Gentamicin protection assay
Encyclopedia
The gentamicin protection assay or survival assay or invasion assay is a method used in microbiology
. It is used to quantify the ability of pathogen
ic bacteria
to invade eukaryotic cells
.
The assay is based on several observations made in the 1970s, in which the ability of internalized bacteria to avoid killing by antibiotic
s was reported. The assay started to be used in biological research in the early 1980s.
of the infected
organism
) in order to replicate
and propagate infection. Many species of Shigella
(causes bacillary dysentery
), Salmonella
(typhoid fever
), Mycobacterium
(leprosy
and tuberculosis
) and Listeria (listeriosis), to name but a few, are intracellular.
Several antibiotics cannot penetrate eukaryotic cells. Therefore, these antibiotics cannot hurt intracellular bacteria that are already internalized. Using such antibiotics enables us to differentiate between bacteria that succeed in penetrating eukaryotic cells and those that do not. Applying such an antibiotic to a culture
of eukaryotic cells infected with bacteria would kill the bacteria that remain outside the cells while sparing the ones that penetrated. The antibiotic of choice for this assay is the aminoglycoside
gentamicin
.
cells are commonly used as eukaryotic cells in the gentamicin protection assay, but other cells can be used as well. As for bacteria, only species susceptible to gentamicin can be assayed.
The assay is performed in polypropylene
plates with round wells, which are commonly used in laboratories
for culturing eukaryotic cells. The cells are allowed to grow in the wells overnight, creating a flat layer. Bacteria are separately grown overnight. On the next day the eukaryotic cells are inoculated
with the bacteria and are incubated together for an hour. Centrifuging
the plates for a few minutes may help bring cells and bacteria in contact and initiate infection.
After infection gentamicin is added to the plates, and they are incubated for an hour, allowing the antibiotic to kill all bacteria that were not able to penetrate the cells and remained outside. The plates are then washed well to remove the dead bacteria. Next the eukaryotic cells are lysed
using a detergent
, most commonly Triton X-100.
The bacteria that penetrated the cells and remained alive are now released, and they are plated on solid medium plates
. Counting the colonies formed on the plates on the next day, and knowing how many bacteria were used in the beginning of the assay, enables the researcher to calculate the percentage of bacteria that were able to invade the eukaryotic cells.
s or protein
s to the bacteria's ability to invade cells can be easily assayed using this method. The gene in question can be knocked out
, and the bacteria's invasiveness compared with that of normal, wild type
bacteria. Environmental conditions, such as pH
level and temperature
, can also be assayed for their effect on invasiveness.
The gentamicin protection assay is very sensitive, as it can detect the internalization of even single bacteria. It has several drawbacks:
To help assess the accuracy of a particular assay, positive and negative controls
should be performed. When performing the assay as described above, bacteria that are known to be entirely invasive (positive control) and bacteria that are known as non-invasive (negative control) should be included in the assay.
An alternative invasion assay is the differential immunostaining
assay, based on the binding of antibodies
to bacteria before and after invasion. The antibodies emit fluorescent
, colored light
, and the results of this assay are viewed under the microscope
.
Microbiology
Microbiology is the study of microorganisms, which are defined as any microscopic organism that comprises either a single cell , cell clusters or no cell at all . This includes eukaryotes, such as fungi and protists, and prokaryotes...
. It is used to quantify the ability of pathogen
Pathogen
A pathogen gignomai "I give birth to") or infectious agent — colloquially, a germ — is a microbe or microorganism such as a virus, bacterium, prion, or fungus that causes disease in its animal or plant host...
ic bacteria
Bacteria
Bacteria are a large domain of prokaryotic microorganisms. Typically a few micrometres in length, bacteria have a wide range of shapes, ranging from spheres to rods and spirals...
to invade eukaryotic cells
Eukaryote
A eukaryote is an organism whose cells contain complex structures enclosed within membranes. Eukaryotes may more formally be referred to as the taxon Eukarya or Eukaryota. The defining membrane-bound structure that sets eukaryotic cells apart from prokaryotic cells is the nucleus, or nuclear...
