Team:BYU Provo/Notebook/Cholera - Enzyme/March-April/Period2/Dailylog

Cholera Enzyme

March-April

May-June

July-August

September-October

Protocols

Professor's Name: I. Phyllis Molobela

Professor's email: mantlophyllis@yahoo.com

Affiliation: Department of Microbiology and Plant Pathology, University of Pretoria, Pretoria, South Africa

Research:

Molobela, I.; Cloete, T.; Beukes, M. Protease and Amylase Enzymes for Biofilm Removal and Degradation of Extracellular Polymeric Substances (EPS) produced by Pseudomonas fluorescens Bacteria. Afr. J. Microbiol. Res. 2010, 4, 1515-1524.

Nicholas J. Shikuma, K. R. D., Jiunn N.C. Fong and Fitnat H. Yildiz (2012). "The transcriptional regulator, CosR, controls compatible solute biosynthesis and transport, motility and biofilm formation in Vibrio cholerae." Environmental Microbiology.

• Inhabits aquatic environments
• Colonizes the human digestive tract
• Adjusts to changes in salinity and osmolarity----produces solutes to counteract extracellular osmotic pressure
• Transcriptional regulator CosR----regulated by ionic strength
• CosR activates biofilm formation and represses motility
• To help maintain osmotic tolerance
• Solute molecules stabilize intracellular levels of water/pressure
• Salinity a significant factor in affecting growth and distribution of Cholera
• Cholera epidemics correlated with increased salinity in riverine and estuarine habitats
• Biofilm formation facilitates cholera survival
• Salinity affects expression of biofilm genes
• CosR represses motility and activates biofilm formation
• Uses CosR to adapt to changing salinity environments
• Cholera depends on its ability to form biofilms for virulence as a human pathogen
• WT and mutant CosR grown in ASW (50% artificial seawater media)
• Mutant defective in biofilm growth
• Grown for 72 hours
• Also grown in LB

M. Kamruzzaman, S. M. N. U., D. Ewen Cameron, Stephen B. Calderwood, G. Balakrish Nair, John J. Mekalanos, and Shah M. Faruque (2010). "Quorum-regulated biofilms enhance the development of conditionally viable, environmental Vibrio cholerae." PNAS 107(4): 1588-1593.

• Cholera diarrheal disease spread through contaminated water
• Exists in water as clumps of cells
• Fully virulent in the intestinal milieu
• Conditionally viable environmental cells
• Biofilm formation dependent on quorum sensing
  • Controlled by cell density----regulatory
• Cholera transmission in vivo-formed biofilms convert to CVEC upon introduction of cholera stools into environmental water
• Cholera transit between intestinal infection and hypotonic aquatic environment
  • Phage predation
• Concentration of pathogenic cholera far less than that required to induce infection
• Clumps of cells referred to as conditionally viable environmental cells
  • Can be cultured in the lab using an enrichment technique
• Genetic regulators of quorum sensing and biofilm formation affect the development of CVECs
• Ileal loops of adult rabbits ideal model
• Mutation in hapR resulted in more biofilm-like clumps
• Enzymes required for EPS synthesis (exopolysaccharide)
• Formation of biofilms necessary for CVEC development

AmyA

• Endohydrolysis of 1->4 alpha-D-glucosidic linkages in polysaccharides containing three or more 1->4 alpha-linked D-glucose units
• Involved in carbohydrate metabolism
• Secreted
• Calcium ligand---can bind 2 calcium ions per subunity
• A glycosidase/hydrolase
• Extracellular compartment
• Involved in alpha glucan bidning and degradation
• A freely secreted putative CGTase
• Help to degrade----results in the formation of maltodextrins

Katrina L. Van Dellen, P. I. W. (2006). "The Vibrio cholerae biofilm: A target for novel therapies to prevent and treat cholera." Drug Discovery Today: Disease Mechanisms 3(2).

