Evaluation of
phagocytosis in human neutrophils using enhanced green fluorescent protein
(EGFP) expressing E. coli
Hosain Aqa Hosaini
1 *, Sayyed Hamid Zarkesh Esfahani 2, Zahra Etemadifar
2, Elahe Mosavi 3
1 Department of Biology, Faculty of Science, University of Herat, Afghanistan
2 Department of Cell and Molecular
biology & Microbiology, Faculty of Biological Sciences, University of
Isfahan, Iran
3 Department of
Sociology, Faculty of Social Science, University of Herat, Afghanistan
*Corresponding
Author: Hosain Aqa Hosaini
* Email: Hosaini.hosain@yahoo.com
Abstract
Introduction: Phagocytosis plays a very important role in innate immunity and helps
the body against bacterial infections. Patients who have defect in phagocytosis
suffer from recurrent bacterial infections that may be life threatening. It is important to detect the defect in
phagocytosis as early as possible in life. Patients who have received
immunosuppressive medication may also have suppressed phagocytosis. There are
different laboratory tests for evaluation of phagocytosis such as NBT (Nitroblue Tetrazolium) and DHR (Dihydrorhodamine)
which use chemical compounds not real bacteria. Nitroblue
Tetrazolium is yellow chemical substance, in NBT test neutrophils are isolated
first and then for Stimulation of respiratory burst in neutrophils add PMA (Phorbol
Myristate Acetate). PMA and NBT are
exposed to neutrophils, if a respiratory burst occurs in neutrophils, the color
of NBT changes from yellow to purple, that purple neutrophils can be seen under
microscope. In DHR test Dihydrorhodamine 1,2,3 was
exposed to neutrophils that were stimulated with PMA. Normal neutrophils
oxidize DHR after ingestion; finally neutrophils will
be fluorescent that can be detected by flow cytometry. The behavior of
neutrophils when exposed to chemicals compounds such as NBT and DHR may be
different and abnormal, so when real bacteria such as E. coli are
exposed to neutrophils the behavior of neutrophils phagocytosis will be normal.
The
aim of this study was to evaluate a simple and quick method for testing
phagocytosis using real bacteria instead of chemical compounds.
Materials
and Methods: An EGFP (Enhanced green fluorescent protein) sequence was cloned into a
pColdI expression vector. E. coli (Bl21
strain) was transformed by EGFP containing vector. EGFP expression in bacteria
was detected by a fluorescent microscope and flow cytometry. EGFP expressing
bacteria were added to the heparinized blood of healthy volunteers.
Phagocytosis and digestion of fluorescent bacteria by neutrophils were detected
using flow cytometry at different time points.
Results: Neutrophils that engulfed the fluorescent bacteria showed high
fluorescent activity and, were identified by flow cytometry. Bacterial
digestion over time led to a decrease in fluorescent of neutrophils.
Conclusion: EGFP expressing bacteria and flow cytometry technique can be used to
evaluate phagocytosis. It can be optimized for clinical as well as research
uses.
Keywords: Phagocytosis, EGFP, E. coli, Fluorescent, Flow cytometry
Introduction
All blood cells, such as red blood cells and platelets, as well as white blood cells, are derived from hematopoietic stem cells in the bone marrow (1). Hematopoietic stem cells divide into lymphoid and myeloid cells. Myeloid cells also differentiate into two cell types including monocytes and granulocytes. Granulocytes are differentiated into three types of cells neutrophils, eosinophils and basophils. Neutrophils have phagocytic properties and are among the first cells to respond to infection (1, 2, 3). In adults, about 1011 neutrophils are produced daily in the bone marrow. Neutrophils cannot proliferate and their lifespan does not exceed a few days. During infection, Neutrophils are activated by bacterial cell wall products such as lipopolysaccharide, (LPS) and muramic dipeptide (MDP) and inflammatory mediators such as interferon-gamma (IFNγ) (4,5). Neutrophils with respiratory burst produce such oxygen radicals, Hydroxyls and their antibacterial peptides (defensins) are able to destroy pathogens (6). Neutrophils have Azerophilic cytoplasmic granules (primary) and have specific granules (secondary). Activation of neutrophils leads to phagocytosis of foreign antigens. Phagocytosis is a type of endocytosis. Endocytosis is a process of removal of environmental substances by the cell. In phagocytosis, the plasma membrane of the cell surrounds a particle. These particles may contain the entire body of a pathogen. Phagocytosis is an active process that uses energy to swallow particles larger than 0.5µm. Phagocytosis has four basic stages, which include chemotaxis, opsonization, swallowing and digestion (7). Neutrophils in the bloodstream circulate naturally, but stimulated by the adhesion of endothelial cells, the blood vessels allow neutrophils to leave. Stimulated endothelial cells produce adhesive proteins that include selectins and integrins. These proteins bind to neutrophils and cause migration of neutrophils into the tissues.
