (2009a) showed that some proteins can be over- or underexpressed in the strains from serovar 1/2a relative to the strains from serovars 1/2b and 4b (Dumas et al., 2008) (Table I.1). Trost et al. (2005) combined two experimental approaches for protein separation and identification, which allowed the identification of a larger number of exoproteins.Nevertheless, many of the identified proteins may not reflect relevant differences among strains. Furthermore, the simultaneous use of different techniques increases the time and cost of the study. The use of a larger number of strains with different degrees of virulence and abilities to persist as in-house strains allows a better understanding of how this species interacts with its milieu. Finally, the relatively low concentration of proteins in the culture supernatants poses a challenge to the analysis of the total exoproteomes.
I.2.2 Strategies to overcome the drawbacks of the exoproteome analysis
The L. monocytogenes exoproteome can be divided into different subsets of exoproteins. Reducing the complexity of the sample will increase the relative amount of each protein to be identified and therefore increases the probability of successful exoprotein detection and identification. The most important and well-studied of these subsets is the subset containing virulence factors, whose expression is controlled by complex networks of several regulators (Chaturongakul et al., 2008; Chaturongakul et al., 2011). The study of the specific virulence factors or regulators of the exoproteomes of mutant strains can aid the discovery of new exoproteins associated with virulence (Port and Freitag, 2007).These authors found that the use of mutationally activated prfA alleles was a useful approach to study the role of proteins such as Lmo0135 and TcsA in bacterial virulence (Table I.1), which had not previously been associated with virulence. Therefore, the use of mutants can be extended to other
regulators of virulence factors for the study of the L. monocytogenes exoproteome, as shown by Abram et al. (2008) and Mujahid et al. (2013).
Virulence factors can also be localized on the bacterial cell surface to promote host-cell interactions. Schaumburg et al. (2004) established a method for the isolation of a low complexity surface proteome and identified 55 proteins by N-terminal sequencing and MS, after serial extraction and 2D electrophoresis. These authors found that the detection and the identification of the cell wall proteins covalently linked by the LPXTG motif were more difficult most likely because of the tight linkage to the peptidoglycan and/or their low expression. To overcome this, Calvo et al. (2005) analyzed the complex peptide mixture obtained from a preparation of peptidoglycan-enriched material by a non-electrophoretic approach such as 2D nLC-MS/MS. These authors identified 19 different proteins for the
L. monocytogenes EGD-e strain (some are indicated in Table I.1), and showed that this highly
sensitive method was effective for studying novel protein-peptidoglycan associations. Recently, non-labeling quantitative approaches have gained prominence over the 2D methods in most proteomic studies, although the 2D methods are still commonly used (Porteus et al., 2011; Armengaud, 2013). The high-throughput identification of proteins by LC-MS/MS after trypsin proteolysis has been widely used to study the sets of proteins expressed under certain conditions and/or in a specific microorganism. Prior to their identification, the whole protein content of the sample is separated in a simple and rapid SDS-PAGE migration is isolate and a single or several protein bands are prepared for protein identification (Clair et al., 2010; Wijte et al., 2010). Gel-free approaches are also used, with prior separation of the proteins based on their isoelectric point (Habicht et al., 2011). The gel-free fractionation of the sample, i.e., by 2D nano-LC, coupled to the prevalent MS/MS technique has been a successful approach for the study of the surface proteomes in several microorganisms (Armengaud, 2013) including L. monocytogenes (Calvo et al., 2005; Pucciarelli et al., 2005; Huang and Hussain, 2012; Nilsson et al., 2013; Zhang et al., 2013).
I.2.3 Comparison of the exoproteins of distinct Listeria strains
Trost et al. (2005) identified 16 proteins in the supernatants of the cultures of
L. monocytogenes strain EGD-e; these proteins have no orthologs in the non-pathogenic
bacterium L. innocua. Nine of these proteins (PlcA, LLO, Mpl, ActA, PlcB, InlA, InlB, InlC and InlH) were virulence factors (Table I.1). These authors also found 43 proteins that were specifically expressed by L. monocytogenes but not by L. innocua. Similarly, a comparative proteomic analysis of the cell wall sub-proteomes in pathogenic and non-pathogenic Listeria identified the molecular components associated with virulence in L. monocytogenes (Calvo
et al., 2005). L. monocytogenes and the non-pathogenic L. innocua were compared to
identify the proteins involved in L. monocytogenes pathogenicity. The mutational activation of the central virulence regulator PrfA allowed the identification of novel potential extracellular virulence factors (Port and Freitag, 2007). These important exoproteomic approaches were based on the study of a single L. monocytogenes strain, generating conclusions that are insufficient to be generally applied to the species. Dumas et al. (2008) compared the extracellular proteins of 12 strains belonging to the serovars 1/2a, 1/2b and 4b, and found that the abundance or scarceness of specific proteins was related to the genetic lineage of the strains. These authors also found proteins that were not considered potential virulence factors by Trost et al. (2005). These results suggest that the environmental conditions and stimuli required for the expression of virulence factors may vary by strain.
