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Stress Response and Virulence in Salmonella Typhimurium: a Genomics Approach

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Stress Response and Virulence in Salmonella Typhimurium: a Genomics Approach Stress response and virulence in Salmonella Typhimurium: a genomics approach Armand P. H. M. Hermans Promotoren Prof. Dr. T. Abee Persoonlijk hoogleraar bij de leerstoelgroep Levensmiddelenmicrobiologie Wageningen Universiteit Prof. Dr. Ir. M. H. Zwietering Hoogleraar Levensmiddelenmicrobiologie Wageningen Universiteit Co-promotor Dr. H. J. M. Aarts Clusterleider Microbiologie en Novel Foods RIKILT – Instituut voor Voedselveiligheid, Wageningen UR Promotiecommissie Prof. Dr. S. Brul Universiteit van Amsterdam Prof. Dr. Ir. A. H. C. van Bruggen Wageningen Universiteit Dr. M. Kleerebezem NIZO food research, Ede Prof. Dr. C. W. Michiels Katholieke Universiteit Leuven, België Dit onderzoek is uitgevoerd binnen de onderzoekschool VLAG Stress response and virulence in Salmonella Typhimurium: a genomics approach Armand P. H. M. Hermans Proefschrift ter verkrijging van de graad van doctor op gezag van de rector magnificus van Wageningen Universiteit, Prof. Dr. M. J. Kropff, in het openbaar te verdedigen op dinsdag 16 januari 2007 des namiddags te half twee in de Aula Hermans, Armand Petrus Henricus Maria Stress response and virulence in Salmonella Typhimurium: a genomics approach - 2007 Thesis Wageningen University, Wageningen, The Netherlands – with summary in Dutch – 182 p. Keywords: Salmonella serovar Typhimurium / stress response / virulence / prophages / transcriptomics ISBN: 90-8504-572-X CONTENTS Abstract 7 Chapter 1 Introduction and outline of the thesis 9 Chapter 2 Identification of novel Salmonella enterica serovar Typhimurium DT104-specific prophage and non-prophage chromosomal sequences among serovar Typhimurium isolates by genomic subtractive hybridization 31 Chapter 3 Distribution of prophages and SGI-1 antibiotic resistance genes among different Salmonella enterica serovar Typhimurium isolates 47 Chapter 4 Development and assessment of a stress and virulence thematic oligonucleotide microarray for Salmonella enterica serovar Typhimurium DT104 65 Chapter 5 Comparative transcriptome analysis of stress and virulence genes in Salmonella enterica serovar Typhimurium DT104 wild type and its LuxS deletion mutant at various growth phases 81 Chapter 6 Comparative transcriptome analysis of Salmonella enterica serovar Typhimurium DT104 stress response under aerobic and anaerobic conditions 101 Chapter 7 Summary, concluding remarks and future perspectives 121 Addendum Supplementary material of Chapter 4 134 Supplementary material of Chapter 5 148 Supplementary material of Chapter 6 157 Samenvatting 169 Dankwoord 173 List of publications 177 Curriculum Vitae 179 ABSTRACT Since 1995 the number of infections caused by multiple-antibiotic-resistant Salmonella serovar Typhimurium phage type DT104 isolates has increased in many parts of the world. Several hypothetical reasons have been proposed to explain this increase. The multiple-antibiotic-resistance and also increased virulence and/or survival of stressful conditions have been mentioned. The current molecular biology research has been focused on the antibiotic resistance genes. In our research, we isolated two novel Salmonella serovar Typhimurium DT104-specific DNA fragments which are associated with virulence. One fragment was identical to irsA, which is suggested to be involved in macrophage survival and the other fragment was homologous to HldD, an Escherichia coli O157:H7 protein involved in a specific lipopolysaccharide (LPS) assembly pathway. While analyzing all novel fragments obtained, we were able to identify a DT104-specific prophage (= bacteriophage integrated into the bacterial genome) remnant that we designated prophage ST104B and that harbors the HldD homologue. We observed that the presence of the genes coding for multiple-antibiotic-resistance, the HldD homologue containing ST104B prophage remnant and phage subtype DT104L revealed to be common features of the most emerging variant within the group of different Salmonella serovar Typhimurium DT104 isolates. Furthermore, a thematic oligo microarray was developed for Salmonella serovar Typhimurium DT104 to be able to study expression of 425 genes involved in stress response and virulence. This microarray was used to measure gene expression in the course of time during growth of a Salmonella serovar Typhimurium DT104 wild type strain and its luxS deletion mutant, since LuxS might play a role in stress response and virulence. Expression of stress response and virulence genes in the course of time appeared to be largely growth-phase-dependent. The most prominent effect of deleting the luxS gene was the increased expression of 15 LPS synthesis and assembly genes at the end-exponential growth phase. Notably, the