Identification and characterisation of the quorum sensing signal of the opportunistic pathogen causing bleaching disease in Halymenia floresii by HPLC/MS method

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Biology of Red Macroalgae

One of the oldest groups of eukaryotic algae, red algae are an ancient group of eukaryotic plants and the majority of them are filamentous (Polysiphonia), pseudoparenchymatous (Ceramium) or parenchymatous (Porphyra/Pyropia, Halymenia). Rhodophyceae vary considerably in their response to different environmental factors, having adapted to living in nearly freshwater to brackish and marine conditions. Members of red algae are found in the tropical, temperate, and arctic waters, but are most abundant in the temperate and tropical regions. The common features of red algae include eukaryotic cells, a complete lack of flagellar structure, food reserves of floridean starch, the presence of phycobiliproteins (details in “Pigments and Proteins”) chloroplasts without stacked thylakoids and no external endoplasmic reticulum (Baweja et al. 2016). The members of Rhodophyta can tolerate a wider range of light levels than any other groups of photosynthetic organisms and this is supported by the presence of additional accessory pigments together with chlorophyll a. These accessory pigments generally known as phycobiliproteins allow them to live in deep waters. The major product of photosynthesis in red algae is floridoside (O-α-d-galactopyranosyl-(1,2)-glycerol), isofloridoside digeneaside or floridean starch (details in ‘Starch’). Another very common feature of red algae is pit connections providing symplastic communication between cells (Lee 2008). Marine red algae contain significant quantities of vitamins, dietary fibers, proteins, polysaccharides, and various micro- and macro elements (Baweja et al. 2016).

Cell wal

The cell wall organization and composition of the Rhodophyta differ significantly from plants and other macroalgae. The cell wall is a rather well-ordered structure as observed by optical and electronic microscopy (Gordon-Mills et al. 1978; Craigie 1990). The cell wall is considered here as including polymeric materials originating from the metabolic activities of the algae. Most investigators recognize polysaccharides, proteoglycans, peptides, proteins, lipids, and associated inorganic constituents as components of the native cell wall of red algae. According to previous studies on biological function, the polysaccharides may be grouped with the more rigid structural (β-linked) glycans such as cellulose, mannans, and xylans, as well as with the more flexible and frequently sulfated glycans that comprise the matrix in which the skeletal fibers are embedded (Rees 1981; Baweja et al. 2016).
The wall of red algae is classically illustrated by two phases or matrices:
1) neutral lower crystalline form with cellulose that forms a weft-like mat (Dawes et al. 1961). Some xylan and mannan have been also characterized in the amorphous embedding matrix.
2) charged phase that contains sulphated galactan polymers, some of which are economically important phycocolloids (carrageenan or agar). The cell wall consists of rigid components such as microfibrils and a mucilaginous matrix. (Fig. 4) (Stiger-Pouvreau et al. 2016).
Under the optical microscope and in electron microscopic studies Gracilaria corticata reveals cells having thick cell walls that have microfibrils arranged in three distinct layers: (i) the inner-most electron-dense (glycoprotein domain), (ii) middle electron-translucent (amorphous matrix) and (iii) outermost electron-dense regions (fibrillar wall). In all three regions, microfibrils are arranged in parallel. The extracellular matrix consists of a cellulose microfibrillar network and an amorphous matrix of cellulose, sulphated galactans, and mucilage.

