Host-interaction effector molecules of Lactobacillus plantarum WCFS1
Lee, I.C. - \ 2016
Wageningen University. Promotor(en): Michiel Kleerebezem, co-promotor(en): P.A. Bron. - Wageningen : Wageningen University - ISBN 9789462576858 - 183 p.
lactobacillus plantarum - molecules - probiotics - immunomodulatory properties - lipoproteins - interactions - molecular interactions - host pathogen interactions - moleculen - probiotica - immunomodulerende eigenschappen - lipoproteïnen - interacties - moleculaire interacties - gastheer-pathogeen interacties
Lactobacillus plantarum is found in various environmental habitats, including fermentation products and the mammalian gastrointestinal tract, and specific strains are marketed as probiotics, which are defined as ‘live microorganisms which when administered in adequate amounts confer a health benefit on the host’. Throughout the studies of the mechanisms underlying probiotic activity, it became apparent that the probiotic effects are often species and/or strain specific. This situation has led more researchers to focus on the molecular characteristics of probiotic strains intending to link specific molecular structures to specific probiotic functions, and thereby deduce the mechanisms of molecular communication of probiotics. This thesis focuses on potential cell envelope effector molecules involved in interaction with the mammalian host cells, including lipoteichoic acid (LTA), lipo- and glyco-proteins, and extracellular polysaccharides (EPS), of L. plantarum WCFS1, a model strain for probiotic lactobacilli with a well-annotated genome sequences and sophisticated genetic engineering tools. First, existing research regarding the potential roles in probiotic functionality of Lactobacillus surface molecules in terms of their biosynthesis pathways and structure variations as well as interaction with host Pattern Recognition Receptors (PRRs) and immunomodulatory properties of these molecules are summarized and compared to provide an overview of the state-of-the-art in probiotic effector molecule research. Subsequently, specific molecules that reside in the cell envelope of L. plantarum WCFS1 were study for their role in bacterial physiology, as well as their role as ligands in Toll-like receptor (TLR) 2 signaling and immunomodulatory properties using human-cell co-incubation models. Our results showed that the deficiency of LTA had a drastic impact on cell division, cell morphology and growth in L. plantarum WCFS1, while LTA-deficient cells also elicited more pro-inflammatory responses in PBMCs rather than the expected loss of pro-inflammatory capacity as was observed with similar mutants of Lactobacillus acidophilus NCFM. Further studies on the signaling capacity of the purified LTA from L. plantarum WCFS1 revealed that these molecules are poor TLR2 activators, which is in clear contrast to the highly potent TLR2 stimulatory capacity of LTA obtained from Bacillus subtilis, implying that structural differences of the LTA produced by different bacteria are prominent determinants of their TLR2 signaling capacity and immunomodulatory properties. Lipoproteins of L. plantarum WCFS1 were studied using a derivative strain that is deficient in prolipoprotein diacylglyceryltransferase (Lgt), which transfers acyl chain moieties onto lipoproteins. The lipid moiety was shown to be important for proper anchoring of lipoproteins and TLR1/2 signaling capacity, but did not affect TLR2/6 signaling, suggesting that lipoproteins of L. plantarum WCFS1 are predominantly (if not exclusively) triacylated. The Lgt deficient strain elicited more pro-inflammatory responses in PBMCs as compared to the wild type, indicating that the native lipoproteins could play a role in dampening inflammation upon host-probiotic interaction. In addition, we explored the protein glycosylation machinery in L. plantarum WCFS1, responsible for the glycosylation of the major autolysin (Acm2) of this bacterium, which was previously shown to be O-glycosylated with N-acetylhexosamine conjugates. Using sequence similarity searches in combination with a lectin-based glycan detection and mass spectrometry analysis, two glycosyl-transferases, GtfA and GtfB (formerly annotated as TagE5 and TagE6, respectively), were shown to be required for the glycosylation of Acm2 and other unidentified L. plantarum WCFS1 glycosylated proteins. These results provide the first example of a general protein-glycosylation machinery in a Lactobacillus species. Finally, extracellular polysaccharides (EPS) in L. plantarum were studied in two strains that produce large amounts of EPS: L. plantarum SF2A35B and Lp90, in comparison to the lowly producing model strain WCFS1. Based on genome sequence comparison, both of the high producer strains were found to possess strain-specific and unique polysaccharide gene clusters. These gene clusters were deleted and the mutants were shown to have lost the capacity to produce large amounts of EPS, and were studied in relation to their properties in host-bacteria interaction. The results illustrate strain-specific and variable impacts of the removal of the EPS in the background of individual L. plantarum strains, supporting the importance of EPS in L. plantarum strains as a strain-specific determinant in host interaction. Overall, this thesis showed that surface molecules not only play important roles in bacterial physiology, but also in the interaction with the host mucosa through pattern recognition receptors expressed by the host cells. With the growing amount of evidence of structural variations in surface molecules, which are influenced by genetic background, physiological status, environmental factors, and other biological processes, these molecules form a unique signature associated with each strain that as a consequence elicits a strain-specific response when interacting with host cells.