• A gram-negative halophilic bacteria
• Surface adhesion of the bacterium to intestinal epithelium
• Different types of cholera biofilms
  • Nutrient scarce environment----attachment to surfaces as single cells
    • Monolayer
    • Consists of type IV pilus, the mannose-sensitive hemagglutinin (MSHA) and chemotaxis
  • Multi-layered biofilms occur when intracellular contacts between bacteria are formed
    • Production of the toxin-co-regulated pilus (TCP)
• Bundle-forming pilus----virulence factor
  • VPS exopolysaccharide
• Encoded by vps genes
  • Environmental calcium----forms salt bridges between negatively charged moieties of the polysaccharide O-antigen

Shuyang Sun, S. K., Diane McDougald (2013). "Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists." PLoS ONE 8(2): e56338.

• Cholera survival depends on its ability to respond to stresses
• Grazing pressure by protozoan organisms can induce biofilm formation
• Vibrio polysaccharide (VPS) facilitates that attachment of cholera cells to the surface
• VPS responsible for biofilm resistance
• VPS positively regulated by VpsR and VpsT but repressed by HapR

Luanne Hall-Stoodley, P. S. (2002). "Developmental regulation of microbial films." Current Opinion in Biotechnology 13(3): 228-233.

• Biofilms complex communities
• To gain an understanding of the developmental processes and regulatory mechanisms involved in biofilm formation
• Biofilm formation is dynamic
• Extracellular microbial structures assist in adhesion and maintenance of structure.
• Initiated by:
  • Redistribution of attached cells by surface motility
  • Motility to assist colonization
  • Protein and polysaccharide adhesins
  • Binary division of attached cells
  • Surface-associated aggregation can also occur by recruitment of single cells
• Bacterial cells in biofilm colonies held together by EPS
  • Includes polysaccharides, nucleic acids and proteins
  • Increased production of EPS in flagellar mutants of V. cholera 0139 correlated with reduced intestinal colonization in an infant mouse model
  • Diversity in EPS production among bacterial species

Jason B. Harris, R. C. L., Firdausi Qadri, Edward T. Ryan, Stephen B. Calderwood (2012). "Cholera." The Lancet 379(9835): 2466-2476.

• Cholera an acute, secretory diarrhea by O1 or O139 serotypes
• Epidemics have been increasing in intensity, duration and frequency-----great need for more effective approaches for prevention and control
• Organisms grow best in presence of salt
• In water, cholera enters a viable but non-culturable form Serotypes based on the O antigen of LPS

We need to focus on how to characterize biofilms, and should alter the NaCl concentrations of growth media for cholera to induce stringent response growth.

4/3/13

Lab Work
Based on our research, cholera biofilms grow best under stringent response. One of the factors affecting this response is salinity. As our cholera cultures are growing fine but aren't really producing a biofilm we decided to raise the salinity of our LB to better simulate the aqueous environment in which cholera biofilms are normally found. We made LB using a synthetic sea water recipe in place of water. The recipe was obtained online from thelabrat.com. Our synthetic seawater (SSW) LB recipe consisted of the following:

Regular LB Mix + 24g NaCl
11.9g MgCl2-6H2O
2.0g CaCl2-2H2O
0.83 g KCl

1 Liter of media was made and the pH was then adjusted to 7.8 and autoclaved.

Discussion
We emailed Dr. Grose the contact information for two research groups that have worked with Bacillus lentus. The first group is based out of South Africa and is working on using Subtilisin Savinase to break down a variety of biofilms. However, Dr. Grose remembered that they attempted to contact this team last year and were unable to get any kind of response. Also, shipping the bacteria samples that we need from South Africa would be tricky so we found a research group at John Hopkin's University that was doing research with Bacillus lentus. They weren't doing anything related to biofilms, but were using the bacteria that we need. I emailed the contact info to Dr. Grose and she sent them an email. We are now just waiting to hear back from them.

Contact info:

Jeffrey J. Gray, Department of Chemical & Biomolecular Engineering, Johns Hopkins University, 3400 N, Charles St., Baltimore, Maryland 21218.

