Measuring Angiotensin-I-Converting Enzyme 2 (ACE2) Activity
in Lactobacillus reuteri (L. reuteri)
by
Airi Gardner
Senior Honors Thesis
Submitted to the
College of Liberal Arts and Sciences Department of Biology
In Partial Fulfillment for the Degree of
Bachelor of Science in Biology
At the
University of Florida
March 27, 2019
©University of Florida 2019. All rights reserved.
University of Florida College of Medicine
Department of Ophthalmology
Principal Investigator: Dr. Qiuhong Li
Research Advisor: Dr. Amrisha Verma
1
Abstract
Angiotensin-I-converting enzyme 2 (ACE2) has recently emerged as a key regulator of
the renin-angiotensin system (RAS) in both health and disease. ACE2 is highly expressed in the
vasculature and kidney tissues, where it degrades Angiotensin II (Ang II) and generates its
physiological antagonist, Angiotensin 1-7 (Ang 1-7). ACE2 deficiency, on the other hand, is
associated with increased levels of Ang II and reduced levels of Ang 1-7, which can cause a
variety of diseases, including hypertension, diabetes, aging, and renal diseases. Interventions,
such as ACE2 replenishment or augmentation of its actions, have proven to be successful in
reducing hypertension, as well as renal and cardiac damage in different models.
Many species of the genus Lactobacillus are components of normal gut microbiota and
known to play an important role in the regulation of the body’s normal microflora and are also
commonly used in production of fermented food. Several species of Lactobacilli have been used
as probiotics to benefit the health of humans and animals. Lactobacillus reuteri (L. reuteri), a
well-studied probiotic bacterium, survive the stomach, when orally administered, and they
proliferate in the intestine, where they are metabolically active. Thus, L. reuteri can be used as a
delivery system for ACE2 replenishment.
This project attempts to genetically engineer L. reuteri for expression and delivery of
ACE2. We hypothesize that the transformed L. reuteri could express high levels of ACE2
activity and can be used for designing potential probiotics. Lactobacillus codon usage optimized
ACE2 gene was transformed into L. reuteri, as well as other Lactobacillus species, L. paracasei,
L. gasseri, and L. plantarum, by electroporation. The transformation method was standardized by
testing with green fluorescent protein (GFP) plasmid, taking advantage of its green fluorescence
to visualize the successfully-transformed cells. Protein extracts were then prepared from the
Lactobacillus bacteria with or without expressing ACE2, and ACE2 activity was measured using
a fluorescent substrate. Our data show that ACE2 is expressed in L. reuteri; however, the ACE2
activity was much lower in L. reuteri compared to other Lactobacillus species. The analysis from
this project will contribute to uncovering the unknown competencies of the Lactobacillus species
for genetic engineering probiotics for delivery of protein therapeutics, as well as suggesting
future applications of these species when engineering ACE2-expressing bacteria.
2
Table of Contents
I. Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
II. Introduction and Literature Survey . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.1 Lactobacillus species as probiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2.2 Bacterial species of Lactobacillus reuteri . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4
2.3 Metabolic disease and its potential causes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.4 Renin-Angiotensin System (RAS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2.5 The importance of ACE2 activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
2.6 Project overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
III. Materials and Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.1 Transformation of L. reuteri . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
3.2 Enzymatic activity assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
IV. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
4.1 Confirmation of transformed GFP electrocompetent cells . . . . . . . . . . . . . . . . . . . . 14
4.2 Extracted protein concentration estimations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
4.3 ACE2 activity assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
V. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
VI. Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
VII. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3
II. Introduction and Literature Survey
2.1 Lactobacillus species as probiotics
Probiotics are defined as “live microorganisms which, when administered in adequate
amounts, confer a health benefit on the host” by the World Health Organization [42]. Different
microorganisms are recognized as probiotics, such as bacteria and yeasts, but most of them fall
into the group of lactic acid bacteria, which are normally found in the food products of yogurt
and other fermented dairy foods [1]. Lactic acid bacteria, also known as the bacterial genus of
Lactobacillus, are also found as part of the normal microbial flora in healthy humans, where they
inhabit the mucosal surfaces such as the gastrointestinal and urogenital tracts [2-5]. These
bacteria grow at optimal temperatures of 37-40 °C and are known to play an important role in the
regulation of the body’s normal microflora [6]; hence, several species of Lactobacilli have been
used as probiotics to benefit the health of humans and animals [2, 7-9]. Researchers are
interested in making genetically-modified Lactobacilli to deliver therapeutic proteins like
vaccine antigens, enzymes, and hormones to the gastrointestinal and urogenital tracts of humans
for prevention and treatment of human diseases. The present project focuses on evaluating the
potential of Lactobacillus reuteri, in comparison to the other Lactobacillus species, for the
expression of angiotensin-I-converting enzyme 2 (ACE2), considering the future applications in
treating many metabolic and cardiovascular diseases.
