6/26/2019

Interference With Cell-to-cell Signaling: a Potential Therapeutic Approach Against Vibrio Species 

Vijaya baskar .P 1* AND Veera ravi.A 2 1. Department of biotechnology Dr. G.R.D.C.S, Coimbatore. 2. Department of biotechnology, Alagappa University, karaikudi.


We suggest that the above said mechanisms in Pseudomonas with quorum sensing principle might have occurred in the present study also. This induces the bacteria Pseudomonas in epibiotic seaweeds to secrete certain active compound against to the competitor Vibrio species.
 




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Whiteley, M., K.M.Lee, and E.P.Greenberg.1999. Identification of genes controlled by quorum sensing in Pseudomonas aeruginosa. Proc.Natl.Acad.Sci.USA.96:13904-13909.

Br B+ /Br B- Br C+/Br C- Br D+/Br D- Br E+/Br E- Br F+/Br F- BrA- Br B- BrC- BrD- BrE- BrF- NIL 39

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Hypnea musiformis 1. BrA+ 2. BrB+ 3. BrC+ 4. BrD+ 5. BrE+ 6. BrF+ BrA- BrB- BrC- BrD- BrE- BrF- Br A+ /Br A-

RESULTS AND DISCUSSION: QUORUM SENSING/CROSS SPECIES INDUCTION ANALYSIS In the present investigation totally 54 isolates were collected out of seaweed species. Among 54 isolates, 17 of them are producer strain, another 17 are the inducer strain rest of 20 isolates is normal and not showing any signs of activity (Table.2).

U2- U3- U4- U1+/U1- U2+/U2- U3+/U3- U4+/U4- U1- U2- U3- U4- NIL NIL NIL NIL Sediment 17. SA+ SA- SA+/SA- SA- 28

Evelyn, A., Y. Liming, K.G. Boyd, P. C. Wright, and J. G. Burgess. 2001. The symbiotic role of marine microbes on living surfaces. Hydrobiologia. 461: 37 - 40

Table 2: The results of Quorum Sensing analysis of epibiotic bacterial isolates from seaweeds. Seaweed sample Producer organism Inducer organism Cross-species producer with inducer Cross-species supernatant test with inducer Zone of clearance (mm)

Microdictyon tenunis Chlorophyceae Chondrococcus hornemonii Chlorophyceae Enteromorpha intestinalis Chlorophyceae Caulerpa cupressoides Chlorophyceae

Ines, M. M., B. Karaigher, U. Cepon, and I. Mahne. 2003. Variability of the Quorum sensing system in natural isolates of Bacillus sp., Food technol. Biotechnol. 41: 23 - 28.

Liming, Y., G. K. Boyd, and J. G. Burgess. 2002. Surface induced attachment induced production of Antimicrobial compounds by marine epibiotic Bacteria using modified Roller Bottle. Mar. Biotechnology. 4: 356 - 366.

Allison, D. G., B. Ruiz, C. Sanjose, A. Jaspe, and P.Gilbert. 1998. Extracellular products as mediators of the formation and detachment of Pseudomonas fluorescens biofilms. FEMS Microbial Lett. 167: 179-184.

Mearns - Spragg, A., K. G. Boyd, and M. O. Hubble. 1997. Antibiotics from surface associated marine bacteria. Proceedings of the fourth underwater science symposium. The society for underwater Technology, London: 147 - 157.

Lemos, M. L, A. E. Toranzod, and L. J. Barja. 1986. Antibiotic activity of epiphytic bacteria isolated from intertidal seaweeds. Microbial Ecology. 11: 149-163.

Liming, Y., K. G. Boyd, and D. R. Adams. 2003. Biofilm specific cross species induction of antimicrobial compounds in Bacilli. AppI.Envi.Microbiol, 69:3719-3727.

Dekievit, T. R., and B. H. Iglewski. 2000. Bacterial quorum sensing in pathogenic relationships. Infect. Immun. 66: 4839-4849. Dimango, E., H. J. Zar, R. Bryan, and A. Prince. 1995. Diverse Pseudomonas aerugniosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J. Clin-lnvestig. 96: 2204-2210.

Bryers, J. D., and W. G. Characklis. 1982. Processes governing primary biofilm formation. Bioeng. 24: 2451-2476 Burgess, J. G., K. Boyd, E. Armstrong, T. Pisacane, and D. R. Adams. 2003. The development of a marine natural product-based antifouling paint. Biofouling. 19: 197 -205.

REFERENCES Alim, I., and K. Yuto. 2003. Mc21-A, a Bacteriocidal antibiotic produced by a new marine bacterium, Pseudoalteromonas phenotica sp. nov. O-Bc 30T, against Methicillin -Resistant Staphylococcus aureus. Antimicro. Agents Chemoth. 47: 480 - 488.

