Molecular analysis of candidate probiotic effector molecules of Lactobacillus plantarum Daniela Maria Remus Thesis committee Promoter Prof. dr. Michiel Kleerebezem Professor of Bacterial Metagenomics Wageningen University Co-promoter Dr. Peter A. Bron Senior Scientist, NIZO food research BV, Ede Other members Prof. dr. Tjakko Abee, Wageningen University Prof. dr. Pascal Hols, University of Lovain (Lovain la Neuve), Belgium Dr. Philippe Langella, National Institute of Agricultural Research (INRA), Paris, France Prof. dr. Roland J. Siezen, Radboud University, Nijmegen This research was conducted under the auspices of the Graduate School VLAG (Advanced studies in Food Technology, Agrobiotechnology, Nutrition and Health Sciences). Molecular analysis of candidate probiotic effector molecules of Lactobacillus plantarum Daniela Maria Remus Thesis submitted in fulfillment of the requirements for the degree of doctor at Wageningen University by the authority of the Rector Magnificus Prof. dr. M.J. Kropff, in the presence of the Thesis Committee appointed by the Academic Board to be defended in public on Tuesday 09 October 2012 at 4 p.m. in the Aula. Daniela Maria Remus Molecular analysis of candidate probiotic effector molecules of Lactobacillus plantarum Ph.D. Thesis, Wageningen University, Wageningen, The Netherlands (2012) With references and summaries in English and Dutch. ISBN 978-94-6173-373-3 Summary Lactobacilli occupy diverse natural habitats, including dairy products and the mammalian gastrointestinal (GI) tract, and several Lactobacillus strains are marketed as probiotics that can interact with host cells in the GI tract by which they are proposed to beneficially influence the health status of their consumers. The discovery of probiotic effector molecules is instrumental to understand the exact modes of probiotic action, which is required for their controlled, safe, and purpose-directed application. This thesis focuses on the molecular characterization of effector molecules of L. plantarum WCFS1, a model organism for probiotic lactobacilli. The molecular mechanism underlying the previously established growth phase-dependent capability of L. plantarum to modulate NF-κB associated pathways in the mucosa of healthy human volunteers was studied by a combined approach of genome- wide transcriptomics and cell surface proteomics. The impact of cell surface trypsinization samples on NF-κB promoter activation was assessed using a dedicated NF-κB reporter derivative of the Caco-2 intestinal epithelial cell (IEC) line. Surface proteome fractions derived from late stationary, but not from mid-logarithmic phase bacterial cells were found to be effective attenuators of NF-κB activation. One of the surface proteins that was found to be up-regulated in the late stationary phase, i.e. StsP, a sortase-dependent protein (SDP), was previously shown to be induced in the intestinal tract of mice and humans. Increased StsP expression was engineered in vitro and was shown to elicit strong NF-κB attenuation in IECs, providing evidence for the role of this cell surface protein in host cell signaling. SDPs were further studied in a sortase-deficient L. plantarum strain (srtA), in which SrtA-deficiency led to decreased amounts but not complete lack of specific SDPs. In addition, SDPs could readily be removed form the cell surface of the SrtA-deficient strain using LiCl-based protein extraction, which was not observed with the wild-type strain. The LiCl extraction impacted strongly on the pro-inflammatory signaling properties of the SrtA-deficient but not of the wild-type strain, supporting a role of one or more SDPs in attenuation of host immune responses. Next to proteins, the role of bacterial surface glycans in host interaction was studied in capsular polysaccharide (CPS)-deficient mutants, revealing that reduced levels of cell surface glycans increase host cell recognition via Toll-like receptor 2. The results presented in this thesis highlight the communication strategies employed by L. plantarum by correlating specific host responses to bacterial effectors that were modulated based on their expression, sub-cellular location, and/or exposure on the bacterial cell surface. Table of contents Chapter 1 General introduction - An intimate tête-à-tête - 1 How probiotic lactobacilli communicate with the host Chapter 2 Late stationary phase surface peptides of 31 Lactobacillus plantarum attenuate NF-κB activation Chapter 3 The impact of Lactobacillus plantarum sortase on 67 target-protein sorting, gastrointestinal persistence, and in vitro immunomodulation Chapter 4 The gastrointestinally induced surface protein 97 StsP of Lactobacillus plantarum attenuates NF-κB responses in intestinal epithelial cells Chapter 5 Impact of 4 Lactobacillus plantarum capsular 123 polysaccharide clusters on surface glycan composition and host cell signaling Chapter 6 General discussion 159 Appendices Nederlandse samenvatting 180 Acknowledgements 182 About the author 185 Co-author affiliations 186 Overview of completed training activities 187 Chapter 1 General Introduction An intimate tête-à-tête - How probiotic lactobacilli communicate with the host Daniela M. Remus Adapted version was published in Remus D.M., Kleerebezem M., and Bron P.A. (2011) An intimate tête-à-tête - How probiotic lactobacilli communicate with the host. Eur J Pharmacol. 