4
2.2 Bacterial species of Lactobacillus reuteri
The species Lactobacillus reuteri is a well-known probiotic bacterium, and its probiotic
properties have been widely studied along with other Lactobacillus species, such as
Lactobacillus acidophilus, L. paracasei, L. bulgaricus, and L. rhamnosus. L. reuteri was first
isolated in 1962. It is a Gram-positive, heterofermentative, and facultative anaerobic bacterium
that can commonly be found in human and animal gastrointestinal tracts [10]. Some have
suggested that L. reuteri could be successful probiotics because of several advantageous
characteristics. This species can tolerate a wide variety of pH environments, possesses multiple
mechanisms that prevent invasions by pathogenic microorganisms, and secretes antimicrobial
substances [11-13]. Moreover, L. reuteri performs impressively in eliminating gut infections of
the host [13].
Since the introduction of a modern lifestyle with antibiotics use, improved hygiene, and
high-calorie diets, the human gut microbial communities have been altered and, in some ways,
disrupted significantly. There has been a decrease in the abundance of healthy microorganisms,
including Lactobacillus species, leaving concerns as to its consequence in the pathogenesis of
various diseases [14-16]. Although the correlation has not yet been fully established, the notable
decrease in healthy gut microbiomes may coincide with increased incidences of inflammatory
diseases. If this appears to be one of the main factors of such diseases, it may be helpful to
supplement patients with L. reuteri to increase its colonization and to take advantage of its
probiotic functions, alongside other effective functions, such as delivering therapeutic agents.
5
2.3 Metabolic disease and its potential causes
Metabolic diseases are complex disorders caused by various interrelated factors that are
both environmental and biological. While the incidents of hyper-metabolic diseases, such as
atherosclerosis, hypertension, kidney diseases, and diabetes, continue to increase worldwide,
clinical researchers and physicians are targeting to develop better therapeutic strategies [17, 18].
One of the biological factors that is thought to be contributing to the pathogenesis of such
disorders is the renin-angiotensin system (RAS). RAS has been studied for over one hundred
years since the discovery of renin in 1897 [19]. Its mechanism was first considered as only in
the regulation of circulatory homeostasis in the 1900s; however, in the 1990s, multiple clinical
studies demonstrated its potential in collaborating in some of the inflammatory processes [20,
21]. RAS is now considered the key mechanism in the development of organ damage, which
would potentially cause complicated cardiovascular disorders [22].
2.4 Renin-Angiotensin System (RAS)
In order to understand the inflammatory process involved in hyper-metabolic diseases,
understanding of the RAS signaling pathway is critical. RAS is a hormonal cascade event which
takes place in the circulatory system. Its evolutionary importance is to regulate fluid and
electrolyte balance, as well as blood pressure in the major loss of blood volume, such as in
dehydration or massive bleeding. It involves multiple components, and each play an important
role in delivering the signal promptly.
The RAS signaling cascade is explained using the mechanistic model established by
Claassen et. al in 2013 [Figure 1]. One of the primary components of the RAS cascade is renin.