E-mail: jgray@jhu.edu

Paper Reference:

Sircar, A.; Chaudhury, S.; Kilambi, K.; Berrondo, M.; Gray, J. A Generalized Approach to Sampling Backbone Conformations with RosettaDock for CAPRI rounds 13-19. Proteins: Struct., Funct., Bioinf. 2010, 3115-3123.

4/5/13

Started 4ml overnights with our SSW LB media

1. 30 degrees celsius with cholera plate culture
2. 30 degrees with 350ul seeded from previous overnights
3. 37 degrees with chlolera plate culture
4. 37 degrees with 350ul seeded from previous overnights

Hopefully, the higher salt content in the growth media will induce strong biofilm growth.

We also identified two labs that have worked with Dispersin B in the past couple of years and sent an email to Dr. Grose to forward to these labs. One was to Dr. Chen at NYUMC, the other was to Dr. Demuth at Louisville.

4/8/13

Our cholera overnights grown in our SLB media shows significant visible difference in the amount of biofilm growth.

We are currently waiting to hear back from several research groups we have contacted to try to obtain samples of the bacteria strains that we need. While we wait, we focused today on doing further research to find other possible sources to obtain the bacteria samples, and on our semester final presentation.

Cholera Final Semester Presentation Outline

1. Background
   1. General overview of quorum sensing
      1. Cholera specifics
   2. Biofilm formation
   3. Degradation enzymes
      1. DspB
      2. AmyA
      3. Subtilin savinase
         1. Propose a few new enzymes----research
2. Methods
   1. Determine cholera biofilm composition
   2. Salt concentrations for biofilm growth
   3. Application of the AmyA to the biofilm
3. Results
   1. Increased biofilm growth with increased salt concentration
      1. Seeded overnight
   2. Sequencing results conclude that AmyA successfully cloned
4. Conclusion

4/10/12

We read the following research articles today: Daniel Nelson, R. S., Peter Chahales, Shiwei Zhu and Vincent A. Fischetti (2006). "PlyC: A multimeric bacteriophage lysin." PNAS 103(28): 10765-10770.

• Lysins----hydrolases introduced by bacteriophages----act on bacterial host cell wall to release progeny phage----PlyC (a lysin from streptococcal phage)
  • Composed of PlyCA and PlyCB
    • 8 PlyCB for every PlyCA
    • PlyCB complex alone can direct cell-wall specific binding
      • Gram positives where as V. cholera is gram-negative
      • Cell wall hydrolase
      • PlyCB contains the cell-wall binding domain
      • 200x more active than other lytic enzymes
  • Holins----perforate bacterial membrane; cleave covalent bonds in the peptidoglycan
  • Most potent bacteriophage-drived enzyme studied
    • Vibriophage?

M. Kamruzzaman, S. M. N. U., D. Ewen Cameron, Stephen B. Calderwood, G. Balakrish Nair, John J. Mekalanos, and Shah M. Faruque (2010). "Quorum-regulated biofilms enhance the development of conditionally viable, environmental Vibrio cholerae." PNAS 107(4): 1588-1593.

• Cholera diarrheal disease spread through contaminated water
• Exists in water as clumps of cells
• Fully virulent in the intestinal milieu
• Conditionally viable environmental cells
• Biofilm formation dependent on quorum sensing
  • Controlled by cell density----regulatory
• Cholera transmission in vivo-formed biofilms convert to CVEC upon introduction of cholera stools into environmental water
• Cholera transit between intestinal infection and hypotonic aquatic environment
  • Phage predation
• Concentration of pathogenic cholera far less than that required to induce infection
• Clumps of cells referred to as conditionally viable environmental cells
  • Can be cultured in the lab using an enrichment technique
• Genetic regulators of quorum sensing and biofilm formation affect the development of CVECs
• Ileal loops of adult rabbits ideal model
• Mutation in hapR resulted in more biofilm-like clumps
• Enzymes required for EPS synthesis (exopolysaccharide)

Ryan D. Heselpoth, D. C. N. (2012). "A new screening method for the directed evolution of thermostable bacteriolytic enzymes." Journal of Visualized Experiments 69: e4216.