Polysaccharides: Structural and Storage a. Carrageenans

Red seaweeds contain large amounts of cell-wall polysaccharides, most of which are sulphated galactans. These galactans are generally built on repeated alternating l,3-linked α-galactopyranose and l,4-linked β-galactopyranose units and differ in the level and pattern of sulphation, in the substitution of methoxyl and/or pyruvate groups and in other sugar residues (galactose, xylose). They also differ in 3,6-anhydrogalactose content and the configuration of the 1-3-linked α-galactopyranose residue (Percival and Mc Dowell 1967; Craigie 1990; Usov 1998). Among these galactans, carrageenans and agars are widely used as gelling or thickening additives by the food industry and in biotechnologies.
The main carrageenans on the market belong to kappa (κ-; G4S-DA), iota (ι-; G4S-DA2S), and lambda (λ-; G2S-D2S,6S) (Fig. 5). Minor types are mu (µ-; G4S-D6S) and nu (ʋ-; G4S-D2S6S), which are κ- and ι-precursors, respectively (Fig. 5). ‘G’ defines the units linked in α (1-3) and ‘D’ for the units linked in β (1-4) and the substituents linked to the units were named as ‘M’ for methyls and ‘S’ for sulphates and they can be linked to the carbons from 1-6. The 3, 6 anhydrogalactose units (3,6 AG) are differentiated by the letters DA (Knutsen et al. 1994). Natural carrageenans usually occur as mixtures of different hybrid types, such as κ/ι-hybrids, κ/μ-hybrids or μ/ι-hybrids, which often form cyclized derivatives. Especially, the repeating unit of κ-carrageenan is composed of a D-galactose with a sulphated group at C4 linked to a hydrogalactose, the repeating unit of λ-carrageenan is constructed by a D-galactose with a sulphated group at C2 linked to a D- galactose sulphated at C2 and C6, and the repeating unit of ι- carrageenan consists of galactose with a sulphated group at C4 linked to an anhydrogalactose sulphated at C2 (Vera et al. 2011).
Carrageenans constitute 30%-75% of the red algal cell wall by their dry weight and are extensively used for stabilizing and texturing products in the food industry, which accounts for 70–80% of the total world carrageenan production estimated at about 60,000 tonnes/year with a value of US$629 million (McHugh 2003; Bixler and Porse 2011; Naseri et al. 2019). Based on this, and on assuming a yield of carrageenan extraction in the industrial scale of 20%, it can be estimated that the total dried macroalgal consumption was at least 300,000 tonnes/year (Naseri et al. 2019).

Agar

Agar consists of a family of compounds mainly constituted by agarose and agaropectin. Agarose is
a polymer of repeating residues of 3-β-D-galactopyranose and 3,6-anhydro-α-L-galactopyranose. Agar possesses the property of being insoluble in cold water. The principal distinguishing feature of the less sulfated agars is the presence of D-galactose, L-galactose, or anhydro L- galactose. The seminal concept of the masked repeating structure first reported for the agar-like porphyran and the repeating units may be substituted or modified in a number of ways to mask the underlying pattern. In that case, the agarobiose repeating structure of agars may be masked by replacing 3,6-anhydro-L- galactose with L-galactose, and/or adding methyl ethers, sulfate hemiesters, and, exceptionally, pyruvic acid ketal at specific sites on either glycosyl unit (Stiger-Pouvreau et al. 2016). Agar is an important phycocolloid, occurs in members of the orders Gelidiales with over 200 species and Gracilariales with 250 taxa (Dawes 2016).

Starch

The main characteristic reserve storage polysaccharide of red macroalgae is floridean starch, first described by Kutzing in 1843 (Stiger-Pouvreau et al. 2016). Floridean starch represents the major sink for photosynthetically fixed carbon and, under certain growth conditions, floridean starch granules can amount to as much as 80% of the total cell volume (Ekman et al. 1991). The starch from red algae also differs from higher plant starches in its apparent lack of amylose (except in some unicellular species) (McCracken and Cain 1981). The floridean starch a branched α-1,4-glucosidic linked glucose homopolymer with α-1,6-branches (Fig. 6) functioning as carbon and energy reserve in the cells (Yu 1992). They were stored in the cytoplasm as a major reserve food and also has an osmoregulatory function (Dawes 2016).
Red algae constitute an exception to this rule, unlike chlorophytes, as they synthesize and store starch as granules outside their plastids in the cytosol (Pueschel 1990). Compositionally, floridean starch granules are constructed from a polymer more similar to amylopectin than glycogen (Peat et al. 1961; Manners and Wright 1962; Craigie 1974) and with similar structural features to higher plant starches, e. g. a radially arranged fibrillar-like pattern and concentric layers (Meeuse et al. 1960; Sheath et al. 1981).
The soluble organic carbon compounds are fairly diverse: floridoside and related compounds. Floridoside (Fig. 7) is the principal low-molecular-weight carbohydrate present in all orders of the Rhodophyta except the Ceramiales, while the isofloridoside (Fig. 8) only occurs at significant concentrations in members of the Bangiales where it may exceed the levels of floridoside. Isofloridoside could be considered as resulting from the isomerization of floridoside rather than as a direct product of photosynthesis. In most members of the Ceramiales, digeneaside (O-α-D-mannopyranosyl-(1-2)- glyceric) (Fig. 9) is the dominant low-molecular carbohydrate (Kremer 1981).