6
Renin is originally synthesized in the kidney from the enzyme precursor prorenin, which is then
secreted into the blood plasma by the granular cells. Renin, that is now an active proteolytic
enzyme, travels a short distance to the juxtaglomerular cells where it is stored. When the cells are
stimulated, renin is released into the plasma, and it converts the hepatically-synthesized inactive
hormone angiotensinogen to Angiotensin I (Ang I). Ang I is reported to have few biological
activities, but is converted by the membrane-bound carboxypeptidase, angiotensin-converting
enzyme (ACE), to Angiotensin II (Ang II). Ang II, the main effector peptide of the RAS, is a
vasoconstrictor that interacts with the angiotensin II receptor type 1 (AT1).
Figure 1: Mechanistic model of the RAS cascade
event [23]; AGT as in angiotensinogen, Ang 1 as in
angiotensin I, Ang 2 as in angiotensin II. ACE as in
angiotensin-converting enzyme, AT1 as in
angiotensin II receptor type 1, and Aldost as in
aldosterone, which is a mineralocorticoid hormone.
7
Ang II increases arterial pressure via two main pathways [24]. One is by directly
affecting arteriolar vasoconstriction. This reaction is recorded to be very rapid and intense and
occurs within seconds. Another is by increasing sodium and water reabsorptions at the kidney
tubules. This increases the extracellular fluid volume, affecting blood volume slowly over hours
and days.
During hemorrhagic shock and in sodium-deficient states, the RAS is activated, and
intrarenal Ang II levels are elevated. Increasing sodium and water reabsorption, thereby
increasing blood pressure, serves an important physiological role in maintaining the circulating
volume and the pressure in such a situation. However, some studies have identified “local” and
“tissue” RAS, suggesting separate biological effects independent of blood-borne Ang II [25-27].
In other words, there are several factors affecting activation of the RAS cascade other than
critical life-threatening situations. Further research identified multifunctional properties of Ang
II, such as altering the expression of adhesion molecules, activation of immune cells, infiltration
of inflammatory cells, and encouraging cell growth [28].
2.5 The importance of ACE2 activity
While Ang II has been implied as a key contributor to some inflammatory responses,
there have been some strategies developed to inhibit the synthesis or the activity of Ang II. One
such strategy is enhancing the activity of Angiotensin-I-converting enzyme 2 (ACE2). ACE2 is a
human homologue of ACE, which act as a carboxypeptidase, like ACE [29-32]. It cleaves Ang II
to produce Angiotensin 1-7 (Ang 1-7), and this simple cleavage can drastically change the RAS
outcome. The product of this reaction, Ang 1-7, exerts opposing effects from Ang II, and it
8
engages in vasodilation and anti-inflammatory as well as anti-fibrogenic effects, leading to
possible prevention of many diseases [33-35].
The discovery of ACE2 and Ang 1-7 has been stimulating high interest. ACE2
upregulation has been shown by several researchers [30, 31, 36] to exert a number of beneficial
effects by decreasing Ang II concentration, and thus several genetic approaches or synthetic
ACE2 activators have been tried to increase ACE2 activity [37-39]. The developed ACE2
activators may further represent potential new therapies for treating cardiovascular, hypertension
kidney diseases, diabetes, and its complications.
2.6 Project overview
This project involved transformation of several Lactobacillus species (L. reuteri, L.
paracasei, L. gasseri, and L. plantarum) with ACE2 and green fluorescent protein (GFP)
plasmids by electroporation. To standardize our transformation method, the GFP plasmid was
utilized to enable visual confirmation with the fluorescence microscope (Leica DMi8 Live Cell
Imaging Microscope) of the successful transformation. Lactobacillus species have a specific
preference of genetic codon usage for gene expression, thus ACE2 gene sequence was codon
optimized to allow high expression level of ACE2 proteins by these species. The main goal of
this study was to evaluate the expression level of this codon-optimized ACE2 gene in L. reuteri
and further compare it to the other Lactobacillus species expressing ACE2, and to their wild-
types. For this purpose, the protein was extracted from wild-type (WT) as well as from
recombinant Lactobacillus species expressing ACE2 and was used to perform ACE2 activity
analysis. The ACE2 enzymatic activity was measured using a fluorescent substrate. This
9
substrate is a self-quenching substrate, once cleaved by the ACE2, it releases fluorescence. The
fluorescence was measured by the reader (SpectraMax M3 microplate reader), and the data was
analyzed. This fluorimetric procedure is extremely sensitive [40], and it requires extra cautions
to obtain accurate measurements.