668, S33-42. 1 Chapter 1 Abstract Pharmaceutical agents are routinely used in the treatment of gastrointestinal disorders and their role as modulators of host cell responses is well characterized. In contrast, the understanding of the molecular mechanisms, which determine the role of probiotics, i.e. health-promoting bacteria, as host cell modulators is still in its infancy. Both in vitro and in vivo studies are just starting to reveal the capability of probiotic lactobacilli to modulate host cell- signaling networks and the associated influences on downstream regulatory pathways, including modulation of mucosal cytokine profiles that dictate host immune functions. The communication between probiotic lactobacilli and intestinal host cells is multifactorial and involves an integrative repertoire of receptors on the host side that recognize multiple effector molecules on the bacterial side, of which most have been found to be cell wall- or cell surface- associated compounds and proteins. This chapter describes the discovery of these bacterial effector molecules and their role in strain- and species- specific modulation of host signaling pathways. Unraveling the mechanisms responsible for probiotic–host interactions will progress this research field towards molecular science and will provide markers for probiotic product quality control as well as host- response efficacy. These developments can ultimately lead to a more dedicated, personalized application of probiotics with strong molecular and scientific support for health promotion. 2 Chapter 1 Introduction The first historical evidence for the consumption of fermented dairy products by humans can be traced back to ancient Egypt as early as 7000 BC (1). Thousands of years later, in 1908, Elie Metchnikoff introduced the idea that fermentation of dairy products brings about more than just extended shelf life and enhanced taste. His work entitled “The prolongation of life” proposed that these products also promote beneficial health effects upon the consumer, which was associated with the consumption of certain lactic acid bacteria (LAB) (2). These findings set the scene for the concept of “probiotic bacteria”. The definition of the term probiotics underwent several changes until the Food and Agriculture Organization of the United Nations and the World Health Organization proposed the currently widely accepted definition that probiotics are “live microorganisms, which, when administered in adequate amounts, confer a health benefit on the host (3). Remarkably, more than a century after the observations by Metchnikoff, LAB, and more specifically certain Lactobacillus species, still represent a substantial proportion of the probiotics marketed today (4). The beneficial effects of probiotic lactobacilli have been exemplified by a manifold collection of in vitro and in vivo studies, which revealed a variety of host effects and generated hypotheses of the mechanisms by which lactobacilli influence their hosts (5). The beneficial strategies of lactobacilli include the reduction of infection risks by strengthening the barrier function of the intestinal epithelium via stimulation of mucin secretion (6) or enhancement of tight-junction functioning (7, 8). Alternatively, probiotics may directly affect the clearance of pathogens by competitive exclusion (9, 10), for example by synthesizing antimicrobial substances such as bacteriocins (11) or lactic acid (12, 13). In addition, the modulation of the host immune response has been proposed as one of the prominent modes of probiotic action accomplished by lactobacilli (5, 14). The tightening of legislation in the European Union for probiotic health claims has led to continued industrial interest to unravel the molecular mechanisms responsible for the observed beneficial probiotic traits. This chapter will discuss the scientific progress that has been made in unraveling the mechanisms involved in the molecular probiotic-host crosstalk. These advances will pave 3 Chapter 1 the way into the next generation of probiotic applications and might stimulate the use of probiotics to support conventional pharmacological therapies or targeted modulation of health