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

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1200

1.5

7.5

13

.5

19

.5

25

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.5

RFU

minutes

60 minutes

LP WT LP ACE2

0

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.5

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.5

RFU

minutes

30 minutes

LP WT LP ACE2

(a) (b)

0

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1.5

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13

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RFU

mintes

60 minutes

G WT G ACE2

0

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.5

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.5

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RFU

minutes

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G WT G ACE2

(c) (d)

(e) (f)

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1.5

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.5

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RFU

minutes

60 minutes

PL WT PL ACE2

0

500

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1.5

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.5

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.5

RFU

minutes

30 minutes

PL WT PL ACE2

19

0

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1.5

7.5

13

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.5

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.5

RFU

minutes

60 minutes

R WT R ACE2

0

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.5

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.5

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.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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