10
III. Materials and Methods
3.1 Transformation of L. reuteri
Preparation of plasmid DNA:
The Escherichia coli expressing pTRKH3-ldh-sc-GFP plasmid was previously made in
the lab. Bacteria were streaked on Luria Broth (LB) plates containing erythromycin (200 µg/ml).
One single colony from a plate was inoculated in liquid culture, and plasmid DNA was isolated
by using a ZymoPURE Plasmid Midiprep kit. The plasmid concentration was checked by a
spectrophotometer and by running an Agarose gel. It was then used to transform L. reuteri by
electroporation.
Preparation of electrocompetent L. reuteri cells:
To begin with, wild-type (WT) L. reuteri was freshly streaked on deMan, Rogosa and
Sharpe agar (MRS) [44] plates without any antibiotics from the -80 °C stock. It was incubated at
37 °C overnight. The next day, a single colony was inoculated in liquid culture (MRS broth, 10
ml) and was grown overnight at 37 °C without shaking. The following day, 800 µl of the grown
L. reuteri was inoculated into 80 ml of MRS media, supplemented with 1% of Glycine. This
culture was grown at 37 °C without shaking until an optical density of 0.6 to 0.8 was reached.
The cells were then harvested by centrifugation at 4 °C at 7000 rpm for 10 minutes. These cells
were subsequently given a wash with 80 ml of cold sterile water and suspended in 3 ml of cold
sterile water. The cells were centrifuged again (15,000 rpm for 2 minutes at 4 °C). The pellet
thus obtained was suspended in 30% cold sterile PEG-8000 reagent. The cells were stored at -
80 °C overnight.
11
Treatment of cells before transformation:
Competent cells were thawed on ice and suspended in 900 µl of cold sterile water for 30
minutes. The cells were then pelleted by centrifugation and were suspended in 30% PEG
solution. These cells were aliquoted as 100 µl suspensions in microfuge tubes to use for
electroporation.
Electroporation:
The electrocompetent cells were placed on ice prior to electroporation. The electroporator
was set to the following settings: voltage to 2.5 kV, capacitance to 25 µF, and resistance to 200
ohms. The electroporation cuvettes were ensured to be dry and clean. DNA was added to and
gently mixed with the electrocompetent cells immediately before delivering the pulse. The
cell/DNA mixture was then transferred into a sterile pre-chilled electroporation cuvette and
shocked by the electric pulse. Immediately after, the cells were added to 1 ml of MRS media.
The same procedures were repeated for four other different DNA concentrations, using a new
electroporation cuvette every time. After electroporation, the cells in MRS media were
incubated for 4 hours at 37 °C without shaking.
Plating transformed L. reuteri cells:
The cells were centrifuged and suspended in 100 µl of MRS media and plated onto MRS
agar plates, supplemented with erythromycin (5 µg/ml) as an antibiotic selection marker. These
plates were then incubated at 37 °C. The colonies were observed after 2 days.
12
3.2 Enzymatic activity assay
Extraction of protein from L. reuteri expressing ACE2 by Lysozyme treatment:
The L. reuteri expressing pTRKH3-ldh-sc-ACE2 plasmid was previously made in the
lab. A 5 ml overnight culture of this bacteria was grown in MRS supplemented with 5 μg/ml
erythromycin at 37 °C for 18 hours. The bacteria were harvested by centrifugation at 5,000 rpm
for 20 minutes at 4 °C and washed with sterile PBS. The extraction of proteins was carried out
according to the method described by Sieo et. al [43]. Briefly, the cell pellet was suspended in
0.15 M Tris/HCl buffer, pH 6.8. Then 100 μl of 10 mg/ml lysozyme was added and incubated on
ice for 90 minutes followed by sonication and centrifugation at 8,000 rpm for 10 minutes at 4 °C.
The supernatant thus obtained was used to estimate protein concentration.