I.2.3.1 The importance of cell wall proteins in strain discrimination
Trost et al. (2005) showed that the “cell surface proteins and metabolism of the cell wall” was the functional category that contributed most to the differences between
L. monocytogenes and L. innocua, followed by proteins in the category of “specific virulence
factors”. It is well known that the degree of pathogenicity of L. monocytogenes under the experimental conditions tested, i.e., its potential, differs among isolates. Despite the association of most human cases with three serovars, differences in the virulence potential
of the strains within each serovar have also been reported (Olier et al., 2003; Cabrita et al., 2004; Neves et al., 2008; Dumas et al., 2009b).
Dumas et al. (2008) performed a proteomic analysis of L. monocytogenes strains from the serovars 1/2a, 1/2b and 4b, and found that the proteins responsible for different levels of virulence were mainly from the “cell surface proteins and metabolism of the cell wall” functional category. These results revealed the importance of this protein set in strain discrimination. A more detailed analysis revealed that more than half of the non-orthologous proteins identified in L. monocytogenes were commonly found in the extracellular milieu of all strains (core exoproteome), whereas some protein spots were specifically present only in a subset of strains. This suggests the existence of partially strain-specific post-translational modifications, as reported for some virulence factors (Dumas et
al., 2009a). Apart from this core exoproteome, 17 proteins (variant exoproteome) appeared
to be expressed only in some L. monocytogenes strains with different levels of virulence, as PlcA in strain EGD-e (Table I.1). However, none of these proteins could be considered a
virulence marker (Dumas et al., 2008; Dumas et al., 2009a). In the extracellular milieu of strain EGD-e, Dumas et al. (2008) found a putative cell-wall protein, Lmo0443, which was generally overexpressed in less virulent strains and underexpressed in more virulent ones (Table I.2).
I.2.3.2. Studies on low-virulence strains
It is generally assumed that all L. monocytogenes strains are strictly pathogenic, but some questions remain unanswered about the virulence in this species. For instance, non-virulent or weakly virulent strains of L. monocytogenes have been reported in the intestine of 1% to 15% healthy individuals among the population (Rocourt et al., 2000; Grif et al., 2003; Olier et
al., 2003; Velge and Roche, 2010). According to some authors, the prevalence of
low-virulence strains isolated from different environmental sources could be greater than 60% (Roche et al., 2009; Velge and Roche, 2010). Mutations in their key virulence genes, including inlA, inlB, plcA, plcB, hly or actA, have been detected (Roberts et al., 2005;
Roche et al., 2012). The transcriptional regulators of L. monocytogenes virulence genes, such as prfA, can also be affected by mutations (Velge et al., 2007; Roche et al., 2012) influencing gene expression and most likely compromising extracellular protein expression. Comprehensive studies showed that the diversity and population structure of
L. monocytogenes according to the virulence level is complex and based on different
mechanisms, which seem to differ according to the genetic lineages of the strains (Roche et
al., 2012). There are few studies on the differential extracellular proteomic expression
and/or differential gene expression between virulent and low-virulence strains of
L. monocytogenes (Olier et al., 2002; Olier et al., 2003; Duodu et al., 2010). Several authors
have proposed that molecular and cellular biological studies to understand the differences between virulent and low-virulence strains would help in the implementation of effective control and prevention measures against L. monocytogenes (Olier et al., 2003; Oevermann et
al., 2010). Cabrita et al. (2010) performed comparisons on the exoproteins from virulent and
low-virulence strains belonging to genetic lineages I, II and III grown in minimal medium at 37 °C and found significant differences in the presence of the InlC and LLO proteins in the culture supernatants of the virulent strains, indicating the correlation of these exoproteins with the virulence potential of the strains (Table I.2). These results emphasize the need for the inclusion of low-virulence strains in proteomic studies to gain new insights into the expression of virulence factors.
