Isolation of symbiotic bacteria Isolation of symbiotic bacteria and bioactive proteins from and bioactive proteins from

the marine sponge, the marine sponge, CallyspongiaCallyspongia diffusadiffusa

NIDHI VAIDYA NIDHI VAIDYA G.E.09016 G.E.09016

Basic idea of the paperBasic idea of the paper Symbiotic associationSymbiotic association PseudomonasPseudomonas aeruginosaaeruginosa,, EscherichiaEscherichia colicoli,,

VibrioVibrio choleracholera,, VibrioVibrio parahaemolyticusparahaemolyticus

Bioactive proteinsBioactive proteins in methanol and in aqueous extractin methanol and in aqueous extract antibacterial propertyantibacterial property hemolytic activity on chicken erythrocyteshemolytic activity on chicken erythrocytes

Protein estimation was carried out.Protein estimation was carried out.

SDS-PAGE was used to know the molecular SDS-PAGE was used to know the molecular weight to the proteins extractedweight to the proteins extracted

Methods and materialsMethods and materials Study area – khardhanda beach near Juhu, Study area – khardhanda beach near Juhu,

Mumbai coastMumbai coast

Handpicking of the Handpicking of the CallyspongiaCallyspongia diffusadiffusa during during low tidelow tide

Preservation at low temperaturePreservation at low temperature

Boiled with concentrated HNOBoiled with concentrated HNO3 3 to extrude the to extrude the

spicules and confirm the identity based on the spicules and confirm the identity based on the characters proposed by Thomascharacters proposed by Thomas

Isolation of the associated Isolation of the associated bacteriabacteriaFresh tissue of spongeFresh tissue of sponge

↓ ↓ cut by sterile bladecut by sterile blade

↓↓Homogenized using pestle and mortarHomogenized using pestle and mortar

↓↓Serial dilutionSerial dilution

↓↓pour in different sterile media platespour in different sterile media plates

[Mac Conkey’s agar, Vibrio agar, Nutrient agar, [Mac Conkey’s agar, Vibrio agar, Nutrient agar, Eosin methyl blue agar]Eosin methyl blue agar]

↓↓Bacterial associates were identified using Bergey’s Bacterial associates were identified using Bergey’s

manualmanual

Extraction of crude toxinExtraction of crude toxin Methanolic extractionMethanolic extraction(bakus and green)(bakus and green)

spongesponge↓↓

dried in air(2days)dried in air(2days)↓↓

10g tissue soaked in 200ml of 10g tissue soaked in 200ml of methanol(5hrs)methanol(5hrs)

↓↓Removal of solvent by squeezing the Removal of solvent by squeezing the

spongesponge↓↓

filter through Whatman filter no.1filter through Whatman filter no.1↓↓

solvent evaporation at low pressure solvent evaporation at low pressure using Buchi Rotavopor at 60°Cusing Buchi Rotavopor at 60°C

↓↓Storage of extract in refrigerator for Storage of extract in refrigerator for

further usefurther use

Aqueous extractionAqueous extraction

Squeezing the sand free sponge Squeezing the sand free sponge specimens in triple distilled waterspecimens in triple distilled water

↓↓Solution obtained is filtered and Solution obtained is filtered and

dialyzed using sigma dialysis dialyzed using sigma dialysis membrane 500 against D-glucose membrane 500 against D-glucose

to remove excess waterto remove excess water↓↓

supernatant obtained is lyophilized supernatant obtained is lyophilized using the Labcono Freeze Dry using the Labcono Freeze Dry

SystemSystem↓↓

Stored at 4°C in refrigerator for further Stored at 4°C in refrigerator for further useuse

Antimicrobial activityAntimicrobial activityPetri dish with sterile nutrient agar Petri dish with sterile nutrient agar

was inoculated with the isolated was inoculated with the isolated bacteriabacteria

↓↓Sponge extract was sterilized by Sponge extract was sterilized by

passing each through a 0.22µm passing each through a 0.22µm Millipore GV filterMillipore GV filter

↓↓Round paper discs were dipped into Round paper discs were dipped into

0.001 ml of each sponge extract0.001 ml of each sponge extract↓↓

Then placed in centre of the Then placed in centre of the inoculated Petri dishinoculated Petri dish