Estimation of protein concentration extracted from Lactobacillus reuteri:
A set of protein standards were prepared by diluting 2 mg/ml Bovine Serum Albumin
(BSA) standards into clean vials (2000, 1500, 1000, 750, 500, 250, 125, and 25 µg/ml). The
standards along with the unknown protein samples were loaded into a 96-well microplate.
Bicinchoninic acid (BCA) reagent was prepared right before use by mixing 50 parts of reagent A
and 1 part of reagent B. The BCA reagent was added to each well and the plate was shaken for
30 seconds. The plate was covered and incubated at 37 °C for 30 minutes. Then, the absorbance
at 562 nm was measured on a SpectraMax M3 microplate reader.
Measurement of ACE2 activity:
The ACE2 fluorescent enzymatic activity assay was performed in a 96-well black
microplate with 50 µM ACE2-specific fluorogenic peptide substrate in a final volume of 100 µl
per well. The standards and samples were run in duplicates along with a negative control. The
13
protein samples were diluted with ACE2 activity buffer (75 mM Tris, 1 M NaCl, 0.5 μM ZnCl2,
pH 7.5), and treated with Captopril (an ACE activity inhibitor). After the addition of fluorogenic
peptide substrate, the plate was covered, and its fluorescence intensity was read using a
SpectraMax M3 fluorescence microplate reader every 90 seconds with excitation at 340 nm and
emission at 400 nm at 37 °C for 2 hours. The results were expressed as relative fluorescent units
(RFU).
14
IV. Results
4.1 Confirmation of transformed GFP electrocompetent cells
The transformation method was standardized by testing five different DNA
concentrations; 100 ng, 200 ng, 400 ng, 600 ng, and 800 ng each in 100 µl of L. reuteri
competent cells; however, the concentration of 800 ng DNA failed to perform electroporation.
There was sparking probably due to the DNA amount being too high. For each concentration,
time constants were recorded; 5.08 for 100 ng, 4.98 for 200 ng, 4.92 for 400 ng, 4.96 for 600 ng,
and 5.02 for cells alone.
After two days of incubation, two colonies were observed on the plate with 600 ng DNA
concentration. No growth was observed on any of the other plates, including the WT control.
Comparing this data, it can be interpreted that the erythromycin-resistant colonies on the 600 ng
plate have the target plasmid, and the WT L. reuteri do not.
The colonies (“Colony 1” and “Colony 2”) were inoculated in MRS media with
erythromycin (5 µg/ml) and incubated at 37 °C overnight. 10 µl of the cells from the overnight
culture were placed onto slides to be tested under the Leica DMi8 Live Cell Imaging
Microscope. GFP served as a marker to confirm electrocompetent cells. The green coloration
was observed on the cells for both colonies; however, the cell number was less. The cells were
then concentrated by centrifugation. This time there were enough number of fluorescent cells.
The green fluorescence was observed on the colony 1 cells, the colony 2 cells, and also on the
positive control (E. coli cells expressing the GFP plasmid). The transformed L. reuteri cells were
stored in glycerol at -80 °C.
15
Previously, we had difficulty in transforming L. reuteri by electroporation. We had been
using competent cells that were stored at -80 °C for a longer period of time, and it can be implied
that the competent cells might have to be freshly made for this kind of electroporation. By
making this GFP construct, the method was standardized, and the ACE2 construct, developed
following this method, was further processed to perform the ACE2 activity assay.
Figure 2: Live cell imaging demonstrating GFP fluorescence: E. coli expressing pTRKH3-ldh-sc-GFP plasmid
as positive control: bright field (a) and fluorescence (b). “Colony 1” of L. reuteri after electroporation
expressing pTRKH3-ldh-sc-GFP plasmid: bright field (c) and fluorescence (d), as well as “Colony 2”
of L. reuteri: bright field (e) and fluorescence (f). WT L. reuteri as negative control: bright field (g)
and fluorescence (h). Leica DMi8 Live Cell Imaging Microscope was used to take the images at 40X.
Scale Bar = 20 µm.