Protein mixtures: interactions and gelation
Ersch, C. - \ 2015
Wageningen University. Promotor(en): Erik van der Linden, co-promotor(en): A.H. Martin; Paul Venema. - Wageningen : Wageningen University - ISBN 9789462574212 - 199
eiwit - wei-eiwit - sojaeiwit - gelering - gelatine - gels - reologie - structuur - moleculaire interacties - protein - whey protein - soya protein - gelation - gelatin - rheology - structure - molecular interactions
Gelation is a ubiquitous process in the preparation of foods. As most foods are multi constituent mixtures, understanding gelation in mixtures is an important goal in food science. Here we presented a systematic investigation on the influence of molecular interactions on the gelation in protein mixtures. Gelatin gels with added globular protein and globular protein gels with added gelatin were analyzed for their gel microstructure and rheological properties. Mixed gels with altered microstructure (compared to single gels) also differed in modulus from single gels. Mixed gels with microstructures similar to single gels were rheologically similar to single gels. Alterations in microstructure were attributed to segregative phase separation between proteins which occurred during gelation. Gelation was treated as a growth process from macromolecule to space spanning network. At conditions where electrostatic interactions were screened the occurrence of phase separation was attributed to the molecular size ratio between gelling and non-gelling proteins before gelation and changes of this size ratio during gelation. Here only mixtures that during gelation passed a region of high compatibility (similar molecular sizes) before entering a region of decreasing solubility phase separated. For applications this implies that whenever the gelling molecule is larger than the non-gelling molecule phase separation during gelation is unlikely while reversely, if the gelling molecules is smaller than the non-gelling molecule phase separation during gelation typically does occur
Berkel, W.J.H. van - \ 2011
Wageningen : Wageningen University - ISBN 9789085858935 - 28
enzymologie - enzymen - moleculaire interacties - enzymology - enzymes - molecular interactions
Analysis of molecular interactions between yoghurt bacteria by an integrated genomics approach
Sieuwerts, S. - \ 2009
Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): J.E.T. van Hylckama Vlieg. - [S.l. : S.n. - ISBN 9789085854654 - 221
streptococcus thermophilus - lactobacillus delbrueckii subsp. bulgaricus - moleculaire interacties - transcriptomics - genexpressieanalyse - molecular interactions - genomics
The lactic acid bacteria (LAB) are a group of Gram-positive bacteria that ferment sugars such as lactose to produce mainly lactic acid. LAB are a group of industrially important microorganisms that are applied for the production of many fermented foods. These include foods produced with substrates from plant origin (e.g. sauerkraut and wine) and animal origin (e.g. fermented meats and dairy products such as yoghurt). The current market trends regarding sustainability and health-promoting foods demand more efficient and a more diverse range of fermentations. Most fermentations are carried out by multiple strains of different species. The interactions between consortium members are at the base of the performances of the individual microorganisms within a microbial ecosystem and therewith of the whole fermentation. These microbial interactions are often poorly understood. Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus are two LAB species that upon fermentation convert (bovine) milk into yoghurt. These two bacteria stimulate each other in growth and acid production. They produce exopolysaccharides (EPS), important for the texture of yoghurt, and characteristic flavor compounds such as acetaldehyde and diacetyl. However, the molecular basis of the mutualistic interactions between these two bacteria was poorly characterized.