↓↓Bacterial colonies were allowed to Bacterial colonies were allowed to

grow overnight at 37°Cgrow overnight at 37°C↓↓

Inhibition zone around the disc was Inhibition zone around the disc was observedobserved

Partial purification of crude Partial purification of crude extractextract

Partial purification Partial purification was carried out using was carried out using DEAE cellulose anion DEAE cellulose anion exchange exchange chromatography chromatography according to the according to the method of Stempionmethod of Stempion

Protein EstimationProtein Estimation(by Lowry & Lopez method)(by Lowry & Lopez method)

Standard used was Bovine albumin serum at Standard used was Bovine albumin serum at concentration ranging from 0.1 to 1 mg/mlconcentration ranging from 0.1 to 1 mg/ml

↓↓5ml of alkaline copper reagent was added 5ml of alkaline copper reagent was added

↓↓Mix well and allow it to stand for 10 minutesMix well and allow it to stand for 10 minutes

↓↓Add 0.5ml of dilute folin’s phenol reagent & Add 0.5ml of dilute folin’s phenol reagent &

mix wellmix well↓↓

Incubate for 30 minutesIncubate for 30 minutes↓↓

Take absorbance at 650nm Take absorbance at 650nm spectrophotometricallyspectrophotometrically

↓↓Protein was estimated using standard graphProtein was estimated using standard graph

Hemolytic activityHemolytic activityPerformed in 96 well ‘v’ bottom microtitre platesPerformed in 96 well ‘v’ bottom microtitre plates

↓↓Selection of different rows for chicken bloodSelection of different rows for chicken blood

↓↓2-fold serial dilution of crude toxin in 100ml of normal saline and repetition of 2-fold serial dilution of crude toxin in 100ml of normal saline and repetition of

above steps up to the last wellabove steps up to the last well↓↓

100µl RBC was added to all wells100µl RBC was added to all wells↓↓

Appropriate controls were includedAppropriate controls were included1% RBC suspension & 100µl normal saline →negative control1% RBC suspension & 100µl normal saline →negative control

↓↓Gentle shaking of platesGentle shaking of plates

And allowed to stand for 2 hrs at room temperatureAnd allowed to stand for 2 hrs at room temperature↓↓

Recorded the results (uniform red color suspension →positive hemolysisRecorded the results (uniform red color suspension →positive hemolysisButton formation at bottom →lack of hemolysis)Button formation at bottom →lack of hemolysis)

↓↓Reciprocal of highest dilution of crude toxin showing pattern was taken as 1 Reciprocal of highest dilution of crude toxin showing pattern was taken as 1

hemolytic unit (HU)hemolytic unit (HU)

SDS-PAGESDS-PAGE

One dimension Sodium dodecyl sulphate One dimension Sodium dodecyl sulphate polyacrylamide gel electrophoresis was carried polyacrylamide gel electrophoresis was carried outout

Proteins were electrophoresed on 12% Proteins were electrophoresed on 12% separating gel (0.75mm thickness) overlaid with separating gel (0.75mm thickness) overlaid with 5% stacking gel.5% stacking gel.

Statistical analysisStatistical analysis

Statistical analysis was carried out using one Statistical analysis was carried out using one way analysis of variance (ANOVA) by SPSS way analysis of variance (ANOVA) by SPSS software package, version-13.0 followed by software package, version-13.0 followed by Duncan’s multiple range test (DMRT).Duncan’s multiple range test (DMRT).

ResultResult Morphological Morphological

and and physiological physiological characteristics characteristics of the bacteria of the bacteria isolated from isolated from the sponge the sponge species are species are given :given :

Bacteria Bacteria Colony charactersColony characters

PP. . aeruginosaaeruginosa Growth on NAM light-grey Growth on NAM light-grey colonies of 1.5mm colonies of 1.5mm diameterdiameter

EE. . colicoli Metallic sheen colonies Metallic sheen colonies on EMB agaron EMB agar

VV. . parahaemolyticuparahaemolyticuss

Grey colonies on NAM, Grey colonies on NAM, MAC, 1-2mm diameter, MAC, 1-2mm diameter, swarm across plateswarm across plate

VV. . choleracholera Colonies are round Colonies are round smooth 2-3mm, greenish smooth 2-3mm, greenish grey on VAgrey on VA

Methanolic extract (500g) of sponge yielded 5.4 g of crude Methanolic extract (500g) of sponge yielded 5.4 g of crude extract, whereas aqueous extract yielded 5.09g.extract, whereas aqueous extract yielded 5.09g.

