Olivier Schiettekatte

Pascale Bourhy

Isolation, Purification and Characterization of Leptophages

Keywords: Leptophages, Polyethylene glycol precipitation, Cesium chloride purification, TEM characterization, One-step growth kinetics

To date, only three bacteriophages of leptospires-leptophages-are known. Nonetheless, numerous prophages have been found in the genus, especially in the genomes of pathogenic species. Thus, some laboratories attempt to isolate leptophage particles from environmental samples or following mitomycin C induction of bacterial cultures. Here, we propose multiple procedures to isolate, purify, and characterize bacteriophages, based on protocols used for LE3 and LE4 characterization.

Introduction

Three bacteriophages of leptospires have been isolated as of yet, and are named "leptophages" [START_REF] Saint Girons | First isolation of bacteriophages for a spirochaete: Potential genetic tools for Leptospira[END_REF]. They have been sequenced [START_REF] Bourhy | Complete Nucleotide Sequence of the LE1 Prophage from the Spirochete Leptospira Biflexa and Characterization of Its Replication and Partition Functions[END_REF][START_REF] Schiettekatte | Characterization of LE3 and LE4, the Only Lytic Phages Known to Infect the Spirochete Leptospira[END_REF] and morphologically characterized [START_REF] Saint Girons | First isolation of bacteriophages for a spirochaete: Potential genetic tools for Leptospira[END_REF][START_REF] Schiettekatte | Characterization of LE3 and LE4, the Only Lytic Phages Known to Infect the Spirochete Leptospira[END_REF]. In the last thirty years, no other leptophage has been isolated, but numerous prophages have been found in the genus, especially in the genomes of pathogenic species [START_REF] Schiettekatte | Characterization of LE3 and LE4, the Only Lytic Phages Known to Infect the Spirochete Leptospira[END_REF][START_REF] Fouts | What makes a bacterial species pathogenic?: Comparative genomic analysis of the genus Leptospira[END_REF], suggesting a role in pathogenicity acquisition [START_REF] Picardeau | Virulence of the zoonotic agent of leptospirosis: still terra incognita?[END_REF]. Thus, some laboratories have attempted to isolate additional phage particles by mitomycin C inductions [START_REF] Zhu | Isolation and Characterization of Two Novel Plasmids from Pathogenic Leptospira interrogans Serogroup Canicola Serovar Canicola Strain Gui44[END_REF], or from environmental samples, using the method of St Girons et al [START_REF] Saint Girons | First isolation of bacteriophages for a spirochaete: Potential genetic tools for Leptospira[END_REF].

We propose, herein, a combination of methods for the extraction of leptophages from environmental samples and induced cultures as well as their isolation, purification, and phenotypic characterization. These protocols are based on classical bacteriophage purification methods-i.e. polyethylene glycol precipitation, plating, and TEM observations-considering Leptospira spp. specific medium requirements and slow growth.

Materials

Prophage inductions

Methods

Prophage inductions in Leptospira spp.

1. Grow the Leptospira sp. bacterial host in EMJH flasks at 30 °C (we suggest 400 mL). 2. Measure the optical density of the culture daily at a wavelength of 420 nm. 3. When the optical density of the culture reaches 0.1 (3 to 7 days depending on the species), divide the culture in two identical volumes. Induce one culture with 0.05 µg/mL final concentration mitomycin C. 4. Measure the optical density of the culture daily at a wavelength of 420 nm. If a significant difference (>50%) in optical density is observed between the control and the induced culture, this is potentially due to prophage expression. 5. Bacteriophages or induced particles are stored at 4 °C.

Bacteriophage extraction and purification

Two protocols exist for leptophage extraction. Tangential flow filtration is available to extract viruses from huge volumes of environmental water (see Note 1), but, due to high density of EMJH, it is not practical for the extraction of viruses from leptospiral lysates. In this case, phage recovery is facilitated by polyethylene glycol precipitation (see Note 2). If working with bacterial lysate, the supernatant should be recovered after pelleting cells and other debris at 6,000 × g for 45 min.

Tangential flow filtration

1. Filter up to 2 liters of environmental water with a 0.22 µm dead-end filter, recover the filtrate. 2. Concentrate this sample up to 400 times with a 100 kDa tangential flow filtration cassette (see Fig 1 for assembly). 3. Add 10% final volume chloroform to eliminate remnant cell contaminants and mix gently. 4. Centrifuge at 1,700 × g for 30 min at 4 °C to pellet chloroform-insoluble material. 5. Filter the supernatant with a 0.22 µm dead-end filter.