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Pigments and Proteins

It is well known that red algae have high protein levels (Galland-Irmouli et al. 2000; Wells et al. 2017). Reports have shown that they have almost 47% w/w of dry matter (Sánchez-Machado et al. 2004). It is noteworthy that the protein content of macroalgae varies not only between species (Fleurence et al. 2018) but also among seasonal periods, geographic location, culture conditions, and processes, biomass storage, and analytical methods used (Mishra et al. 1993; Wells et al. 2017). Although the structure and biological properties of algal proteins are still relatively poorly documented, the amino acid composition among several species of algae is known (Harnedy and FitzGerald 2011). Most red algae contain all the essential amino acids and are a rich source of the acidic residues asparatic and glutamic acid (Fleurence et al. 2018). The predominance of acidic over basic amino acids is typical of red algae (Galland-Irmouli et al. 2000), their high levels being responsible for the algal flavor and taste (Mabeau et al. 1992). Threonine, lysine, tryptophan, cysteine, methionine, and histidine have been shown to be present at low levels in macroalgal proteins. Phycobiliproteins (PBS), the water-soluble proteins (MacColl 1998) are the main proteins of the red algae, representing up to 50% of the total protein content. They are a family of fluorescent proteins covalently linked to tetrapyrrole groups, known as bilins, a prosthetic group (Niu et al. 2007). These proteins act as antennae, absorbing energy in the portions of the visible spectrum where chlorophyll barely does (Sekar and Chandramohan 2008). Unlike carotenoids and chlorophylls, phycobiliproteins are not part of the photosystems located in the lipid bilayer but constitute a structure attached to the cytoplasmic surface of thylakoid membranes named phycobilisomes.

Table of contents :