The protein content from the sponge was found to be 1.62 The protein content from the sponge was found to be 1.62 mg/ml in methanolic extract and 1.43 mg/ml in aqueous mg/ml in methanolic extract and 1.43 mg/ml in aqueous extract.extract.

The 2 extract were tested at different concentration(5,10 and The 2 extract were tested at different concentration(5,10 and 15 mg/ml) against the isolated bacteria and none of the 15 mg/ml) against the isolated bacteria and none of the extract inhibited the test bacteria except the aqueous extract extract inhibited the test bacteria except the aqueous extract did inhibit did inhibit VV. . choleracholera..

The crude methanolic extract induced hemolysis on chicken The crude methanolic extract induced hemolysis on chicken erythrocytes and its hemolytic titre was 14 and also its erythrocytes and its hemolytic titre was 14 and also its hemolytic activity was estimated at 8.46 HU/mg of protein hemolytic activity was estimated at 8.46 HU/mg of protein whereas the hemolytic titre of aqueous extract was 10 and its whereas the hemolytic titre of aqueous extract was 10 and its activity was estimated at 6.99 HU/mg of proteinactivity was estimated at 6.99 HU/mg of protein

SDS-PAGE SDS-PAGE →→6 bands in aqueous extract and 8 6 bands in aqueous extract and 8 bands in methanolic extract ranging from 14.4 to 116 bands in methanolic extract ranging from 14.4 to 116 kDa molecular weight.kDa molecular weight.

3 well defined bands of 19.5, 39.0, 66.2 kDa in both 3 well defined bands of 19.5, 39.0, 66.2 kDa in both extractextract

DiscussionDiscussion 500g sponge yielded 5.09g(methanol) and 5.4g (water) of crude 500g sponge yielded 5.09g(methanol) and 5.4g (water) of crude

extractextract↓ ↓ comparecompare

Richet →5.8g of crude extract from 490g fresh weight of marine Richet →5.8g of crude extract from 490g fresh weight of marine sponge, sponge, SubereaSuberea preatensepreatense

Protein content→ corresponding data on protein content of Protein content→ corresponding data on protein content of sponge is not available in literature for comparisonsponge is not available in literature for comparison

Hemolytic activity of both extract are in accordance with earlier Hemolytic activity of both extract are in accordance with earlier studies [Stempein→ halitoxin (studies [Stempein→ halitoxin (HaliclonaHaliclona), Fusetanie →sterol ), Fusetanie →sterol derivatives viz., halistanol sulphate and sokotrasterol sulphate derivatives viz., halistanol sulphate and sokotrasterol sulphate ((halichondriidaehalichondriidae sponge), Mebs →haemagglutination and sponge), Mebs →haemagglutination and hemolytic activity of aqueous extract (48 tropical sponge hemolytic activity of aqueous extract (48 tropical sponge species).species).

ConclusionConclusion Hemolytic activity indicates cytotoxicity of the extractHemolytic activity indicates cytotoxicity of the extract→ →

worthwhile for studies on their antitumour/anti-neopastic worthwhile for studies on their antitumour/anti-neopastic activities →detection of anticancer compoundsactivities →detection of anticancer compounds

Antibacterial agents produced by sponge →enhancing Antibacterial agents produced by sponge →enhancing efficiency with sponge retains bacterial food.efficiency with sponge retains bacterial food.

Marine sponge have anti microbial activity against marine Marine sponge have anti microbial activity against marine bacteria but no activity against bacteria has been observed bacteria but no activity against bacteria has been observed thus confirming the isolated bacteria are symbiotic to marine thus confirming the isolated bacteria are symbiotic to marine sponge studied.sponge studied.

The 66.2 kDa band obtained in SDS-PAGE analysis → in The 66.2 kDa band obtained in SDS-PAGE analysis → in present study might be stress induced protein and this type present study might be stress induced protein and this type of stress induced protein has been reported in marine cat-of stress induced protein has been reported in marine cat-fish.fish.

Thank YouThank You