Sequesteration of lead, cadmium and arsenic by Lactobacillus

154 Pages·2017·1.53 MB·English

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Marc A. Monachese, The University of Western Ontario
Supervisor: Dr. Gregor Reid, The University of Western Ontario
A thesis submitted in partial fulfillment of the requirements for the Master of Science degree in
Microbiology and Immunology
© Marc A. Monachese 2012
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Monachese, Marc A., "Sequesteration of lead, cadmium and arsenic by Lactobacillus species and
detoxication potential" (2012). Electronic Thesis and Dissertation Repository. 729.
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SEQUESTRATION OF LEAD, CADMIUM AND ARSENIC BY
LACTOBACILLUS SPECIES AND DETOXICATION POTENTIAL

(Spine Title: Sequestering Metals Using Lactobacilli)
(Thesis Format: Monograph)

by
Marc Alberto Monachese

Graduate Program in
Microbiology and Immunology

Thesis submitted in fulfillment
of the requirements for the degree of
Master of Science

The School of Graduate and Postdoctoral Studies
The University of Western Ontario
London, Ontario, Canada

© Marc Alberto Monachese, 2012
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THE UNIVERSITY OF WESTERN ONTARIO
School of Graduate and Postdoctoral Studies

CERTIFICATE OF EXAMINATION

Supervisor Examiners

______________________________ ______________________________
Dr. Gregor Reid Dr. Peter Cadieux

Supervisory Committee ______________________________
Dr. Martin McGavin

______________________________
Dr. Jack Bend ______________________________
Dr. Sheila Macfie

______________________________
Dr. Gordon Southam

The thesis by

Marc Alberto Monachese

entitled:

Sequestration of Lead, Cadmium and Arsenic by Lactobacillus Species and
Detoxication Potential

is accepted in partial fulfillment of the
requirements for the degree of

Master of Science

______________________ _______________________________
Date Chair of the Thesis Examination Board
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ABSTRACT AND KEYWORDS

Toxic metals are a class of elements with no biological role but with extreme
toxicity. On average only 50% of ingested toxins are absorbed into the human body, for
reasons still unknown. It was hypothesized that the gut microbiota plays a role in
reducing toxin absorbance. The aim of this study was to determine if constituents of the
gut, namely Lactobacillus species, are able to sequester arsenic, lead and cadmium from
the environment. Lactobacilli were incubated with the metals, both in vitro and with a
Caco-2 cell line. Analysis of metal concentrations was conducted to determine if these
were reduced by lactobacilli and if this in turn lowered mortality of the Caco-2 cells, and
to establish if lactobacilli could block transport of metals across a cell layer. In vitro
studies and electron microscopy showed that lactobacilli had significant binding potential
to the metals lead and cadmium. In addition, they conferred protection of Caco-2 cells
from toxicity with less metal binding to the epithelial cell surfaces. This supports my
hypothesis and raises the concept of using probiotic lactobacilli, perhaps in a food
formulation, to reduce heavy metal exposure and poisoning. A yogurt formulation was
created using two strains with metal-binding potential, and this could be tested in the
future.

Keywords: Arsenic, lead, cadmium, lactobacilli, gut microbiota, detoxication/
detoxification

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“All we have to decide is what to do with the time that is given to us.”
― J.R.R. Tolkien, The Fellowship of the Ring

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ACKNOWLEDGEMENTS

I would first like to thank Dr. Gregor Reid for allowing me to work in his lab and
for giving me this project from which I was able to learn so much. His support, guidance
and friendship have been invaluable and I cannot thank him enough for all he has
provided me.
I thank my advisory committee, Drs. Jack Bend and Gordon Southam, who were
always available to provide expertise and assistance during the course of my project, and
Dr. Jeremy Burton for giving me helpful insights into my project on a daily basis and Dr.
Charlie Wu for assistance with all metal measurement.
I thank the University of Western Ontario Nanofabrication Facility, specifically
Tim Goldhawk and Todd Simpson for equipment training and assistance with image
capturing and X-ray measurements.
I also thank Mohammed Soltani for assistance in making the probiotic yogurt and
Julie McDonald for assistance in maintaining the chemostat.
Finally, I would like to thank the Reid lab: Jordan Bisanz, Lesley Carmichael,
Jean Macklaim, Rod Macphee, Amy McMillan, Shannon Mifflin and Camilla Urbaniak.
Your friendship and kindness cannot be put into words.