(b) (a) (c) (d)
(e) (f) (h) (g)
16
4.2 Extracted protein concentration estimations
The protein extracts were obtained for WT L. reuteri and its ACE2 construct, as well as
for other Lactobacillus species, L. paracasei, L. gasseri, and L. plantarum, which were already
available in the lab. The extracted protein concentrations were estimated using BSA standards
with concentrations of 2000, 1500, 1000, 750, 500, 250, 125, and 25 µg/ml. The standard curve
is provided [Figure 3].
4.3 ACE2 activity assay
50 µg of each extracted protein sample was calculated based on the protein estimation
data, and it was loaded into a 96-well black microplate. Figure 4 represents the entire readout
recorded by a SpectraMax M3 fluorogenic microplate reader. The first observation that can be
made is that there are differences between the species in terms of ACE2 activity. Some showed
higher ACE2 activities than others. All the WT had lesser ACE2 activity as compared to their
y = 0.0007x + 0.0304R² = 0.9957
0.000
0.200
0.400
0.600
0.800
1.000
1.200
1.400
1.600
0 500 1000 1500 2000 2500
OD
56
2
BSA (ug)
Protein standard curve
Figure 3:
BSA standards measured using SpectraMax M3 microplate reader at optical
density of 562 nm.
17
ACE2 constructs. L. gasseri and L. plantarum displayed higher ACE2 activities in comparison to
L. paracasei and L. reuteri. The L. reuteri WT and its ACE2 construct showed minimum
activity.
The data from each species was organized for further analysis. Figure 5 below provides
the magnified view of each bacterium activity for the first 30 minutes and 60 minutes.
Accumulative fluorescence for the periods of 2 hours was calculated and organized within Figure
Figure 4: Relative fluorescent unit (RFU) measured with ACE2-specific fluorogenic substrate for every 90
seconds over 2 hours period, using SpectraMax M3 microplate reader. LP as for L. paracasei; G as for
L. gasseri; PL as for L. plantarum; R as for L. reuteri.
0
500
1000
1500
2000
2500
3000
3500
1.5 6
10
.5 15
19
.5 24
28
.5 33
37
.5 42
46
.5 51
55
.5 60
64
.5 69
73
.5 78
82
.5 87
91
.5 96
10
0.5
10
5
10
9.5
11
4
11
8.5
RFU
minutes
LP WT LP ACE2 G WT G ACE2 PL WT
PL ACE2 R WT R ACE2 standard
18
6 to show the differences among different species for the ACE2 activity level, assessing which is
more and which is less activating.
0
200
400
600
800
1000
1200
1.5
7.5
13
.5
19
.5
25
.5
31
.5
37
.5
43
.5
49
.5
55
.5
RFU
minutes
60 minutes
LP WT LP ACE2
0
200
400
600
800
1000
1.5
4.5
7.5
10
.5
13
.5
16
.5
19
.5
22
.5
25
.5
28
.5
RFU
minutes
30 minutes
LP WT LP ACE2
(a) (b)
0
500
1000
1500
2000
2500
1.5
7.5
13
.5
19
.5
25
.5
31
.5
37
.5
43
.5
49
.5
55
.5
RFU
mintes
60 minutes
G WT G ACE2
0
500
1000
1500
1.5
4.5
7.5
10
.5
13
.5
16
.5
19
.5
22
.5
25
.5
28
.5
RFU
minutes
30 minutes
G WT G ACE2
(c) (d)
(e) (f)
0
500
1000
1500
2000
1.5
7.5
13
.5
19
.5
25
.5
31
.5
37
.5
43
.5
49
.5
55
.5
RFU
minutes
60 minutes
PL WT PL ACE2
0
500
1000
1500
1.5
4.5
7.5
10
.5
13
.5
16
.5
19
.5
22
.5
25
.5
28
.5
RFU
minutes
30 minutes
PL WT PL ACE2
19
0
200
400
600
800
1000
1.5
7.5
13
.5
19
.5
25
.5
31
.5
37
.5
43
.5
49
.5
55
.5
RFU
minutes
60 minutes
R WT R ACE2
0
200
400
600
800
1000
1.5
4.5
7.5
10
.5
13
.5
16
.5
19
.5
22
.5
25
.5
28
.5
RFU
minutes
30 minutes
R WT R ACE2
Figure 5:
ACE2 activity comparing between the WT and the ACE2 construct according to each
species: L. paracasei at the first (a) 60 minutes and (b) 30 minutes, L. gasseri at the first (c)
60 minutes and (d) 30 minutes, L. plantarum at the first (e) 60 minutes and (f) 30 minutes, L.
reuteri at the first (g) 60 minutes and (h) 30 minutes durations.