• bacteriophage encode and express endolysins to hydrolyze critical covalence bonds in peptidoglycan
• Elevated temperature treatment can increase enzymatic activity
• Engineered phage to acquire enhanced kinetic stability
• mutations increase thermostability

Jinki Yeom, J.-H. S., Ji-Young Yang, Jungmin Kim, Geum-Sook Hwang (2013). "H NMR-based metabolite profiling of planktonic and biofilm cells in Acinetobacter baumannii 1656-2." PLoS ONE 8(3): e57730.

• Acinetobacter baumannii: aerobic gram-negative bcateria; highly resistant to antibiotics
• Metabolite profiling of biofilms performed using NMR spectroscopy
• Principal metabolite components included: acetates, pyruvate, succinate, UDP-glucose, AMP, glutamate, lysine
  • Help to better characterize metabolic changes
  • Changes in metabolism are dependent on the growth state
  • Changes in acetyl-glucosamine level may act as markers for biofilm infections
  • The higher levels of succinate, pyruvate and acetate indicate a reduction in energy production
• Higher levels of UDPG, AMP and NAD+
  • UDPG a possible precursor of polysaccharides----production of exopolysaccharide (EPS)----helps to maintain the biofilm
  • High levels of AMP to maintain biofilm structure
  • Oxidative stress can promote biofilm formation (high levels of NAD+ as a cofactor for oxidative stress defense protein)
  • Steady valine amino acid production observed in biofilm cells---contributes to biofilm matrix maintenance
  • EPS critical for biofilm formation----a mixture of sugar polymers
  • N-acetyl-D-glucosamine

Berche, S. V. a. P. (2000). "NhaA, an Na+/H+ antiporter involved in environmental survival of Vibrio cholerae." Journal of Bacteriology 182(10): 2937-2944.

• Vibrio cholerae inhabits aquatic environments----wide range of pH and salinity
• the nhaA gene encodes a protein that results in formation of the Na+/H+ anti-porter
  • Contributes to the Na+/H+ homeostasis
• Most epidemic strains belong to serogroup O; O139 pandemic (also a new epidemic strain)
• pH of seawater between 7 and 8
• optimal pH ranges from 7-9 depending on salinity
• Salt concentrations between .2 and 2%; in vitro .25-3%
  • optimal salinity 2%
• Na+/H+ establishes electrochemical potential of Na+ across the membrane; flagellar rotation, extrusion of Na+, intracellular pH regulation
• activity of NhaA dependent on pH

Mekalanos, S. M. F. a. J. J. (2012). "Phage-bacterial interactions in the evolution of toxigenic Vibrio cholerae." Virulence 3(7).

• evolution from environmental non-pathogenic strains through acquisition of virulence genes
• phage predation to control cholera epidemics
  • CTX phage

Byung-Doo Lee, R. T., Sachidevi Puttaswamy, Brandon M. Smith, Keshab Gangopadhyay, Shubhra Gangopadhyay and Shramik Sengupta (2013). "Ultra-rapid elimination of biofilms via the combustion of a nanoenergetic coating." BMC Biotechnology 13(30).

• a biofilm: microbially derived community characterized by cells that irreversibly attach; can occur spontaneously

Shuyang Sun, S. K., Diane McDougald (2013). "Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists." PLoS ONE 8(2): e56338.

• Cholera survival depends on its ability to respond to stresses
• Grazing pressure by protozoan organisms can induce biofilm formation
• Vibrio polysaccharide (VPS) facilitates that attachment of cholera cells to the surface
• VPS responsible for biofilm resistance
• VPS positively regulated by VpsR and VpsT but repressed by HapR

Luanne Hall-Stoodley, P. S. (2002). "Developmental regulation of microbial films." Current Opinion in Biotechnology 13(3): 228-233.