In the present investigation, it was aimed to produce the antibiotics from the seaweed epibionts through quorum sensing principle. The bacterial isolates of seaweed epibionts were identified as species of Pseudomonas and Vibrio from seaweeds Hypnea musiformis and Gracillaria edulis. In this study the Pseudomonas acts as a producer strain and Vibrio as inducer strain. The recent finding says that the seaweed epibionts having potential to control the metabolic activity of competitor organisms. Allison et al., 1998 reported that many bacterial strains up on attaching to a surface produce exopolysaccharides or exopolypeptides. In addition, it has been postulated that exopolysaccharides could mediate the attachment of the bacteria to the surface and induce metabolic changes.

*Corresponding author e-mail: vijay10bas@yahoo.co.in INTRODUCTION We are living in a microbial planet. About 71 % of the surface of this planet is covered by sea water. A typical milliliter of seawater contains 103 fungal cells, 106 bacteria, and 107 viruses, including pathogens that cause widespread mortalities and microbes that initiate fouling of host surfaces (Rheinheimer, 1992). Thus, marine plants and animals are continually exposed to high concentrations of potentially harmful microbes. These microorganisms in nature exists as free living planktonic mode of life in sea water or it may exist as epibiotic organisms in various living and nonliving surfaces. Among living organisms, seaweeds and invertebrates act as suitable substrate for the establishment of epibiotic organisms Seaweeds are known to release a large amount of organic carbon into the surrounding environment providing a nutrient rich habitat for microorganisms like bacteria. Bacteria are generally considered to be independent unicellular organisms. One cell accomplishes all of the tasks of feeding, locomotion, 'reproduction, respiration and all other processes necessary to keep an organism alive. There are several classes of bacteria such as primary film forming bacteria, sediment bacteria, symbiotic bacteria, and epibiotic bacteria in various aquatic organisms. The marine surface environment is a site of intense composition for living space by a wide variety of organisms. Bacteria are generally recognized as primary colonizers of this habitat and are able to form biofilm on marine surface such as invertebrates and algae (Bryers, et al., 1982). Bacteria may also be abundant on the surfaces of some algae as an important epibiotic organism. In many cases, the bacterial population found to be specific, with changes occurring throughout the year or life span of the algal surface. This algal-bacterial relationship is symbiotic in most cases; the epibiotic bacteria in seaweed play a protective role by releasing secondary metabolites into the surrounding seawater that help preventing extensive fouling of the surface. Epibiotic bacteria are therefore attracting attention as a source of new natural products. Bacteria from the larvae of some crustaceans protect them from fungal infection by the production of simple antimicrobial compounds. Bacteria isolated from the surface of a tunicate prevented the settlement of barnacle and tunicate larvae exposed to the bacteria as biofilm in petridishes (Evelyn et al., 2001).

GcC- GcD- GcE- GcF- Gc A+/GcA- Gc B+/GcB- Gc C+/GcC- Gc D+/GcD- Gc E+ /GcE- Gc F+/GcF- GcA- GcB- GcC-

ANTIBIOTIC ASSAYS Antibiotic activity was performed in duplicate using a standard paper disc diffusion method as well as well assay. In well assay 10mm in diameter wells were made in marine agar plates and the plates were swabbed with 16 hours old inducer strain. To these wells 200ul of cell free supernatant were added to each well. In paper disc assay the Watmann no.1 filter paper discs (6mm in diameter) were saturated with 200ul of cell free supernatant. The impregnant discs were Dlaced in the centre of the plates swabbed with test organisms. The plates were Incubated at 37??C overnight and observed for inhibition zone. The zone of inhibition was measured as the distance from the border of paper disc to the edge of the clear zone and expressed in mm.

Mckenney, D., K. E. Brown, and D. G. Allison. 1995. Influence of Pseudomonas aeruginosa exoproducts on virulence factor production in Burkholderia cepacia: evidence of interspecies communication. J. Bacterial 177: 6989 - 6992.

In concluding this discussion, the quorum sensing is wider spread among bacterial population then was previously thought, (In Gram positive, Gram negative bacterial communication). Current assays for antimicrobial activities are inadequate because some antibiotic producing bacteria may require the presence of another bacterial species. These findings have important implication for the discovery of novel antimicrobial compounds from marine bacteria and may allow the development of new methods for screening novel compounds active against multidrug resistant bacteria.

All the 17 strains were named as PRODUCERS STAINS BrA+, BrB+, BrC+, BrD+, BrE+, BrF+ Hypnea musiformis GcA+, GcB+, GcC+, GcD+, GcE+, GcF+ Gracillaria edulis

Wagner, V. E., D. Bushneil, and L. Passador. 2003. Micro array analysis of Pseudomonas aeruginosa quorum sensing regulons. J. Bacteriol, 185: 2080 - 2095.