luxS deletion mutant showed higher in vitro adhesion and invasion capacity into human epithelial cells, although the expression levels of invasion genes appeared to be similar to that in the wild type. We concluded that the loss of luxS results in overexpression of LPS genes and most likely also the LPS molecules, thereby affecting in vitro virulence characteristics of this DT104 luxS deletion mutant. In addition, gene expression was measured and compared for a Salmonella serovar Typhimurium DT104 wild type strain cultured under aerobic and anaerobic conditions and subjected to heat, oxidative, and acid stress. Stress genes such as the RpoS and PhoPQ regulon, chaperones and universal stress proteins were mainly induced or repressed in a similar manner in both aerobically or anaerobically grown cells when subjected to the different stresses. Furthermore, the virulence(-associated) LPS, PhoPQ, Spv, SPI-1, and SPI-2 encoded genes were differentially regulated by the different stresses. The thematic microarray developed allows assessment of the impact of stresses and combinations thereof on the expression of stress and virulence genes for Salmonella serovar Typhimurium DT104. Finally, the results of our molecular biology research enlarges the current understanding of characteristics of emergence of Salmonella serovar Typhimurium DT104 by harboring additional virulence factors or by surviving stress conditions. Chapter 1 Introduction and outline of the thesis Chapter 1 INTRODUCTION The foodborne pathogen Salmonella. Salmonellae are Gram-negative, rod shaped, facultatively anaerobic, nonspore- forming, usually motile bacteria (Fig. 1). In general, salmonellae are able to infect a wide range of hosts from cold-blooded animals to humans resulting in diseases ranging from mild diarrhea and gastroenteritis to severe systemic infections such as typhoid fever. Some Salmonella types can infect a wide variety of animals, while others only one. For example, the serovars Typhimurium and Enteritidis infect humans as well as chickens and mice. Other serovars are host-adapted, infecting only a few species such as serovar Dublin which primarily infects cattle and serovar Choleraesuis which primarily infects swine. Finally, some serovars are host-specific, infecting only one single animal host, such as serovar Typhi that only infects humans and serovar Pullorum that only infects chickens. Although Salmonella serovars share some virulence factors needed for intestinal infections, unique virulence factors among serovars also occur that are responsible for host specificity and specific disease symptoms (28). Salmonellae infections in humans are generally foodborne but can also be contracted through contact with infected animals. Food products from farm animals such as poultry, pigs, and cattle have been identified as an important source of human salmonellae infections (81). Estimations are made that worldwide each year up to 1.3 billion cases of acute gastroenteritis and diarrhea are caused by non-typhoid salmonellosis, resulting in 3 million deaths annually. Furthermore, about 16 million cases of typhoid fever occur each year with a fatal outcome of around 0.6 million. Although fatal salmonellae infections mainly occur in developing countries, mortality due to acute salmonellae infections also has an important impact in industrialized countries. FIG. 1. Transmission electron Especially children and elderly people have a higher risk micrograph (false coloured) of Salmonella serovar Typhimurium by P. of dying because of the dehydrating effects of diarrhea Gunnig and R. Bongaerts IFR Norwich (39, 60). In The Netherlands approximately 50,000 (http://www.foodandhealthnetwork.com/ infections with salmonellae occur annually (83). predictive.html) Salmonellae taxonomy. To identify salmonellae among the large number of different bacteria occurring in the environment, several biochemical reactions are performed. The following general reaction pattern represents salmonellae apart from a few exceptions. Salmonellae are able to ferment and form gas on glucose and produce H2S. Lactose and sucrose are not fermented and urea is not hydrolyzed. They form lysine-decarboxylase and do not form indole and acetyl- methylcarbinol. Finally, salmonellae do not show ß-galactosidase activity (26, 54). Based on differences in these biochemical reactions (the few exeptions) and surface proteins, salmonellae can be divided into the two species Salmonella enterica and Salmonella bongori (Fig. 2), and subsequently S. enterica into six subspecies (ssp.): ssp. houtenae, ssp. 10 Introduction FIG. 2. Schematic representation of salmonellae classification and diversity. arizonae, ssp. diarizonae, ssp. salamae, and ssp. indica. In addition, more than 2,000 serovars can be assigned to ssp. enterica. Examples of well known serovars are Paratyphi, Typhi and Typhimurium
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