• Biofilms complex communities
• To gain an understanding of the developmental processes and regulatory mechanisms involved in biofilm formation
• Biofilm formation is dynamic
• Extracellular microbial structures assist in adhesion and maintenance of structure.
• Initiated by:
  • Redistribution of attached cells by surface motility
  • Motility to assist colonization
  • Protein and polysaccharide adhesins
  • Binary division of attached cells
  • Surface-associated aggregation can also occur by recruitment of single cells
• Bacterial cells in biofilm colonies held together by EPS
  • Includes polysaccharides, nucleic acids and proteins
  • Polymer alginate, from P. aeruginosa strains----helps to determine biofilm structure
  • Increased production of EPS in flagellar mutants of V. cholera 0139 correlated with reduced intestinal colonization in an infant mouse model
  • Diversity in EPS production among bacterial species

Nicholas J. Shikuma, K. R. D., Jiunn N.C. Fong and Fitnat H. Yildiz (2012). "The transcriptional regulator, CosR, controls compatible solute biosynthesis and transport, motility and biofilm formation in Vibrio cholerae." Environmental Microbiology.

• Inhabits aquatic environments
• Colonizes the human digestive tract
• Adjusts to changes in salinity and osmolarity----produces solutes to counteract extracellular osmotic pressure
• Transcriptional regulator CosR----regulated by ionic strength
• CosR activates biofilm formation and represses motility
• To help maintain osmotic tolerance
• Solute molecules stabilize intracellular levels of water/pressure
• Salinity a significant factor in affecting growth and distribution of Cholera
• Cholera epidemics correlated with increased salinity in riverine and estuarine habitats
• Biofilm formation facilitates cholera survival
• Salinity affects expression of biofilm genes
• CosR represses motility and activates biofilm formation
• Uses CosR to adapt to changing salinity environments
• Cholera depends on its ability to form biofilms for virulence as a human pathogen
• WT and mutant CosR grown in ASW (50% artificial seawater media)
• Mutant defective in biofilm growth
• Grown for 72 hours
• Also grown in LB

M. Kamruzzaman, S. M. N. U., D. Ewen Cameron, Stephen B. Calderwood, G. Balakrish Nair, John J. Mekalanos, and Shah M. Faruque (2010). "Quorum-regulated biofilms enhance the development of conditionally viable, environmental Vibrio cholerae." PNAS 107(4): 1588-1593.

• Cholera diarrheal disease spread through contaminated water
• Exists in water as clumps of cells
• Fully virulent in the intestinal milieu
• Conditionally viable environmental cells
• Biofilm formation dependent on quorum sensing
  • Controlled by cell density----regulatory
• Cholera transmission in vivo-formed biofilms convert to CVEC upon introduction of cholera stools into environmental water
• Cholera transit between intestinal infection and hypotonic aquatic environment
  • Phage predation
• Concentration of pathogenic cholera far less than that required to induce infection
• Clumps of cells referred to as conditionally viable environmental cells
  • Can be cultured in the lab using an enrichment technique
• Genetic regulators of quorum sensing and biofilm formation affect the development of CVECs
• Ileal loops of adult rabbits ideal model
• Mutation in hapR resulted in more biofilm-like clumps
• Enzymes required for EPS synthesis (exopolysaccharide)
• Formation of biofilms necessary for CVEC development

Katrina L. Van Dellen, P. I. W. (2006). "The Vibrio cholerae biofilm: A target for novel therapies to prevent and treat cholera." Drug Discovery Today: Disease Mechanisms 3(2).

Shuyang Sun, S. K., Diane McDougald (2013). "Relative Contributions of Vibrio Polysaccharide and Quorum Sensing to the Resistance of Vibrio cholerae to Predation by Heterotrophic Protists." PLoS ONE 8(2): e56338.

Luanne Hall-Stoodley, P. S. (2002). "Developmental regulation of microbial films." Current Opinion in Biotechnology 13(3): 228-233.

Jason B. Harris, R. C. L., Firdausi Qadri, Edward T. Ryan, Stephen B. Calderwood (2012). "Cholera." The Lancet 379(9835): 2466-2476.

4/12/13

Today all of the separate groups in our team presented our current project plans and our progress so far. Because of finals and the break between semesters, we are adjourning until May 1st.