Polyethylene glycol precipitation

1. Filter 200 mL of leptospiral lysate (see section 3.1) with a 0.22 µm dead-end filter. 2. Add 8% polyethylene glycol 8000 and 0.3 M NaCl (final concentrations). 3. Mix thoroughly to dissolve polyethylene glycol. [START_REF] Fouts | What makes a bacterial species pathogenic?: Comparative genomic analysis of the genus Leptospira[END_REF]. Incubate overnight at 4 °C. 5. Centrifuge at 24,000 × g for 45 min at 4 °C. 6. Solubilize the pellet in a minimal volume of TN buffer for higher yields, we recommend 5 mL.

Phage amplification

1. Grow Leptospira sp. bacterial host in EMJH flasks at 30 °C (we suggest 400 mL).

2. When the optical density of the culture reaches 0.2, divide the culture in two identical volumes. Induce one culture with 5 mL of previously extracted phage (see section 3.2.1 or 3.2.2), and the other culture with the same volume of TN buffer (control culture). 3. Measure the optical density of the culture daily at a wavelength of 420 nm. If a significant difference (>50%) in optical density is observed between the control and the induced culture, this is potentially due to phage amplification. 4. Repeat the PEG extraction and / or isolate phages with double agar overlay plaque lysis assay (see section 3.3).

Cesium chloride gradient ultracentrifugation

A purification step by cesium chloride gradient ultracentrifugation is recommended for particle characterisation (TEM or mass spectrometry) but should be avoided prior to infection of bacteria because cesium chloride is toxic to leptospires.

1. Add 45% final concentration cesium chloride to sample. 2. Ultracentrifuge between 110,000 to 260,000 × g for 24 h at 10 °C in an ultracentrifuge tube. 3. Recover formed bands with a syringe (see Note 3). [START_REF] Fouts | What makes a bacterial species pathogenic?: Comparative genomic analysis of the genus Leptospira[END_REF]. Dialyze the sample with TN buffer with a 3.5 kDa cassette.

Double agar overlay plaque lysis assay

Phage isolation and counting

1. Prepare Petri dishes containing 20 mL EMJH with 1.2% agar and wait for complete solidification. 2. Prepare an equivalent number of Falcon tubes containing 5 mL EMJH with 0.6% agar, stored in 43 °C water bath to prevent solidification before plating.

3. Incubate 500 µL of exponential phase Leptospira sp. culture with 10 µL of different dilutions of an extract of leptophages, at 30 °C for 15 min. 4. Mix these co-cultures with 5 mL EMJH with 0.6% agar and pour immediately over 1.2% agar EMJH plates. Work efficiently, as the overlays harden quickly. 5. Incubate at 30 °C and check every 2 days for the appearance of lysis plaques (see Fig 2). The number of plaques formed is proportional to the bacteriophage concentration in the extract, considering the dilution used.

Fast dropping

1. Prepare Petri dishes containing 20 mL EMJH with 1.2% agar and allow for complete solidification. 2. Add 5 mL EMJH with 0.6% agar as an overlay and wait for complete solidification.

Store dishes at 4 °C up to one month. 3. Spread 1 mL of exponential phase Leptospira sp. culture on a room temperature overlay medium. 4. Incubate at 30 °C for 2 h. 5. Remove any remaining liquid with a micropipette. 6. Leave the dish open under a sterile biosafety cabinet up to 15 min to allow the small volume of remnant liquid to dry. 7. A -For screening: Drop 10 µL of each purified phage sample to test for clearing.

B -For raw counting: Drop 10 µL of serial dilutions of the bacteriophage extract to titer. 8. Incubate at 30 °C and check every 2 days for the appearance of lysis plaques. The number of plaques is proportional to the concentration of bacteriophages in the extract, considering the dilution used (see Note 5).

Leptophage transmission electron microscopy

Induced particles morphological characterization

1. Deposit a carbon-coated copper grid on a 15 µL drop of phage sample for 15 min. 2. Deposit the grid on a 30 µL drop of 4% uranyl acetate solution for 3 seconds for washing and staining. 3. Deposit the grid on another 30 µL drop of 4% uranyl acetate solution for 8 seconds for staining. 4. Dry the grid with a filter paper; take care to remove any remnant liquid. 5. Observe the grid with a transmission electron microscope at a magnification of 23,000 for screening, and 49,000 for characterization (see Fig 3A).

Infected bacteria observation

1. Deposit a carbon-coated copper grid on a 15 µL drop of phage-infected Leptospira culture (we suggest a MOI of 10) at the desired infection time, for 15 min.