.
The assay is based on several observations made in the 1970s, in which the ability of internalized bacteria to avoid killing by antibiotic
Antibiotic
An antibacterial is a compound or substance that kills or slows down the growth of bacteria.The term is often used synonymously with the term antibiotic; today, however, with increased knowledge of the causative agents of various infectious diseases, antibiotic has come to denote a broader range of...
s was reported. The assay started to be used in biological research in the early 1980s.
Background and principle
Intracellular bacteria need to enter host cells (cellsCell (biology)
The cell is the basic structural and functional unit of all known living organisms. It is the smallest unit of life that is classified as a living thing, and is often called the building block of life. The Alberts text discusses how the "cellular building blocks" move to shape developing embryos....
of the infected
Infection
An infection is the colonization of a host organism by parasite species. Infecting parasites seek to use the host's resources to reproduce, often resulting in disease...
organism
Organism
In biology, an organism is any contiguous living system . In at least some form, all organisms are capable of response to stimuli, reproduction, growth and development, and maintenance of homoeostasis as a stable whole.An organism may either be unicellular or, as in the case of humans, comprise...
) in order to replicate
Cell division
Cell division is the process by which a parent cell divides into two or more daughter cells . Cell division is usually a small segment of a larger cell cycle. This type of cell division in eukaryotes is known as mitosis, and leaves the daughter cell capable of dividing again. The corresponding sort...
and propagate infection. Many species of Shigella
Shigella
Shigella is a genus of Gram-negative, nonspore forming, non-motile, rod-shaped bacteria closely related to Escherichia coli and Salmonella. The causative agent of human shigellosis, Shigella causes disease in primates, but not in other mammals. It is only naturally found in humans and apes. During...
(causes bacillary dysentery
Bacillary dysentery
Bacillary dysentery is a type of dysentery, and is a severe form of shigellosis.Bacillary dysentery is associated with species of bacteria from the Enterobacteriaceae family. The term is usually restricted to Shigella infections....
), Salmonella
Salmonella
Salmonella is a genus of rod-shaped, Gram-negative, non-spore-forming, predominantly motile enterobacteria with diameters around 0.7 to 1.5 µm, lengths from 2 to 5 µm, and flagella which grade in all directions . They are chemoorganotrophs, obtaining their energy from oxidation and reduction...
(typhoid fever
Typhoid fever
Typhoid fever, also known as Typhoid, is a common worldwide bacterial disease, transmitted by the ingestion of food or water contaminated with the feces of an infected person, which contain the bacterium Salmonella enterica, serovar Typhi...
), Mycobacterium
Mycobacterium
Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. The genus includes pathogens known to cause serious diseases in mammals, including tuberculosis and leprosy...
(leprosy
Leprosy
Leprosy or Hansen's disease is a chronic disease caused by the bacteria Mycobacterium leprae and Mycobacterium lepromatosis. Named after physician Gerhard Armauer Hansen, leprosy is primarily a granulomatous disease of the peripheral nerves and mucosa of the upper respiratory tract; skin lesions...
and tuberculosis
Tuberculosis
Tuberculosis, MTB, or TB is a common, and in many cases lethal, infectious disease caused by various strains of mycobacteria, usually Mycobacterium tuberculosis. Tuberculosis usually attacks the lungs but can also affect other parts of the body...
) and Listeria (listeriosis), to name but a few, are intracellular.
Several antibiotics cannot penetrate eukaryotic cells. Therefore, these antibiotics cannot hurt intracellular bacteria that are already internalized. Using such antibiotics enables us to differentiate between bacteria that succeed in penetrating eukaryotic cells and those that do not. Applying such an antibiotic to a culture
Cell culture
Cell culture is the complex process by which cells are grown under controlled conditions. In practice, the term "cell culture" has come to refer to the culturing of cells derived from singlecellular eukaryotes, especially animal cells. However, there are also cultures of plants, fungi and microbes,...
of eukaryotic cells infected with bacteria would kill the bacteria that remain outside the cells while sparing the ones that penetrated. The antibiotic of choice for this assay is the aminoglycoside
Aminoglycoside
An aminoglycoside is a molecule or a portion of a molecule composed of amino-modifiedsugars.Several aminoglycosides function as antibiotics that are effective against certain types of bacteria...
gentamicin
Gentamicin
Gentamicin is an aminoglycoside antibiotic, used to treat many types of bacterial infections, particularly those caused by Gram-negative organisms. However, gentamicin is not used for Neisseria gonorrhoeae, Neisseria meningitidis or Legionella pneumophila...