1. General Introduction
1.1 Part I – Introduction to Macroalgae
1.1.1 Macroalgae
1.1.2 History and Classification
1.1.3 Ecology
1.1.4 Valorization of Macroalgae
1.1.5 Macroalgae in Mexico
1.2 Part – II– Red Macroalgae
1.2.1 Introduction
1.2.2 Biology of Red Macroalgae
i. Cell wall
ii. Polysaccharides: Structural and Storage
a. Carrageenans
b. Agar
c. Starch
iii. Pigments and Proteins
iv. Lipids
1.2.3 Species under Study – Halymenia floresii
1.3 Part – III- Abiotic and Biotic parameters
1.3.1 Introduction
1.3.2 Definition and Types – Abiotic parameters
i. Hydrodynamism
ii. Nutrients
iii. Desiccation
iv. Temperature
v. Light
vi. Salinity
1.3.3 Definition and Types of Biotic parameters
i. Herbivory
ii. Biofilm
iii. Epiphytism
1.4 Part – IV – Influence of the stressors (abiotic and biotic parameters) Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020
1.4.1 Introduction
1.4.2 Chemical Defence
i. Oxidative bursts
ii. Halogenation of volatile and non-volatile compounds
iii. Distance signaling
1.4.3 Microbial Defence
1.5 Part – V – Consequence of Holobiont break-up – Macroalgal Disease
1.5.1 Introduction
1.5.2 Definition
1.5.3 Algal diseases
1.5.4 Nature of pathogens
1.5.5 Mechanisms of action of pathogen
1.5.6 Impact of disease on the aquaculture sector
1.6 Context and Aims of the Thesis
2. Chapter I – Defence on Surface: Macroalgae and their associated-microbiome
2.1 Abstract
2.2 Introduction
2.3 Seaweed surface-microbe interactions – biofilm development
2.3.1 Seaweed Surface: as a substratum
i. Cell wall structure
ii. Surface topographical features
2.4 Macroalgae defence
2.4.1 Removal of surface layers
2.4.2 Production of Reactive Oxygen Species
2.4.3 Antimicrobial compounds
2.5 Microbiome on the surface
2.5.1 Bacteria
2.5.2 Fungi
2.5.3 Microalgae
2.5.4 Virus
2.6 Role of Microbiome: Microbial Interactions and their Hosts Response
2.6.1 Quorum Sensing
2.6.2 Quorum Quenching
2.6.3 Positive Effects: Symbiotism Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020
2.6.4 Negative Effects: Pathogenism
i. Spatial effects
ii. Temporal Effects
2.7 Future Perspectives
2.8 References
3. Chapter II – Screening of surface-associated bacteria from the Mexican red alga Halymenia floresii for Quorum Sensing activity
3.1 Abstract
3.2 Introduction
3.3 Materials and methods
3.3.1 Algal material
3.3.2 Isolation of surface-associated bacterial strains
3.3.3 Characterization of selected bacterial strains
i. Morphological and biochemical characterization
ii. Molecular characterization
iii. Phylogenetic analyses
3.4 Screening of the selected bacterial for QS signals – Reporter assay
3.4.1 Reporter strains
3.4.2 Cross-feeding assay
3.4.3 Bioluminescent assay
3.5 Statistical analysis
3.6 Results
3.6.1 Isolation and identification of surface-associated bacteria
i. Morphological and biochemical characterization
ii. Molecular characterization
a. Alphaproteobacteria
b. Gammaproteobacteria
c. Bacteroidetes
d. Firmicutes
3.6.2 Detection and screening of QS signal production
3.7 Discussion
3.7.1 Isolation and identification of selected surface-associated bacteria of H. floresii
3.7.2 Detection and screening of QS signals
i. Cross-feeding assay
ii. Bioluminescence assay
Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020
3.8 Conclusions
3.9 Future perspectives
3.10 Acknowledgements
3.11 References
4. Chapter III – Chemical defense against microfouling by allelopathic active metabolites of Halymenia floresii (Rhodophyta)
4.1 Abstract
4.2 Introduction
4.3 Materials & Methods
4.3.1 Algal material
4.3.2 Optimization of Selective Extraction of surface-associated metabolites
i. Surface-associated metabolites (DIP) extraction – Determination of Immersion solvent and Immersion period
ii. Whole-Cell Metabolites (WCM) extraction
4.3.3 Quorum Quenching activity by Bioluminescent reporter assay
i. Bacterial strains – Isolation from the surface of H. floresii
ii. Identification – Taxonomic affiliation of the surface-associated bacteria
iii. Bioluminescence – Reporter Assay
a. Preparation of supernatants
b. Reporter strain
c. Bioluminescent assay
4.3.4 Untargeted Metabolomic profiling – Identification of the compounds by LC-MS
i. LC-MS conditions
ii. Pre-processing and processing of data: XCMS Online and the Madison Metabolite Consortium Database
4.4 Statistical Analysis
4.5 Results
4.5.1 Algal material – Culture of H. floresii
4.5.2. Optimization of Selective Extraction of surface-associated metabolites
i. DIP Extraction
ii. WCM Extraction
4.5.3. Quorum Quenching activity of DIP extracts by Bioluminescence – Reporter Assay
i. Identification – Taxonomic affiliation of bacterial strains
ii. Bioluminescence – Reporter Assay
4.5.4. Untargeted metabolomic profiling Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020
4.6 Discussion
4.6.1 Selective extraction of surface-associated metabolites
4.6.2 Influence of DIP n-hexane extract on bacterial communication
4.6.3 Untargeted Metabolomic Profiling
4.7 Acknowledgements
4.8 References
5. Chapter IV – Identification and characterisation of the quorum sensing signal of the opportunistic pathogen causing bleaching disease in Halymenia floresii by HPLC/MS method
5.1 Materials and Methods
5.1.1 Algal material
5.1.2 Bacterial isolates
5.1.3 Tip bleaching assay with single epibacterial strains
5.1.4 Extraction of HomoSerine Lactones (HSLs) from the ‘significant pathogen’
5.1.5 Chromatographic conditions – LC-MS QTOF analysis
5.2 Results
5.3.1 Tip bleaching assay with single epibacterial strains
8.3.2 Identification of homoserine lactones (HSLs) from the ‘significant pathogen’
5.3 Discussion
5.5 Conclusion and perspectives
6. Chapter V – Screening Halymenia floresii secondary metabolites which underly its surface defence mechanisms
6.1 Abstract
6.2 Introduction
6.3 Materials and Methods
6.3.1 Algal material and extractions
6.3.2 LC-MS conditions and data pre-processing
6.3.3 LC-MS data processing – Madison Metabolomics Consortium Database
6.3.4 Data post-processing – SMILES
6.3.5 Data Validation & Interpretation – Statistical Analysis
6.4 Results
6.4.1 LC-MS analyses
6.4.2 Identifying the H. floresii secondary metbolites
6.4.3 H. floresii secondary metabolites
6.4.4 Hydrophobicity of the secondary metabolites
6.5 Discussion
Defence on surface of Rhodophyta Halymenia floresii: metabolomic profile and interactions with its surface-associated bacteria Shareen Arockiasamy 2020
6.6 Conclusion
6.7 Acknowledgements
6.8 References
7. General Discussion
7.1 Origin of the Thesis
7.2 Holobiont: Halymenia floresii and the associated bacterial community
7.3 The partner: associated bacteria
7.3.1 Characterisation of bacterial community
7.3.2 Influence of the epibacterial community
7.4 The Host – H. floresii
7.5 Interactions among bacteria and the Host’s Interference
7.5.1 Analysis of positive relation– symbiotism
7.5.2 Putative opportunistic pathogenicity
7.6 Aquaculture/Disease/Global Change
7.7 Future perspectives
7.8 Future possible experimentations
8. Experimentation Setup
8.1 Media composition
8.2 Chemicals
8.3 Antibiotics
8.4 DNA Extraction Kits
8.5 PCR Amplification – 16S

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