Running Head: TEAM MTB PROPOSALAmerican Psychology Association, 6th ed.

Inhibition of Protein-protein Interactions in Mycobacterium

Tuberculosis through Drug Screening

Team MTBUniversity of Maryland, College Park, MD

Gemstone Honors Program

Mentor: Dr. Volker Briken

Authors: Malik Antoine, Paige Chan, He Chun, Elizabeth Corley, Isaac Jeong, Christopher Kim, Carolyn Lane, Ari Mandler, Nathaniel Nenortas, Michelle Nguyen, Ian Qian,

Pradip Ramamurti, James Tuo, Jimmy Zhang

______________________________________________________________________________

“We pledge on our honor that we have neither given nor received any unauthorized assistance on this assignment.”

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Table of Contents

Abstract 3Chapter 1: Introduction 4

Specific Aim 4

Specific Aim 1: Establishing high-throughput liquid assays 4

Specific Aim 2: Constructing desired fusion proteins for the Mycobacterial Protein Fragment Complementation system to test for protein-protein interaction 5

Specific Aim 3: Performing drug screenings on a multi-compound drug panel with our Mycobacterial Protein Fragment Complementation assay 5

Specific Aim 4: Validating our results by testing false negatives and false positives

assay 6

Chapter 2: Literature Review 7

Epidemiology 7

Type VII Secretion Systems 11

ESX-1 12

ESX-5 15

The Nuo Operon of Type I NADH Dehydrogenase of Mtb 17

Conclusion 19

Chapter 3: Methodology 20

Background 20

Origin of the Compound Library Source 20

Advantages of using Mycobacterium smegmatis as a model system 21

Establishing a high-throughput liquid screening assay 21

Mycobacterial Protein Fragment Complementation assay 21

Additional methods to quantify protein-protein interaction 23

Constructing desired fusion proteins for the Mycobacterial Protein Fragment Complementation system to Test for Protein-Protein Interaction 24

Drug screening to determine interference of drug interactions 28

Validation 30

False positives30

False negatives 31

Human cell cytotoxicity 33

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Alternative methods 33

Compound structure analysis 33

Dose response relationship 34

Compounds are inert against protein interaction 34

Future research 35

Logistics 36

Conclusion 36

Appendix A: Glossary 37

Appendix B: Timeline 40

Appendix C: Budget 41

Appendix D: NIH Clinical Collection 42

Appendix E: Protocols 43

References 55

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Abstract

Tuberculosis is a highly contagious, infectious disease that kills about 1.3 million people

annually. Currently, the disease only has suboptimal treatment due to the rise of multidrug

resistant strains of Mycobacterium tuberculosis (Mtb), the causative bacterial agent of

tuberculosis. Therefore, we aim to identify novel drugs that may interfere with Mtb virulence

mechanisms. We will use the mycobacterial protein fragment complementation (M-PFC) assay

to screen a 446 compound drug panel to find candidate drugs that interfere with type VII

secretion systems or type I NADH dehydrogenase systems of Mtb. Furthermore, select candidate

drugs will be screened for toxicology in human liver and kidney cells. We hope to discover novel

drug candidates for the treatment of tuberculosis that can be transitioned into animal and

hopefully clinical trials.

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Chapter 1: Introduction

Tuberculosis (TB), caused by the bacillus, Mycobacterium tuberculosis (Mtb), is a highly

contagious and infectious disease that kills about 1.3 million people annually (World Health

Organization, 2012). Despite current research and treatments, TB is the second leading cause of

death in several regions, mainly in East Asia and Africa, and worldwide, one in three people are

carriers for the disease. The TB epidemic is magnified by the emergence of multidrug resistant

strains such as mycobacteria MDR-TB and XDR-TB, which are resistant to rifampicin and

isoniazid, two of the leading treatments (CDC, 2007).

Specific Aims

The goal is to discover a drug that disrupts virulence pathways within the Mtb cell with a

focus on the Nuo operon of the Nicotinamide adenine dinucleotide (NADH) dehydrogenase

system, and/or ESX-1 or ESX-5 secretion systems of the type VII secretion systems. This elicits

the following research question: what established drugs can be repurposed to disrupt vital

virulence pathways within Mycobacterium tuberculosis? We hypothesize that at least one of the

446 drug compounds purchased from the National Institute of Health will inhibit the protein-

protein interactions in either the ESX 1 or ESX 5 secretion systems or the Nuo operon.

Specific Aim 1: Establishing a High-Throughput Liquid Screening Assay

The mycobacterial protein fragment complementation (M-PFC) assay is specifically

designed to detect protein-protein inhibition in mycobacterium cells, allowing us to confirm the

effects of the drugs on the proteins. To assess the level of inhibition, we will be using Alamar

Blue-Trimethoprim (AB-TRIM), a colorimetric and fluorescent assay that signifies the level of

growth through color transition from blue to fluorescent yellow. This will be measured in plate

reading spectrophotometer. Then, we will also assess protein-protein interaction by using a

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spectrophotometer, which will determine the optical density of Mycobacterium smegmatis

(Msm) over time using a wavelength of 600 nm. Similar to the MPFC-assay, we will be utilizing

the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, which

establishes the level of growth through quantifying a change of color. Of the three methods

mentioned, our primary method of quantification will be AB-TRIM assay.