.
Procedure
HeLaHeLa
A HeLa cell is a cell type in an immortal cell line used in scientific research. It is the oldest and most commonly used human cell line. The line was derived from cervical cancer cells taken on February 8, 1951 from Henrietta Lacks, a patient who eventually died of her cancer on October 4, 1951...
cells are commonly used as eukaryotic cells in the gentamicin protection assay, but other cells can be used as well. As for bacteria, only species susceptible to gentamicin can be assayed.
The assay is performed in polypropylene
Polypropylene
Polypropylene , also known as polypropene, is a thermoplastic polymer used in a wide variety of applications including packaging, textiles , stationery, plastic parts and reusable containers of various types, laboratory equipment, loudspeakers, automotive components, and polymer banknotes...
plates with round wells, which are commonly used in laboratories
Laboratory
A laboratory is a facility that provides controlled conditions in which scientific research, experiments, and measurement may be performed. The title of laboratory is also used for certain other facilities where the processes or equipment used are similar to those in scientific laboratories...
for culturing eukaryotic cells. The cells are allowed to grow in the wells overnight, creating a flat layer. Bacteria are separately grown overnight. On the next day the eukaryotic cells are inoculated
Inoculation
Inoculation is the placement of something that will grow or reproduce, and is most commonly used in respect of the introduction of a serum, vaccine, or antigenic substance into the body of a human or animal, especially to produce or boost immunity to a specific disease...
with the bacteria and are incubated together for an hour. Centrifuging
Centrifuge
A centrifuge is a piece of equipment, generally driven by an electric motor , that puts an object in rotation around a fixed axis, applying a force perpendicular to the axis...
the plates for a few minutes may help bring cells and bacteria in contact and initiate infection.
After infection gentamicin is added to the plates, and they are incubated for an hour, allowing the antibiotic to kill all bacteria that were not able to penetrate the cells and remained outside. The plates are then washed well to remove the dead bacteria. Next the eukaryotic cells are lysed
Lysis
Lysis refers to the breaking down of a cell, often by viral, enzymic, or osmotic mechanisms that compromise its integrity. A fluid containing the contents of lysed cells is called a "lysate"....
using a detergent
Detergent
A detergent is a surfactant or a mixture of surfactants with "cleaning properties in dilute solutions." In common usage, "detergent" refers to alkylbenzenesulfonates, a family of compounds that are similar to soap but are less affected by hard water...
, most commonly Triton X-100.
The bacteria that penetrated the cells and remained alive are now released, and they are plated on solid medium plates
Growth medium
A growth medium or culture medium is a liquid or gel designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens.There are different types of media for growing different types of cells....
. Counting the colonies formed on the plates on the next day, and knowing how many bacteria were used in the beginning of the assay, enables the researcher to calculate the percentage of bacteria that were able to invade the eukaryotic cells.
Usage, advantages and caveats
The gentamicin protection assay is commonly used in pathogen research. The contribution of specific geneGene
A gene is a molecular unit of heredity of a living organism. It is a name given to some stretches of DNA and RNA that code for a type of protein or for an RNA chain that has a function in the organism. Living beings depend on genes, as they specify all proteins and functional RNA chains...
s or protein
Protein
Proteins are biochemical compounds consisting of one or more polypeptides typically folded into a globular or fibrous form, facilitating a biological function. A polypeptide is a single linear polymer chain of amino acids bonded together by peptide bonds between the carboxyl and amino groups of...
s to the bacteria's ability to invade cells can be easily assayed using this method. The gene in question can be knocked out
Gene knockout
A gene knockout is a genetic technique in which one of an organism's genes is made inoperative . Also known as knockout organisms or simply knockouts, they are used in learning about a gene that has been sequenced, but which has an unknown or incompletely known function...