2. Deposit the grid on a 15 µL drop of 2% glutaraldehyde in PHEM buffer for 10 min. 3. Deposit the grid successively on four 30 µL drops of distilled water for washing. 4. Deposit the grid on a 30 µL drop of 4% uranyl acetate solution for 3 seconds for washing and staining. 5. Deposit the grid on another 30 µL drop of 4% uranyl acetate solution for 8 seconds for staining. 6. Dry the grid with a filter paper; take care to remove any remnant liquid. 7. Observe the grid with a transmission electron microscope at a magnification of 23,000 for screening, and 49,000 for characterization (see Fig 3B).

Leptophage kinetics

Phage adsorption assays

1. Grow Leptospira sp. bacterial host in EMJH flask at 30 °C. 2. When the culture reaches exponential phase, dilute the bacteria to 10 8 /mL in EMJH. 3. Infect this culture with a final concentration of 10 4 bacteriophages per mL (MOI of 0.0001), or with the same volume of buffer (control culture). 4. Prepare a control dilution of the bacteriophage at 10 4 bacteriophages per mL (without bacteria). 5. Sample 1 mL of the co-culture every 30 min for up to 6 hours and filter immediately with a 0.22 µm dead-end filter. Sample 1 mL of control culture and control dilution at times 0 and 6 hours and filter with a 0.22 µm dead-end filter. 6. Count the lytic bacteriophages from this sample with the double agar overlay plaque lysis assay (see 3.3). 7. The number of phages found in the non-infected control culture should stay at 0, and the concentration of phages in the control dilution should remain close to 10 4 /mL and corresponds to the percent lost in the filter. 8. In the experimental co-culture, the number of recovered phages should decrease until the first burst. This decrease corresponds to the proportion of phages adsorbed on host cells (and retained in the 0.22 µm filter).

One-step growth kinetics

1. Grow Leptospira sp. bacterial host in EMJH flask at 30 °C. 2. When the culture reaches exponential phase, standardize dilute with EMJH to 10 8 bacteria/mL in a final volume of 10 mL. 3. Infect this culture with a final concentration of 10 7 bacteriophages per mL (MOI of 0.1). 4. Incubate at 30 °C for 30 min. 5. Centrifuge the mixture at 6 000 × g for 30 min to remove non-adsorbed phages. 6. Resuspend the supernatant in 100 mL EMJH. 7. Each 30 min, sample 1 mL of each culture or control and filter immediately with a 0.22 µm dead-end filter. 8. Count the lytic bacteriophages with the double agar overlay plaque lysis assays. 9. The number of phages should remain approximately 10 6 /mL (considering the dilution at step 6) as long as the latency period lasts. The burst is observed when this number grows, and the percent increase corresponds to the number of phages produced per infected cell (burst size).

Notes

1. To date, leptophages have only been isolated from water sources contaminated with Leptospira species (1). But soils [START_REF] Thibeaux | Seeking the Environmental Source of Leptospirosis Reveals Durable Bacterial Viability in River Soils ». Édité par Claudia Munoz-Zanzi[END_REF] and organs of infected animals should also be considered. 2. For environmental samples, ultrafiltration can handle larger volumes than PEG purification with higher yields 3. If no band is found, or if bacteriophages are not recovered from the bands, a systematic sampling and TEM observation of different layers of the gradient may be necessary. 4. The double agar overlay plaque lysis assay is required to isolate phage clones, but samples can be pre-tested in a fresh liquid culture as a preliminary assay. 5. Fast dropping is a quick counting or screening method, especially when testing numerous samples, but can only be used to elucidate the order of magnitude of the virus concentration.

Acknowledgments

This work was part of the PhD thesis of O. S. who received financial support from "Université Paris Diderot" and "Sorbonne Paris Cité". We thank Soizick Lucas-Staat and Nicolas Dufour for technical assistance during leptophages extractions, Olivier Gorgette for technical assistance during the TEM observations and Robert A. Gaultney for proofreading the manuscript.

Figure 1 .

 1 Figure 1. Tangential flow filtration assembly.Figure 2. LE4 leptophage plaque lysis on Leptospira biflexa overlay.Figure 3. LE4 bacteriophages (A) in a lysate and (B) with the bacterial host.

Figure 2 .

 2 Figure 1. Tangential flow filtration assembly.Figure 2. LE4 leptophage plaque lysis on Leptospira biflexa overlay.Figure 3. LE4 bacteriophages (A) in a lysate and (B) with the bacterial host.

Figure 3 .

 3 Figure 1. Tangential flow filtration assembly.Figure 2. LE4 leptophage plaque lysis on Leptospira biflexa overlay.Figure 3. LE4 bacteriophages (A) in a lysate and (B) with the bacterial host.