*Corresponding author e-mail: vijay10bas@yahoo.co.in INTRODUCTION We are living in a microbial planet. About 71 % of the surface of this planet is covered by sea water. A typical milliliter of seawater contains 103 fungal cells, 106 bacteria, and 107 viruses, including pathogens that cause widespread mortalities and microbes that initiate fouling of host surfaces (Rheinheimer, 1992). Thus, marine plants and animals are continually exposed to high concentrations of potentially harmful microbes. These microorganisms in nature exists as free living planktonic mode of life in sea water or it may exist as epibiotic organisms in various living and nonliving surfaces. Among living organisms, seaweeds and invertebrates act as suitable substrate for the establishment of epibiotic organisms Seaweeds are known to release a large amount of organic carbon into the surrounding environment providing a nutrient rich habitat for microorganisms like bacteria. Bacteria are generally considered to be independent unicellular organisms. One cell accomplishes all of the tasks of feeding, locomotion, 'reproduction, respiration and all other processes necessary to keep an organism alive. There are several classes of bacteria such as primary film forming bacteria, sediment bacteria, symbiotic bacteria, and epibiotic bacteria in various aquatic organisms. The marine surface environment is a site of intense composition for living space by a wide variety of organisms. Bacteria are generally recognized as primary colonizers of this habitat and are able to form biofilm on marine surface such as invertebrates and algae (Bryers, et al., 1982). Bacteria may also be abundant on the surfaces of some algae as an important epibiotic organism. In many cases, the bacterial population found to be specific, with changes occurring throughout the year or life span of the algal surface. This algal-bacterial relationship is symbiotic in most cases; the epibiotic bacteria in seaweed play a protective role by releasing secondary metabolites into the surrounding seawater that help preventing extensive fouling of the surface. Epibiotic bacteria are therefore attracting attention as a source of new natural products. Bacteria from the larvae of some crustaceans protect them from fungal infection by the production of simple antimicrobial compounds. Bacteria isolated from the surface of a tunicate prevented the settlement of barnacle and tunicate larvae exposed to the bacteria as biofilm in petridishes (Evelyn et al., 2001).

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MATERIALS AND METHODS: SAMPLE COLLECTION: Seaweed samples were collected from Gulf of Mannar Marine Biosphere Reserve and identified up to species level by using CMFRI bulletin (14) as follows:

Table 1. List of Seaweeds species collected for the present study SPECIES NAME FAMILY Halimeda gracilis Chlorophyceae Ulva lactuca Chlorophyceae

In the gram negative bacteria AHSL is an active principle of quorum sensing. Our producer strain is also been identified as Pseudomonas sps. So in these organisms also active principle must falls under the AHSL. The cell-cell signaling mechanism can either require import of the signal and subsequent interaction with intracellular effectors or a two-component signaling system that transducers the information across the membrane. In V. harveyi genetic analysis of the density sensing apparatus has two independent density-sensing systems, and each is composed of a sensor-auto inducer pair; system one is composed of sensor I and Al -1, and system two is composed of sensor 2 and AI-2. The two densities - sensing system are redundant, because a null mutation in either system alone results in expression of hidden genes (Bassler, et al.,1999.).

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Boyd, K.G., A. Mearns -Soragg, G. Briedley, K. Hatzidimitriou, A. Rennie, M. Bregu, M. 0. Hubble, and J. G. Burgess. 1998. Antifouling potential of epiphytic marine bacteria from the surface of marine algae. Plouzane, France: 128 -136.

Bassler, B. L., E. P. Greenberg, and A. M. Sterens. 1997. Cross species induction of luminescence in the quorum - sensing bacterium Vibrio harveyi. J. Bacteriology. 179: 4043-4045.

U1+, U2+, U3+, U4+ Ulva lactuca SA+ Sediment INDUCER STRAIN BrA-, BrB-, BrC-, BrD-, BrE-, BrF- Hypnea musiformis

In the present investigation, it was aimed to produce the antibiotics from the seaweed epibionts through quorum sensing principle. The bacterial isolates of seaweed epibionts were identified as species of Pseudomonas and Vibrio from seaweeds Hypnea musiformis and Gracillaria edulis. In this study the Pseudomonas acts as a producer strain and Vibrio as inducer strain. The recent finding says that the seaweed epibionts having potential to control the metabolic activity of competitor organisms. Allison et al., 1998 reported that many bacterial strains up on attaching to a surface produce exopolysaccharides or exopolypeptides. In addition, it has been postulated that exopolysaccharides could mediate the attachment of the bacteria to the surface and induce metabolic changes.