, and the bacteria's invasiveness compared with that of normal, wild type
Wild type
Wild type refers to the phenotype of the typical form of a species as it occurs in nature. Originally, the wild type was conceptualized as a product of the standard, "normal" allele at a locus, in contrast to that produced by a non-standard, "mutant" allele...
bacteria. Environmental conditions, such as pH
PH
In chemistry, pH is a measure of the acidity or basicity of an aqueous solution. Pure water is said to be neutral, with a pH close to 7.0 at . Solutions with a pH less than 7 are said to be acidic and solutions with a pH greater than 7 are basic or alkaline...
level and temperature
Temperature
Temperature is a physical property of matter that quantitatively expresses the common notions of hot and cold. Objects of low temperature are cold, while various degrees of higher temperatures are referred to as warm or hot...
, can also be assayed for their effect on invasiveness.
The gentamicin protection assay is very sensitive, as it can detect the internalization of even single bacteria. It has several drawbacks:
- Gentamicin can sometimes penetrate eukaryotic cells and kill the internalized bacteria. This may happen if the permeabilitySemipermeable membraneA semipermeable membrane, also termed a selectively permeable membrane, a partially permeable membrane or a differentially permeable membrane, is a membrane that will allow certain molecules or ions to pass through it by diffusion and occasionally specialized "facilitated diffusion".The rate of...
of the cells somehow increased during the assay, sometimes due to poor handling of the cells. - Internalized bacteria may sometimes not be entirely protected from the outside environment, such as when the phagosomePhagosomeIn cell biology, a phagosome is a vacuole formed around a particle absorbed by phagocytosis. The vacuole is formed by the fusion of the cell membrane around the particle. A phagosome is a cellular compartment in which pathogenic microorganisms can be killed and digested...
(the vacuoleVacuoleA vacuole is a membrane-bound organelle which is present in all plant and fungal cells and some protist, animal and bacterial cells. Vacuoles are essentially enclosed compartments which are filled with water containing inorganic and organic molecules including enzymes in solution, though in certain...
surrounding the bacterium inside the cell) is defective in some way. Gentamicin may kill those bacteria. - Gentamicin may fail to kill all the bacteria that remained outside the cells.
To help assess the accuracy of a particular assay, positive and negative controls
Scientific control
Scientific control allows for comparisons of concepts. It is a part of the scientific method. Scientific control is often used in discussion of natural experiments. For instance, during drug testing, scientists will try to control two groups to keep them as identical and normal as possible, then...
should be performed. When performing the assay as described above, bacteria that are known to be entirely invasive (positive control) and bacteria that are known as non-invasive (negative control) should be included in the assay.
An alternative invasion assay is the differential immunostaining
Immunostaining
Immunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. The term immunostaining was originally used to refer to the immunohistochemical staining of tissue sections, as first described by Albert Coons in 1941...
assay, based on the binding of antibodies
Antibody
An antibody, also known as an immunoglobulin, is a large Y-shaped protein used by the immune system to identify and neutralize foreign objects such as bacteria and viruses. The antibody recognizes a unique part of the foreign target, termed an antigen...
to bacteria before and after invasion. The antibodies emit fluorescent
Fluorescence
Fluorescence is the emission of light by a substance that has absorbed light or other electromagnetic radiation of a different wavelength. It is a form of luminescence. In most cases, emitted light has a longer wavelength, and therefore lower energy, than the absorbed radiation...
, colored light
Light
Light or visible light is electromagnetic radiation that is visible to the human eye, and is responsible for the sense of sight. Visible light has wavelength in a range from about 380 nanometres to about 740 nm, with a frequency range of about 405 THz to 790 THz...
, and the results of this assay are viewed under the microscope
Microscope
A microscope is an instrument used to see objects that are too small for the naked eye. The science of investigating small objects using such an instrument is called microscopy...
.