Evidence of a large diversity of N-acyl-homoserine lactones in symbiotic Aliivibrio fischeri strains associated to the squid Euprymna Scolopes

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QS associated phenotypes

It is now clear that communication is widely widespread and controls many physiological processes in Vibrio. QS allows bacteria to act socially as a cohesive unit in order to synchronize their behaviour like multicellular organisms. The group detects and respond to the signal molecule by a population-wide alteration of gene expression. These processes are predicted to be effective when undertaken by a group of bacteria but detrimental for single cells (Bassler and Losick, 2006; Waters and Bassler, 2005). However, recent studies have shown that this model might be oversimplistic and revealed that a non- homogeneous response is observed for the same QS stimuli. In fact numerous other genetic elements and environmental conditions can control these phenotypes and it appears that heterogeneous populations are sometimes more efficient in taking actions (Anetzberger et al., 2009; Miyashiro and Ruby, 2012; Pérez and Hagen, 2010).
In Vibrio, QS associated phenotypes have been widely studied in well described species along with QS pathways and the regulatory mechanisms. A non-exhaustive list of functions controlled by different QS pathways or molecules can be found in Table 1.4. In  addition to the perfectly described mechanism of bioluminescence, QS is an important component of the pathogenicity of Vibrio species and controls many virulence factors as well as some proteases allowing a considerable advantage for host colonization. QS is also determinant for the access to different ecological niches by regulating biofilm formation, exopolysaccharide production, motility or membrane protein expression. The impact of QS has also been observed on the production of extracellular products involved in nutrient acquisition (siderophore production), defence mechanisms or survival strategies such as the VBNC state (Ayrapetyan et al., 2014), pigment production or metabolism (Goo et al., 2015). For less studied Vibrio species much work is needed for the characterization of QS pathways and the identification of signal molecules, prior to the study of QS associated phenotypes.

Interference with bacterial QS

Vibrio species can have symbiotic relationship with higher organisms, where they can be pathogenic, commensal or in mutualistic association (Austin, 2010; Nishiguchi and Nair, 2003; Ruby, 1996). As some of these higher organisms have developed defence mechanisms in order to compete for ecological niches or to protect themselves against bacterial colonisation. QS can be disrupted by diverse metabolites and enzymes. All the QS inactivation mechanisms are connected behind the term Quorum quenching (QQ) and include the disruption of QS signalling pathways by antagonistic/agonistic molecules or the inactivation of QS molecules by enzymatic degradation (Rolland et al., 2016). The global interest on QQ has started with the discovery of the red algae Delisea pulchra and its halogenated furanones able to inhibit the signalling pathways by displacing AHLs from theirreceptors and inactivating the AI-2 synthase LuxS (Givskov et al., 1996; Manefield et al., 1999; Zang et al., 2009).

Link between AHL production and genetic diversity of A. fischeri strains

We examined the relationship between AHL production patterns and the genetic diversity of the associated Aliivibrio fischeri strains. First, it is worth noting that while the strains KB4B5, ES114, KB2B1, and ES213 are phylogenetically distinct based on whole- genome comparisons, these strains all presented the same AHL pattern(Bongrand et al., 2016). Interestingly, these data reveal the absence of a link between whole genome diversity and the AHL production patterns of these strains. Furthermore, the clustering of these strains according to their AHL production patterns did not correlate with the colonization capacities (D- types: ES213 and KB2B1 or S- types: ES114, KB4B5 and MB13B1) or the sampling location of host squids (Kaneohe Bay, KB: ES114, KB4B5 and KB2B1 or Maunalua Bay, MB: ES213 and MB13B1; Wollenberg and Ruby, 2009). In a second step, we then examined the genetic diversity of synthases and receptors involved in AHL meditated QS across A. fischeri strains by focusing our work on five key proteins: AinS, AinR, LuxI, LuxR and LitR. These five genes were identified by BLAST in the strains ES213, KB2B1, MB13B1 and KB4B5 (genome sequenced by Bongrand et al., 2016) using the ES114 strain as a reference (NCBI Accession number: NC_006840). First, we observed a clear separation between MB13B1 and the other strains in AinS, LuxR and LitR- based phylogenies, while no correlation was observed with LuxI and AinR (Figure S2). Such observations imply that AHL production through the AHL synthase AinS as well as signal reception by LuxR and LitR are likely to have substrate specificity, which may vary depending on genetic diversity. As suggested by Collins et al., 2004, LuxR is evolutionary pliable and not fixed in its response to AHLs of different acyl-side-chain lengths. However, we showed that the separation of MB13B1 in a LuxR phylogeny is explained by a difference of 3 amino acids, including 2 located in the amino-terminal domain responsible for the sensitivity to a broad spectrum of AHLs (Table 2, Collins et al., 2004; Fuqua and Greenberg, 2002). Some works have identified the key amino-acids for LuxR specificity towards historical signaling molecules but data are missing for other AHLs as described in the present study (Collins et al., 2004, 2006; Hawkins et al., 2007). The AinS synthase is still poorly studied, but Gilson et al., 1995 have demonstrated that the full-length AinS is necessary to produce AHL. This implies that the four observed amino-acid changes in MB13B1 can possibly have an impact on AHL synthesis. The study of LitR sequences indicates a change of polarity on one amino acid in MB13B1; however, the lack of structural information prevents any conclusions from being drawn (Table 2).