When neutrophils are drawn to the source of foreign objects, First the pseudopod and then the bulk of the cell are pushed forward. The neutrophil pseudopod cytoplasm consists of a filamentous network containing actin and myosin proteins. The status of this network determines the fluidity of the cytoplasm. When the neutrophil encounters the bacteria, the pseudopod neutrophil is drawn to the bacteria and encloses it and the opsonins on the organism and the neutrophil surface receptors bind to each other. Eventually, the bacteria are drawn into the cell and then the bacteria are trapped in a vacuole called a phagosome. Neutrophils include or produce deadly molecules, including a number of active oxygen proteases and mediators that are able to destroy pathogens in the body (8). Destruction of the swallowed bacteria is accomplished through two distinct processes: first, the production of oxygen radicals, (process of respiratory burst) and second, the release of decomposing enzymes and antimicrobial peptides from inside the cell. One of the most important steps in responding to inflammation by neutrophils is respiratory burst, which is characterized by high oxygen consumption and the production of active oxygen species (9). There are different laboratory tests for evaluation of phagocytosis such as NBT (Nitroblue tetrazolium) and DHR (Dihydrorhodamin) which use chemical compounds, not real bacteria. To study the behavior of phagocytic cells during phagocytosis, it is more appropriate to use real bacteria such as E. coli. These bacteria can be transformed to expressed Enhanced Green Fluorescent Protein (EGFP). The phagocytosis of fluorescent bacteria can be detected by flow cytometry.
NBT test is used to measure the production of active oxygen
mediators in a respiratory burst, in the short time that neutrophils attach to
the bacterium, oxygen consumption increases by about 100 times; this, increase
in consumption is the result of the activation of a cellular enzyme called
NADPH oxidase. This enzyme causes the transfer of electrons to oxygen in the
cell wall. By the action of the enzyme myeloperoxidase, hydrogen peroxide is
converted to its bactericidal compound (10). In this study, the E. coli
was transformed using an EGFP containing plasmid. Expression of EGFP by E.
coli was detected using fluorescent microscopy and flow cytometry.
Phagocytosis of EGFP expression bacteria by neutrophils then was assessed using
flow cytometry at different time points.
Materials and Methods
The steps of material and method for this study are divided into
two parts. The first step was to prepare E. coli strain BL21 that can
express a bright green fluorescent protein (EGFP). The second step was to use
fluorescent bacteria to evaluate phagocytosis and obtained the optimal time for
the stages of ingestion and digestion of phagocytosis. Preparation of bacterial
competent cells from E. coli strain BL21were done with the Calcium
chloride chemical method. For this purpose inoculated
bacteria to 10ml LB (Luria Bertani) media incubated at 37 ºC for 16 hours. Take
some colony and inoculated to 20ml LB media and use shaker incubator at 37 ºC
to OD600=0.4-0.6. Culture media was
transferred to 1.5ml microtubules and placed on ice for 10 minutes. Centrifuge
microtube in refrigerated centrifuge at 4 ºC for 20 minutes at 4000 rpm and
transferred to ice. Supernatant out and added 0.75 ml of CaCl2 100Mm to each
microtube. Microtubes were placed on ice for an hour and then continue like
centrifugation step. Finally supernatant out and added 200 µl glycerol stock
solution and store -70 ºC.
PEGFP Transformed to Pcold Expression
vector with Electroporation Method. For this propose, 5µl of recombinant vector
was added to 100ml competent bacterial cell and placed on ice for 30 minutes.
The thermal shock was caused by plasmid passing through the bacterial membrane
so that microtubes containing competent bacteria were put in Bain Marie at 42
ºC for 90-120 seconds and then microtubes were placed on ice for 10 min. 400μl
sterile LB culture medium was added to the contents of the microtube and
pipette and was placed at 37 ºC in shaker incubator for bacterial reproduction
for an hour. After an hour, the microtubes were centrifuged for 10 minutes at
5000 rpm. The supernatant was out and 100µl of LB medium was added to the
bacterial sediment and pipetted. The contents of the microtube were transferred
to a plate containing LB agar culture medium and incubated overnight at 37 ºC.
Reproduction and plasmid extraction for evaluating the attendance of EGFP was
done by GenetBio kit. For plasmid extraction from the
bacteria that contain the EGFP plasmid, cultured the bacteria overnight then
the following steps are performed.