In this thesis research, a combination was used of screening, mixed culture transcription profiling, whole-genome metabolic modeling, experimental evolution and next-generation sequencing. This was done to unravel the molecular basis of the interactions between S. thermophilus and L. bulgaricus in milk. The results showed that interactions were primarily based on the exchange of metabolites (see Figure 1). Moreover, it was shown which genes or pathways were affected. Evidence was found that S. thermophilus provided L. bulgaricus with formic acid, folic acid (both involved in purine metabolism), long-chain fatty acids (by the action of lipolytic enzymes to break down milk fat) and CO2. The proteolysis by the exoprotease of L. bulgaricus, in turn, provided both species peptides, which are taken up by the cell and broken down into amino acids (AA) by intracellular peptidases. However, this probably did not yield a sufficient supply of branched-chain and sulfur AA, leading to a higher expression of the genes for biosynthesis of these AA in both species when grown in mixed culture. Moreover, EPS biosynthesis genes were induced in the mixed culture, leading to increased EPS production and a higher viscosity of the yoghurt.
Figure 1. Schematic representation of the mutualistic interactions between S. thermophilus and L. bulgaricus in yoghurt. Solid arrows indicate interactions; dotted arrows indicate pathways that are affected by the interactions. Pathways that were for the first time shown to be regulated at the transcriptome level upon co-culture are indicated in bold. Pathways that were confirmed in our study to be regulated at the transcriptome level upon co-culture are underlined. EPS is hypothesized to promote the exchange of both bacteria. There was no evidence at the transcriptome level for the exchange of putrescine and ornithine. AA, amino acids; BCAA, branched-chain AA; EPS, exopolysaccharides; LCFA, long-chain fatty acids.
A mixed culture genome-scale metabolic model confirmed that cross-feeding interactions between the yoghurt bacteria were based on purine and AA metabolism. Moreover, this model was used to show that the interactions provided a significant benefit to both bacteria, i.e. their biomass yield on lactose increased by around 50% in mixed culture.
Experimental evolution revealed that it is possible to co-adapt a novel combination of strains of S. thermophilus and L. bulgaricus. It was shown that their mutual stimulation increased by optimizing their interactions by fine-tuning expression of pathways involved in the interactions. Furthermore, as little as ~1000 generations of co-culture was sufficient to transform the relatively slow growing mixed culture into one that showed similar performance as commercial starters with respect to key characteristics such as acidification rate and viscosity.
Improved understanding of the described interactions that are at the base of the yoghurt fermentation provides us targets for the rational optimization of existing mixed culture fermentations and the rational development of industrially relevant mixed cultures, such as those containing probiotics. Moreover, the results are in particular interesting for the field of microbial ecology as they show how mutual nutritional dependencies evolve and structure the microbial composition of this ecosystem.
RxLR-effectors en avirulentiefactoren van Phytophthora infestans
Govers, F. ; Weide, R.L. - \ 2009
phytophthora infestans - moleculaire biologie - resistentiemechanismen - resistentieveredeling - moleculaire interacties - molecular biology - resistance mechanisms - resistance breeding - molecular interactions
Characterization of genes coding for small hypervariable peptides in Globodera rostochiensis
Bers, N.E.M. van - \ 2008
Wageningen University. Promotor(en): Jaap Bakker, co-promotor(en): Geert Smant; Aska Goverse. - [S.l.] : S.n. - ISBN 9789085049579 - 229
globodera rostochiensis - plantenparasitaire nematoden - peptiden - genen - genexpressie - solanum tuberosum - arabidopsis - nicotiana - gensplitsing - gastheer-pathogeen interacties - moleculaire interacties - plant parasitic nematodes - peptides - genes - gene expression - gene splicing - host pathogen interactions - molecular interactions
Plant parasitic nematodes secrete a cocktail of effector molecules, which are involved
in several aspects of the interaction with the host, eg. in host defense suppression, in
migration and in feeding cell formation. In this thesis, we performed the first study on
10 novel peptide genes, believed to be important for parasitism of the potato cyst
nematode, Globodera rostochiensis. Nine of the peptide genes described here belong
to the SECPEP gene family. The SECPEP genes are all expressed in the dorsal
esophageal gland, which is one of the main sites for the production of effector
molecules. This, together with the predominant expression in preparasitic and early
parasitic juvenile nematodes, makes it very likely that the SECPEPs code for effector
peptides essential for succesful infection and feeding site formation.