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TABLE OF CONTENTS
TITLE PAGE ................................................................................................................................. i
CERTIFICATE OF EXAMINATION ....................................................................................... ii
ABSTRACT AND KEYWORDS ............................................................................................... iii
ACKNOWLEDGEMENTS ......................................................................................................... v
LIST OF FIGURES ...................................................................................................................... x
LIST OF TABLES ...................................................................................................................... xii
LIST OF APPENDICES ........................................................................................................... xiii
LIST OF ABBREVIATIONS ................................................................................................... xiv
CHAPTER 1-INTRODUCTION ................................................................................................. 1
1.1 Focus of Thesis ......................................................................................................................... 2
1.2. Heavy Metal Contamination .................................................................................................... 2
1.2.1. Arsenic .................................................................................................................. 3
1.2.2. Lead ....................................................................................................................... 6
1.2.3. Cadmium ............................................................................................................... 9
1.3. Bacterial Interactions with Metals ......................................................................................... 11
1.3.1. Geomicrobiology ................................................................................................. 11
1.3.2. Bioremediation .................................................................................................... 12
1.4. Host Interaction with Metals .................................................................................................. 14
1.4.1. Metal Biosorption in the Human Body ............................................................... 14
1.4.2. Metals and the Gut Microbiota ............................................................................ 17
1.5. Detoxification of Metals by Lactobacilli ............................................................................... 18
1.5.1. Uses and Applications of Lactobacilli ................................................................ 18
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1.5.2. Arsenic Detoxification ........................................................................................ 19
1.5.3. Lead and Cadmium Detoxification ..................................................................... 20
1.6. Rationale for Studying Lactobacilli Interaction with Toxic Metals ...................................... 22
1.7. Hypothesis and Objectives ..................................................................................................... 23
CHAPTER 2-MATERIALS AND METHODS ....................................................................... 24
2.1. Bacterial Cultures and Conditions ......................................................................................... 25
2.1.1. Metal Tolerance Growth Curve ........................................................................... 25
2.1.2. DNA Isolation and Verification of Working Strains .......................................... 28
2.1.3. DNA Purification ................................................................................................ 31
2.1.4. Sequencing .......................................................................................................... 31
2.2. Bacterial DNA Preparation for Ion Torrent Sequencing ....................................................... 31
2.3. Isolation of Cell Wall-Associated Proteins ............................................................................ 33
2.3.1. Isolation of Bacterial Supernatant Proteins ......................................................... 34
2.3.2. SDS-PAGE Protein Purification ......................................................................... 34
2.4. Metal Binding Assays – Live Cells ....................................................................................... 37
2.4.1. Metal Binding Assays – Nonviable Cells ........................................................... 38
2.5. Autoaggregation ..................................................................................................................... 39
2.6. Bacterial Potentiometric Titration .......................................................................................... 40
2.7. Tissue Culture Maintenance and Conditions ......................................................................... 41
2.7.1. Caco-2 Cell Viability and Lactobacilli Testing ................................................... 42
2.7.2. Transwell Metal Transport Studies ..................................................................... 43
2.8. Quantitative Real-Time PCR ................................................................................................. 44
2.8.1. RNA extraction and clean up .............................................................................. 44
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2.8.2. Reverse transcription ........................................................................................... 45
2.8.3. Quantification of CYP3A4 and GSTP1 Expression ........................................... 46
2.8.4. Data Analysis ...................................................................................................... 46
2.9. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Analysis (EDX)........ 49
2.10. Transmission Electron Microscopy (TEM) and Ultra Thin Sectioning ............................... 50
2.11. Yogurt Production ................................................................................................................ 51
2.12. Statistical Analysis ............................................................................................................... 51
CHAPTER 3-RESULTS ............................................................................................................ 52
3.1. Lactobacilli Growth/Tolerance in Toxic Metal Containing Media ....................................... 53
3.2. Metal Binding Assays ............................................................................................................ 56
3.3. Metal Binding Kinetics .......................................................................................................... 60
3.4. Potentiometric Titrations ....................................................................................................... 64
3.5. Autoaggregation ..................................................................................................................... 68
3.6. Metal Binding Mechanism ..................................................................................................... 70
3.6.1. Heat Treatment .................................................................................................... 70
3.6.2. Gamma Irradiation .............................................................................................. 72
3.6.3. Proton Motive Force Inhibition ........................................................................... 74
3.7. Specificity and Effectiveness of Toxic Metal Binding by Lactobacilli ................................. 76
3.8. Confirmation of Metal Sequestration by Lactobacilli............................................................ 78
3.9. Transmission Electron Microscopy (TEM) and Ultra Thin Sectioning ................................. 82
3.10. Quantitative-PCR CYP3A4 Induction by Lactobacilli and Toxins ..................................... 86
3.11. Caco-2 Cell Toxicity Protection Assay ................................................................................ 88
3.12. Caco-2 Cell Metal Translocation Study ............................................................................... 90
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3.13. Gut Microbiota and Chemostat Analysis ............................................................................. 92
CHAPTER 4-DISCUSSION ...................................................................................................... 95
4.1. Lactobacilli Toxic Metal Tolerance and Detoxication Potential ........................................... 96
4.2. Detoxication Kinetics: Where is the Metal Going? ............................................................. 101
4.3. Lactobacilli Beneficial Effects and Human Application ..................................................... 106
4.4. Gut Microbiota and Chemostat Population Analysis ........................................................... 109
4.5. Future Directions ................................................................................................................. 111
4.6. Conclusions .......................................................................................................................... 113
CHAPTER 5-REFERENCES .................................................................................................. 115
APPENDICES ........................................................................................................................... 134
CURRICULUM VITAE ........................................................................................................... 137

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