Specific Aim 2: Constructing Desired Fusion Proteins for the Mycobacterial Protein

Fragment Complementation System to Test for Protein-Protein Interaction.

In order to test for protein-protein interaction in our assay, we need to incorporate the

genes of the suspected proteins and protein components of the M-PFC system into the plasmids,

pUAB300 and pUAB400, so that there is one gene of a protein of interest and one component

gene of our reporter protein in each plasmid. The plasmids will be transformed into E. coli and

then grown on selectivity plates to kill off failed transformations. Upon purification of the

plasmids from E. coli, they will be used in gel electrophoresis and sequencing to confirm

successful plasmid construct. Finally, they will be transformed into Msm using electroporation

and are ready for use in the M-PFC assay.

Specific Aim 3: Performing drug screenings on a multi-compound drug panel with

our Mycobacterial Protein Fragment Complementation assay.

The transformed Msm cells will contain both plasmids at once, each expressing a specific

protein fused to one of the domains of the enzyme murine dihydrofolate reductase (mDHFR),

which protects against the antibiotic, trimethoprim (TRIM). The formation of mDHFR requires

the interaction between the three-polypeptide domains of mDHFR. It is important to note that the

protein domains of mDHFR do not have a natural affinity for one another and so only the

interactions between the proteins of interest will produce immunity to trimethoprim. After

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successful incorporation of the plasmids into the Msm cell line, the mutant cells will be isolated

and screened against the 446 drug panel. The drugs at the minimum effective concentrations will

be inserted into triplicate wells with the mutant Msm cells and TRIM. Along with these

experimental wells, there will be two sets of triplicate controls: one containing Msm, media, and

TRIM; the other containing Msm, media, TRIM, and DMSO, which controls for the cytotoxicity

of the solvent. No growth indicates that the drug succeeded in disrupting the protein-protein

interaction as the mDHFR enzyme did not assemble causing the cell to not possess immunity to

trimethoprim.

Specific Aim 4: Validating our results by testing for potential false negatives and

false positives.

It is possible that one of the compounds tested could have direct microbicidal activity and

not affect the protein-protein interaction within Mtb. To account for direct cytotoxic activity

causing a positive result, we will screen our compounds without TRIM. Drugs that are directly

cytotoxic will demonstrate equal killing with or without TRIM. Additionally, drugs may directly

interfere with the assembly of mDHFR, but not the interaction of our proteins of interest, which

would also result in a false positive of no growth. Screening the drug of interest against another

set of known interacting proteins will address this issue. A false negative occurs when a cell

grows in media containing TRIM, but the drug interferes with the protein-protein interaction,

which can result from well to well contamination. As a result, repeated trials will be conducted to

confirm the positive results.

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Chapter 2: Literature Review

Epidemiology

TB is a highly contagious, airborne, infectious disease that kills about 1.3 million people

annually (World Health Organization, 2013). More than half of these patients reside in China and

India, two of the world’s most populous countries. Despite current research and treatments, TB is

the second leading cause of death in several regions, mainly in East Asia and Africa, and one in

three people are carriers for the disease. The bacillus Mtb affects the lungs resulting in

pulmonary TB. The disease is highly contagious due to its mechanism of transmission. It is

spread when infected people cough or sneeze and project droplets of liquid into the air, which

contain Mtb (WHO, 2013). The probability of developing the disease is much higher for patients

who are immunocompromised prior to their contact with tuberculosis. TB is also statistically

proven to be much more common in men than in women. It affects mostly adults, with 8,070,000

cases in 2012 alone (WHO, 2013).

Once Mtb enters a human host, it will usually infect one or both lungs (Schluger, 1988).

The host immune system recruits macrophages to phagocytize the bacilli. Normally, the

Figure II-1. Worldwide map depicting TB incidence rates (2012). (WHO, 2013)

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phagosomes will fuse with the lysosomes, and then the digestive enzymes in the lysosome

eventually kill the bacilli (Flynn, 2001). The macrophages then travel to the nearby lymph nodes

and present the remaining antigen to CD4 T-cells (Helper T-cells) (Müller, 1987). This activates

a cascade of protein pathways that cause an increase of nitric oxide and reactive oxygen species

(Chan, 1999). These two molecules are capable of both destroying bacterial cell walls and

causing apoptosis of macrophages. A granuloma then forms at the site of infection as a circle of

immune cells isolating a center of caseous necrosis. This is the breaking down of tissue in the

center of a granuloma, where the bacilli are exterminated (Bean, 1999).