CONCLUSION: The present investigation was aimed to study the quorum sensing principle among seaweed epibiotic bacterial organisms. In the past few decades there was no findings of new novel antibacterial class compounds were identified. But, the pathogenic microorganisms show much higher rate of resistant development even to the potential antibiotics. So, there is an urgent need to discover new novel antibiotic compounds. The marine inhabitants such as microorganisms, seaweeds, invertebrates, etc., act as an undepleted source of wide range of natural products among which the seaweeds act as a potential source of antibiotic compounds. Currently the cross species induction / quorum sensing attracts the total attention of researchers in finding new novel drugs against multidrug resistant pathogenic microorganisms. So, the present study aim to find out the capability of seaweed epibiotic bacterial organisms to produce novel drugs against the animal and plant pathogens

Hypnea valentiae Rhodophyceae Hypnea pannose Rhodophyceae Hypnea esperi Rhodophyceae Acanthophora spicifera Rhodophyceae ISOLATION OF EPIPHYTIC BACTERIA

In culture system B 200ul of 16 hours old producer strain alone was inoculated. In culture system C 200ul of 16 hours old inducer strain alone was inoculated.

All the cultures were incubated at 28??C for 5 days. After the incubation period the cultures were centrifuged at 10,000 rpm for 15mins. The supernatant was collected and subjected to antibacterial assay with respective inducer strain.

Patterson, G. L., and C. Ml. Bolis. 1997. Fungal cell - wall poly sacchraides elicit an antifungal secondary metabolite in the Cyanobacterium Scytonema ocellatum. J. Phycology. 33: 54 - 60.

In culture system B 200ul of 16 hours old producer strain alone was inoculated. In culture system C 200ul of 16 hours old inducer strain alone was inoculated.

Spray and concentrate products then complete the manor in which pheromones are made available to the public. For women though, it seems manufacturers are starting to realize they may have been leaving money on the table when they first began their quest to turn the human population into attraction magnets.

Cappuccino J. G., and N. Shermann. 1999. Microbiology, A Laboratory manual, Fourth edition, Addison Wesley, New York. Davies, D. G., M. R. Parsek, J. P. Pearson, B. H. Iglewski, J. W. Costerton, and E. P. Greenberg. 1998. The involvement of cell to cell signals in the development of a bacterial biofilm. Science. 280: 295-298.

c) The normal 20 bacterial strains isolated from 20 algal species were crossed with terrestrial bacteria such as E-coli, Staphylococcus aureus separately

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Holmstrom, C, and S. Kjelleberg. 1994. The effect of external biological factors on, settlement of marine invertebrates and new antifouling technologies. Biofouling. 8: 147-160.

Bassler, B. L., E. P. Greenberg, and A. M. Sterens. 1997. Cross species induction of luminescence in the quorum - sensing bacterium Vibrio harveyi. J. Bacteriology. 179: 4043-4045.

Burgess, J. G., H. Miyashita, H. Sudo, and T. Matsunaga.1991. Antibiotic production by marine photosynthetic bacterium Chromatium purpuratum NKPB 031704: Localization of activity to the chromatophores. FEMS Microbiol. Lett. 84: 301 - 306.

Hypnea valentiae Rhodophyceae Hypnea pannose Rhodophyceae Hypnea esperi Rhodophyceae Acanthophora spicifera Rhodophyceae ISOLATION OF EPIPHYTIC BACTERIA

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The earlier genetic analysis in Pseudomonas reveals the Pseudomonas consist of two quorum sensing systems as Las R1-I and Rh1R-l and have linked with R and I genes, in addition recently a third Lux R homolog that is advanced to a cluster of quorum sensing - controlled (qsc) genes were detected. Las R is a transcriptional regulator that responses primarily to the Las I - generated signal and Rh1R is a transcriptional regularly that responses best to the Rhl -generated signal. In Pseudomonas auriginosa, at low population densities Las I produce a basel level of 3-O-C12-HSL. As density increases, 3-0-C12-HSL builds to a critical concentration, at which point interacts with LasR. This Las R -3-0-012-HSL complex that activates transcription of a number of genes [Whileley, et al., 1999].

Trischman, J.A., D.M.Tapiolas, P.R.Jensen, R.Dwight, W.Fenical, T.C.Mckee, C.M.Ireland, T.J.Stout, J.CIarely. 1994. Salinamide - A and salinamide - B - anti -inflammatory depsipeptides from a marine streptomycete. J.Am.Chem.Soc. 116: 757 -758.

b) Among these 17-inducer strain 6 strains were isolated from Hypnea musiformis, 6 from Gracillaria edulis, 4 from Ulva lactuca & 1 from sediment.


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Author Dave Thomson
 
 
 


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