I.2.4. Key exoproteins produced at 37 °C
I.2.4.1 Lmo0443, a protein with a putative role in cell-wall structural maintenance
Dumas et al. (2008) found that the Lmo0443 protein, encoded by lmo0443, was overexpressed in the less virulent L. monocytogenes strains and underexpressed in the more virulent ones (Table I.2) and is part of the core exoproteome of L. monocytogenes (Table I.1) (Dumas et al., 2009a). This protein is similar to the Bacillus subtilis transcription regulator LytR, which belongs to LytR/CpsA/Psr family and has a putative role in cell-wall structural maintenance, possibly through autolysin regulation (Chatfield et al., 2005). The members of this family consist of membrane-anchored proteins with a short cytoplasmic N-terminal
sequence, a transmembrane helix and a large extracellular region containing the LytR/CpsA/Psr domain. Although the main function of these proteins remains unclear, they are known to influence the clinically relevant attributes of various gram-positive pathogens, such as cell division and septum formation (Chatfield et al., 2005; Hübscher et al., 2008; Johnsborg and Havarstein, 2009; Over et al., 2011). Recently, Minami et al. (2012) found that the lytR mutant strain of the gram-positive bacterium Streptococcus pyogenes had a higher activity of the cysteine protease SpeB and was more virulent than the wild-type strain. Consistent with the results of Dumas et al. (2008), the LytR protein of S. pyogenes (25% similar to the Lmo0443 protein of L. monocytogenes) seems to play an important role in the virulence of S. pyogenes. The transcription of lmo0443 is controlled by CesRK, an antibiotic-sensing two-component system in L. monocytogenes, and is up-regulated by cell wall- targeting antibiotics such as ampicillin, vancomycin, cefuroxime (Kallipolitis et al., 2003; Gottschalk et al., 2008; Nielsen et al., 2012), amoxicillin, penicillin and teicoplanin (Knudsen
et al., 2012). Knudsen et al. (2012) also tested protein- and DNA/RNA-synthesis-targeting
antibiotics and found that lmo0443 expression is downregulated by tetracycline and upregulated by co-trimoxazole. The gene encoding Lmo0443 has an ortholog in L. innocua; however, it is only expressed in L. monocytogenes (Schaumburg et al., 2004; Trost et al., 2005). The expression of lmo0443 is co-regulated by PrfA and B; in the absence of B, PrfA suppresses the transcription of lmo0443, whereas in the presence of B, PrfA promotes the transcription of lmo0443 (Ollinger et al., 2009; Chaturongakul et al., 2011).
I.2.4.2 Pleiotropic effects of listeriolysin O
LLO, a member of the cholesterol-dependent cytolysin (CDC) family of bacterial toxins, was first described as essential for the intracellular escape of L. monocytogenes from the internalization vacuole and subsequent growth within infected cells (Gaillard et al., 1987; Sun
et al., 1990) (Table I.2). LLO is positively regulated by PrfA (Leimeister-Wachter et al., 1990).
LLO is also required for the exit of L. monocytogenes from the double-membrane vacuole that is formed during cell-to-cell spreading (Gedde et al., 2000; Dancz et al., 2002) (Fig. I.1).
Table I.2 Exoproteins identified in the supernatants of 37 C-grown culture of different strains of
L. monocytogenes
Characteristics / functional description References
Lmo0443
Similar to B. subtilis transcription regulator LytR protein Chatfield et al., 2005 The expression of lmo0443 is significantly co-regulated
by PrfA and B
Ollinger et al., 2009; Chaturongakul et al., 2011 Overexpressed in less virulent L. monocytogenes strains
and underexpressed in more virulent ones
Dumas et al., 2008 In S. pyogenes, the lytR mutant was more virulent than
the wild-type strain.
Minami et al., 2012 Putative role in cell-wall structural maintenance, that
could influence clinically important attributes of pathogens
Chatfield et al., 2005; Hübscher et al., 2008; Johnsborg and Havarstein, 2009; Over et al., 2011 Transcription is controlled by CesRK, an antibiotic-sensing
two-component system in L. monocytogenes.
Gottschalk et al., 2008 Up-regulated by several cell wall-acting antibiotics and
proteins and DNA/RNA synthesis-acting antibiotics.
Kallipolitis et al., 2003; Nielsen et al., 2012; Knudsen et al., 2012
Down-regulated by tetracycline. Knudsen et al., 2012
Listeriolysin O
Inside host cells
Essential for intracellular L. monocytogenes escape from the internalization vacuole and from the double-membrane vacuole
Gaillard et al., 1987; Sun et al., 1990; Gedde et al., 2000; Dancz et al., 2002 A pore-forming toxin that suppresses host mechanisms
against pathogen
Hamon et al., 2012 Outside host cells
LLO, a cholesterol-dependent cytolysin, is positively regulated by PrfA, and induces bacterial internalization, modulation of host immune response and actions on host cell organelles
Leimeister-Wachter et al., 1990;
Kayal and Charbit, 2006; Stavru et al., 2011; Hamon et al., 2012; Pillich et al., 2012 Induces mucin exocytosis from GCs and induces
upregulation of mucin genes
Lievin-Le Moal et al., 2005
Internalin C
One of the major protein targets of host immune response against L. monocytogenes. The gene inlC is absent in some strains from lineage III. Deletion mutants showed reduced virulence.
Engelbrecht et al., 1996; Domann et al., 1997; Grenningloh et al., 1997; Doumith et al., 2004; Tsai et al., 2006; Liu et al., 2007
Higher levels of InlC are related to higher pathogenic potential
Dumas et al., 2008; Dumas et al., 2009a; Cabrita et al., 2010
Harbors an “internalin domain” which comprise a LRR-IR domain
Ooi et al., 2006 The gene inlC is positively regulated by PrfA at 37 C,
but is not regulated by B
McGann et al., 2007b; McGann et al., 2007a; Port and Freitag, 2007; McGann et al., 2008