LB medium containing bacteria from overnight culture was
transferred to 1.5 ml microtubes and centrifuged for 3 minutes at 8000 rpm.
Supernatant out and added 250µl suspension solution and pipetted. Added 250µl
of lysis buffer to each microtube and each microtube was slowly turned upside
down about 4 to 6 times for 5 minutes. After that add 350µl neutralizing
solution to each microtube was slowly turned upside down about 4 to 6 times.
The sample was centrifuged at 12500 rpm for 10 minutes; supernatant was
carefully transferred to the purification column,
purification column was centrifuged at 12500 rpm for 1 minute and was out micro
tube sediment. After that added 700µl washing solution to column and
centrifuged at 12500 rpm for 30 seconds and was out supernatant. In order to
remove the residual wash buffer, which contains ethanol, the column was
centrifuged again for 2 minutes and placed at room temperature for 2 minutes to
completely remove the alcohol. The column was transferred to a new microtube
and 50 µl of the dissolution buffer was added to the center of the purification
column to separate the plasmid DNA extracted from the column and incubated for
2 minutes at room temperature. In the last step, the column was centrifuged at
12500 rpm for 2 minutes. The collected solution was stored in the microtube in
a freezer at -20 °C. After plasmid extraction to check the accuracy of plasmid
extraction, 2µl of the plasmid extraction product was electrophoresed on 1%
agarose gel. In order to quantitatively and qualitatively verify the expression
of EGFP in transform bacteria with pEGFP, samples
were first prepared for observation by fluorescent microscope and then read by
flow cytometry. Evaluation of protein expression by bacteria done with
different concentrations of IPTG (Isopropyl β-D-1-thiogalactopyranoside
). IPTG with a concentration of 0.3mmol was selected to express high
volume EGFP protein. Evaluation of phagocytosis of neutrophils, Performed by
DHR(Dihydrorhodamine) test. Select 3 FACS tubes and
add 100µl heparinized blood and added 0.15ngr/ml DHR just to the second and third tube
and incubate at 37ºC for 20min. After incubation added 10ngr/ml PMA (Phorbol
Myristate Acetate) just to third tube and incubate at 37ºC for 20min. Added to
tree tubes 2ml aluminum chloride lysis buffer 1x. In this study flow cytometry
techniques were performed on a BD FACSCalibur flow
cytometry (Partec PAS, Germany). The number of
bacteria selected for each sample was 10µl from the medium with OD600 =
0.4-0.6, which contains about 6×106 bacteria. To determine the optimal time for
phagocyte bacteria by neutrophils, samples with different duration of time
(from 20 minutes to 90 minutes) were incubated in the presence of EGFP
bacteria, and used flow cytometry techniques for evaluating optimal time of phagocytosis. Preparation of samples for reading by flow
cytometry similar to the steps performed with DHR but difference is that
instead of DHR in the tubes, was used the E. coli bacteria strain Bl21,
which contains EGFP plasmid, was stimulated expression by IPTG. Selected
2.5hours to determine the optimal time to digest phagocyte bacteria by
neutrophils.
Results
PEGFP plasmid extraction was done with Kit Company and the result
of gel electrophoresis shows band PEGFP plasmid in figure 1.
Figure 1. Plasmid extraction of EGFP. Column
1: 1Kb DNA Ladder. Column 2: 2µl of plasmid extraction product. Column 3: 5µl
of plasmid extraction product. The pcoldI vector is
4407 bp, which contains the EGFP = 32.7 bp.
Expression Confirmation of EGFP done with Flow cytometry and
Fluorescent Microscope. Figure 2 shows negative control of bacteria that
includes dot diagram (Dot Plot) of the FSC and SSC bacterial characteristics.
The FSC shows the size of the EGFP bacteria and the SSC shows their granulocyte
characteristics. As can be seen in the figure, most of the bacteria are present
inside the gate. The majority of them are in the same range in terms of size
and granulocyte. The upper and left sides of the histogram show auto
Fluorescent. These bacteria don’t have the EGFP gene and don’t have
fluorescents, and their auto Fluorescent is shown on their histograms. The autofluorescent shown in their histogram is the result of
the device absorbing the laser beam and emitting it in the same range. The RN1
gate is the negative fluorescent range and the RN2 gate is the positive
fluorescent range. Numerical data for RN1 gate calculated by FlowMax software.
Mean x for RN1 represents the evaluation index, the value of which is
0.21 and doesn’t have any fluorescent.