Double agar overlay plaque lysis assay

	1. Incubator. 2.5 Leptophage kinetics
	2. Ellinghausen-McCullough-Johnson-Harris (EMJH) medium: dissolve 2.3 g of Difco
	Leptospira Medium Base EMJH (Becton Dickinson) into 900 mL of distilled water, 1. Incubator.
	sterilize by autoclaving, and add 100 mL of Difco Leptospira Enrichment EMJH 2. EMJH medium.
	(Becton Dickinson). 3. 50 mL flasks.
	3. 400 mL flasks. 4. Fresh Leptospira cultures.
	4. Fresh Leptospira cultures. 5. Petroff-Hauser counting chamber.
	5. Spectrophotometer. 6. TN buffer.
	6. Spectrophotometry cuvettes. 7. Centrifuge.
	7. Mitomycin C: 1 mg/mL solution in sterile water. 8. 0.22 µm dead-end filter.
	2.2 Bacteriophage purification
	1. Polyethylene glycol 8000.
	2. Sodium chloride.
	3. Cesium chloride.
	4. TN buffer: 10 mM Tris-HCl, pH 7.5, 100 mM NaCl.
	5. Chloroform.
	6. Centrifuge.
	7. Ultracentrifuge.
	8. Beckman Coulter rotor SW41.
	9. 13 mL thin-walled polypropylene ultracentrifuge Coulter tubes.
	10. Peristaltic pump and flexible plastic tubing.
	11. Ultrafiltration cassette (100 kDa).
	12. 3.5 kDa cutoff dialysis cassettes.
	13. 0.22 µm dead end filters.
	14. Syringes (1-5 mL) with 18-gauge needles.
	2.3 1. EMJH medium supplemented with 0.6% and 1.2% agar.
	2. Water bath (43 °C).
	3. Petri dishes (90×15 mm).
	4. Polypropylene Falcon tubes (15 mL).
	2.4 Leptophage observation
	1. Electron microscope.
	2. Microscopy carbon-coated copper grids.
	3. Uranyl acetate: 4% solution in water.
	4. Glutaraldehyde: 25% solution in water (stock solution) and 2% solution
	(working solution).
	5. PHEM buffer (2×): 60 mM PIPES, 25 mM HEPES, pH 7.0, 10 mM EGTA, 4 mM
	MgSO4.
	6. Filter paper.


domain: sdv sdv.aen spi

text:
Hélène Martin-Yken

Etienne Dague

Christophe Furger

H T Darius

M Chinain

From the Yeast cell wall to new strategies in the fight against Ciguatera

From the yeast cell wall to new strategies in the fight against Ciguatera.

H. Martin-Yken I will attempt to explain my scientific journey, from studying the cell wall and stress response mechanisms of yeasts, up to the development of innovative approaches to fight Ciguatera, a major cause of food poisonings by seafood worldwide.

Yeasts are eukaryotic microorganisms traditionally used by man to make bread, beer and wine and thus very well studied. Their cells are surrounded by a thick cell wall composed of glycans and proteins organized in a complex multilayered architecture. Essential for the survival of yeasts and fungi, this highly dynamic organelle gets constantly remodeled during the cell cycle to allow morphogenetic events as well as upon environmental changes. It is a key player in cellular adhesion and resistance to antifungal compounds. While studying the mechanisms of cell wall adaptations to various stresses, I came across the different cellular signaling pathways involved notably MAP kinase cascades and the Calcineurin pathway which are conserved with higher eukaryotes.

Ciguatoxins (CTXs) are lipid-soluble, highly neurotoxic, polyether compounds produced by dinoflagellates from the genus Gambierdiscus spp.. mostly found in tropical and subtropical zones. CTXs bind to Voltage Gated Sodium Channels at the surface of human sensory neurons where they remain, causing Ciguatera Fish Poisoning or CFP. This severe disease is characterized by with a variety of gastrointestinal, cardiovascular and neurological symptoms (paresthesia, ataxia, cold allodynia), including persistent neurological effects. Despite the importance and prevalence of CFP, there is so far no simple and quick way of detecting these toxins in contaminated samples. Currently, only heavy and expensive laboratory methods are available to detect them: LC-MS/MS, receptorbinding assays by competition with labeled compounds, and neuroblastoma cell-based assays performed on mammalian neurons. We have started to engineer biosensors based on the detection of a transcriptional signal in the model yeast Saccharomyces cerevisiae, using the very good conservation of its signaling pathways with higher eukaryotes.

Finally, I will also present a smaller project (NeuroSens) based on a different strategy, to study and characterize the binding of Ciguatoxins on mammalian neuronal cells by Single Molecule Force Spectroscopy using Atomic Force Microscopy.