(g) (h)
Figure 6:
Accumulative fluorescence in RFU by different Lactobacillus species for 2 hours.
0
20000
40000
60000
80000
100000
120000
140000
160000
180000
LP WT LP ACE2 G WT G ACE2 PL WT PL ACE2 R WT R ACE2
RFU
Lactobacillus species
20
V. Discussion
Overall implications of the results
Taken together, the data obtained from the ACE2 activity assay suggest that L. reuteri
expressing ACE2 shows low ACE2 activity in comparison to the other species that were tested.
Regardless of all the constructs being processed by the same procedures, each demonstrated
different ACE2 expression levels.
Biological reasons behind L. reuteri low ACE2 activity
The ACE2 cDNA in the plasmid used to transform L. reuteri in this experiment was
previously codon optimized based on codon usage preference in Lactobacillus species. This
specific ACE2 design has not yet been thoroughly tested among different species of
Lactobacillus, and L. reuteri was one of the first species to be examined. It is possible that
further codon optimization may be required for L. reuteri to achieve best results.
There are a few obstacles that one may encounter when transforming a mammalian gene
into a prokaryotic genome. One issue is that if the encoded protein is too large, it will be difficult
for the prokaryotic organism to handle, thus, it spontaneously reduces the expression of the
protein-encoding gene. A factor that may be associated with this matter is the promoter strength
of the gene. The gene promoter is a great determinant of the gene transcription initiation, and it
influences the transcription rate by activating or repressing it. It is actively different across
species, indicating that each species has its own preferred design of the promoter. That is to say,
the lactate dehydrogenase promoter design, that works perfectly for the L. paracasei species,
may be acting weaker in L. reuteri; thus, lowering the production of ACE2 protein.
21
Follow-up experiments to confirm the results
Another reason for the low ACE2 activity could be potential technical errors that might
have been conducted during the experiment. One possibility is that the protein was lost during
the extraction procedure, which may suggest a required modification to the extraction protocol.
There are several challenges that can be presented; one is that the proteins became denatured
from the chemical buffers and hence the ACE2 activity was lost. The response to the harshness
of the chemicals can vary by the types of protein and its surrounding environment, therefore,
buffers with more gentle chemical conditions are sometimes needed to be used as the
replacement.
Another possibility is that ACE2 protein was secreted into the media by L. reuteri, which
then was lost in the discarded supernatant after the centrifugation process to obtain protein
extracts. An immediate experiment that can be conducted to answer the question is to verify
whether ACE2 protein is secreted into the culture medium. To successfully obtain the protein of
interest from the media which Lactobacillus grows in, the procedure of trichloroacetic acid
(TCA) precipitation may be appropriate [41]. Precipitated protein can be run on sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to estimate the size, so that the protein
of interest can be identified. The protein thus concentrated from the media can be used to
estimate ACE2 activity. These experiments may be able to address the potential causes of the
low ACE2 activity seen in L. reuteri.
Significance of this study
In this study, we found that there were differences in the ACE2 activities of different
Lactobacillus species expressing the human ACE2 protein. This shows that it may be important
22
to standardize the gene expression conditions and protein extraction protocols for each species
separately.
23
VI. Acknowledgement
I would like to thank Dr. Qiuhong Li for giving me the valuable opportunity to work on
an exciting project in her laboratory. I am also deeply grateful to Dr. Amrisha Verma for her
patient guidance and careful teaching as my research advisor. Without their continued support, I
would not have been able to learn not only how to do research, but also to become a better
scientist.
24
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