Vibrio isolation and characterization

Water samples (0.1, 1, 10 mL) were filtered onto 0.45μm pore-size nitrocellulose filters (Millipore) and plated on the selective medium thiosulfate-citrate–bile–sucrose (TCBS) (Pfeffer and Oliver, 2003) for the enumeration of total Vibrio CFUs after 24 h incubation at 20°C. Vibrio strains were isolated from two fractions: less than 50 μm assumed to represent Vibrio in the water column and greater than 50 μm corresponding phytoplankton and zooplankton-associated Vibrio. The <50 μm fraction consisted in the concentration of 20 L of water prefiltered on 50 μm using a model HF80S hollow fiber filter (Hemoflow). The concentrated water was back-washed with 500 mL of a wash solutions (0.01% sodium hexametaphosphate, and 0.5% Tween 80; Sigma-Aldrich). The >50 μm fraction consisted of the plankton net tow concentrate split into five subsamples of 15 mL and homogenized by very gentle sonication for 5 min in order to unbind phytoplankton-attached bacteria. For both fractions, a ten-fold serial dilution was made in artificial seawater and 100 μL of each was spread and plated in triplicate on TCBS agar. Thirty colonies by fraction and by sampling date were randomly picked using a gridded Petri dish after 24 h of incubation at 20°C.

Table of contents :

List of Figures
List of Tables
List of Abbreviations
Publications & Communications
CHAPTER 1: Introduction 
1. The genus Vibrio
1.1 Metabolism.
1.2 Classification.
1.3 Pathogenic Vibrio species.
2. Methods for the identification and quantification o
2.1.1 Phenotypical and biochemical identification.
2.1.2 Whole genome based methods.
2.1.3 Gene sequence based methods. Sequencing of housekeeping genes. MultiLocus Sequence Analysis or Typing.
2.2 Quantification.
2.2.1 Culture-based quantification.
2.2.2 Fluorescence In Situ Hybridization (FISH).
2.2.3 Real-time Polymerase Chain Reaction (PCR)
3. Ecology of Vibrio.
3.1 Abiotic parameters.
3.1.1 Temperature.
3.1.2 Salinity.
3.1.3 Others abiotic parameters.
3.2 Biotic parameters.
3.2.1 Zooplankton.
3.2.2 Phytoplankton.
3.3 Population as ecological unit
4. Quorum sensing in Vibrio.
4.1 The Aliivibrio fischeri historical model.
4.2 Multichannel QS system in Vibrio.
4.3 Molecules, biosynthesis and signalling pathways.
4.3.1 Autoinducer-1 or N-Acyl-Homoserine-Lactone Quorum sensing
4.3.2 Autoinducer-2 or Furanosyl borate diester Quorum sensing.
4.3.3 Cholerae autoinducer-1 Quorum sensing.
4.3.4 Other molecules.
4.4 QS associated phenotypes.
4.5 AHL diversity.
4.6 Interactions with other organisms.
4.6.1 Interference with bacterial QS.
4.6.2 Effects of QS molecules in the interactions with other organisms.
5. Objectives of this thesis.
6. References.
CHAPTER 2: AHL Diversity.
Publication 1: Characterization of N-Acyl Homoserine Lactones in Vibrio tasmaniensis LGP32 by a Biosensor-Based UHPLC-HRMS/MS Method.
Supplementary Materials.
Publication 2: Evidence of a large diversity of N-acyl-homoserine lactones in symbiotic Aliivibrio fischeri strains associated to the squid Euprymna Scolopes.
Supplementary Information.
CHAPTER 3: Temporal dynamic of AHL production phenotypes. 
1. Salses-Leucate lagoon.
1.1 Characteristics.
1.2 Monitoring programs.
1.3 Preliminary study.
2. References.
Publication 3: Genetic Diversity and Phenotypic Plasticity of AHL Mediated Quorum Sensing in Environmental Strains of Vibrio mediterranei.
Supplementary Information.
CHAPTER 4: Importance of Quorum Sensing in ecological population of Vibrio. 
Publication 4: Quorum sensing properties of Ecological units of Vibrio.
Supplementary Information
CHAPTER 5: Discussion & Perspectives 
1. AHL diversity
1.1 Signal specificity: How AHL signalling can be species specific?
1.2 Factors affecting AHL production:
How diverse AHL production patterns for a single species can be explained?
2. Prevalence of QS in Vibrio spp.
3. In situ QS studies: Complexity of signal production and transmission in the environment.
4. Concluding Remarks.
5. References 


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