In chapter 2, we show that diversifying selection is a likely driver of the molecular
evolution of the SECPEPs. The sequences of the mature peptides appear to be highly
diverse, while the non)coding 3’UTR and intronic regions as well as the region coding
for the signal peptide for secretion are relatively conserved. In fact, a pairwise
comparison of the SECPEPs reveals no significant sequence similarity between family
members at all. In chapter 5 we further speculate on a possible role for RNA)editing as
a mechanism to yield hypervariability in the SECPEPs, because the sequence diversity
at the transcript level significantly exceeds that of the genomic locus. Chapter 5 further
elaborates on the analysis of trans)splicing in SECPEP1 transcripts. We show that
SECPEP1 transcripts are trans)spliced to a surprising diversity of novel spliced)leader
sequences. The first approach to unravel the role of the members of the SECPEP family
in plant parasitism, is described in chapter 4. We generated transgenic potato and
Arabidopsis plants expressing SECPEP3 while using the CaMV 35S promotor. The
phenotype associated with SECPEP3 in both potato and Arabidopsis plants includes a
reduction of root growth and an alteration of the leaf morphology. The SECPEP3
peptide harbors several sequence motifs first found in the cyclin)dependent kinase
inhibitors ICK1/KRP1, SIM and Smr1. We, therefore, suggest a role for SECPEP3 in cell
cycle alteration in nematode feeding site formation. Although the SECPEP genes show
only a low level of primary sequence similarity, all code for positively charged,
hydrophilic peptides with a C)x)G γ)core motif (chapter 2). These are characteristics
typical for host defense peptides, and in chapter 6 we investigate whether these
characteristics are also found for other peptides involved in plant)parasite interactions.
We show that a considerable number of these effector peptides share a positive
charge, hydrophilicity and C)x)G γ)core motif with the SECPEPs, and we speculate on a
role for the positive charge in peptide)ligand interaction.
In chapter 3 we describe the NEMPEP peptide, secreted by G. rostochiensis. NEMPEP
is also a positively charged, hydrophilic peptide with a C)x)G γ)core motif, although it is
genetically unrelated to the SECPEP gene family. During the life cycle of G.
rostochiensis, the expression pattern of NEMPEP reveals a striking regulation. NEMPEP
is highly expressed in preparasitic juveniles and in the parasitic life stages after initial
feeding cell formation. However, NEMPEP expression was hardly detectable in the
juveniles just after entering the plant root. Several disease resistance genes condition
nematode resistance at the onset of parasitism. The downregulation of NEMPEP at
exactly this timepoint could be a strategy to avoid recognition by the host’s immune
system. In planta expression of NEMPEP, as a fusion to GFP, shows that NEMPEP
accumulates in the nucleolus of tobacco cells. Potato plants transformed with
35S::NEMPEP were slow at forming roots and the internodes between the leaflets were
shortened. This, together with a reduced transformation efficiency, led us to
hypothesize a role for NEMPEP in cytokinin signaling (Chapter 3).
Currently, there are two models regarding the functional role of the SECPEPs and
NEMPEP. The first one concerns a role as an antimicrobial peptide, which could protect
the host plant against secondary infections by opportunistic microbes. As a competing
hypothesis, the high hydrophilicity of the peptides may point to a role as peptide
hormone. As such, they may be involved in redirecting cell cycle or hormonal regulation
upon feeding cell formation.