Alternatively, Mtb enters the macrophage and proliferates within and around the

phagosomes that are holding the bacilli. The bacterium has methods of preventing lysosomal

markers from being added to the phagosomal membrane. Thus, the phagolysosome never forms

(Stanley, 2013). Additionally, virulent strains of Mtb have ways to inhibit apoptosis of

macrophages. The anti-apoptotic characteristics of the bacilli are a key factor for virulence as it

permits further growth of the bacteria within the host. This results in a primary infection of Mtb,

leading to lesions in the lungs and caseation in the lymph nodes. The bacilli will likely

disseminate through the host’s blood and cause both fibrosis and calcification in affected organs

(Kumar et al., 2012).

Figure II-2. Flow chart depicting pathology of Mtb infection (Kumar et al., 2012).

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Once the host recovers from the primary infection, either a secondary infection may

occur immediately or the bacteria may remain latent for an indefinite period of time. A

secondary infection occurs if remnants of the bacteria remain in open lesions or if Mtb is

reintroduced through aerosols into the host. Because the host’s immune response is already hyper

sensitized to Mtb, the tissue at the site of infection could experience drastic caseating necrosis.

Consequently, enough tissue in the lungs will erode so that the mycobacteria will spread

throughout the airways, leading to a contaminated sputum and susceptibility for transmission. In

addition, Mtb could eventually gain access to both the pulmonary pathways from the heart as

well as the systemic pathways; this would lead to lesions throughout the lungs and on any organs

to which Mtb has spread. Mtb is well equipped to evade the adaptive immune response, so

medicinal treatment is highly important for control of the disease (Kumar et al., 2012).

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The TB epidemic is magnified by the emergence of multidrug resistant strains, such as

MDR-TB and XDR-TB, which are resistant to rifampicin and isoniazid, two of the leading drugs

for treating TB. Due to this, new drugs are needed in order to cure TB (CDC, 2007). In 1943,

Selman Waksman developed streptomycin, the first known effective antimicrobial agent (Farmer

& Keshavjee, 2012). Although many patients claimed to be cured of the disease, many have

relapsed due to mycobacterial resistance to streptomycin. This led to the development of two

new antimicrobial agents, thiacetazone and para-aminosalicylic acid. These agents hold a

synergistic effect with streptomycin that leads to more effective treatments and a decreased

resistance to antibiotics. Isoniazid is now an integral part of the current recommended treatment

for new cases of drug resistant TB. Rifampicin, ethambutol, and pyrazinamide are the current

first-line drugs, and are part of a 6-month regimen used to eradicate the bacteria (Kaufmann,

2013). Since 1990, TB incidence rates have not fluctuated considerably, which is especially

evident in developing countries where there have been no improvements in reducing incidence

rates of the disease (WHO, 2013).

Currently, there is a safe and effective vaccine named Bacille Calmette-Guerin (BCG),

which was developed by Albert Calmette and Camille Guerin in the early 20th century

(Kaufmann, 2013). This vaccine was first tested on a human subject in 1921 and still exists as

the only licensed TB vaccine although it is only effective in children. It has been given to

roughly 4 billion people worldwide to date, and it is usually administered soon after birth

(Farmer & Keshavjee, 2012). However, it has been found that BCG provides insufficient

protection against the disease in adults, rendering the need for newer, more efficient treatments.

It has been shown that BCG- induced immunity neither prevents nor eliminates Mtb infection

(Kaufmann, 2013).

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As a dominant issue in developing countries and immunocompromised communities, TB

is rapidly becoming more difficult to treat due to the increased resistance against a multitude of

drugs. The increasing number of cases outpaces current vaccines and treatments, but there are

plenty of unexplored niches in the bacterium’s mechanism that can be targets for new and novel

drugs. We will elucidate on possible drug targets, namely, type VII secretion systems (T7SS) and

type I NADH dehydrogenase.

Type VII Secretion Systems

All mycobacterial species use T7SS to secrete proteins across their cell envelope

(Houben et al., 2013). Moreover, pathogenic mycobacteria require the T7SS to transport

virulence factors through their protective and unique cell membranes into infected host cells

(Daleke et al., 2012).  Pathogenic mycobacteria such as Mtb form a unique order of bacteria

called Corynebacteriales characterized by the presence of mycolic acids within their cell wall

(Houben et al., 2013). Corynebacteriales tend to be strong, resilient organisms due to their

protective outer membrane (Houben et al., 2013).  This protective membrane restricts protein

transport and is one of the main reasons why pathogenic mycobacteria require T7SS (Houben et

al., 2013). Once T7SS substrates are recognized in the cytosol, it is thought that they are targeted

to the inner membrane and transported over the mycobacterial cell envelope (Houben et al.,

2013). This transport is partially mediated by T7SS membrane components that form a

translocation channel (Houben et al., 2013). There are still many unknowns within the T7SS

including the method of substrate recognition, but it is known that substrates are normally

secreted as folded dimers and have similar bundles of 4 alpha-helices followed by a secretion

motif (Houben et al., 2013).  

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Within the T7SS, the core of the secretion mechanisms is composed of four conserved

proteins (Houben et al., 2013). There can be up to five different loci on a single species in the

system with the number of genes and overall size of the ESX loci varying significantly. Of the

five systems within T7SS, named ESX-1 through ESX-5, three of these systems have been

shown to be essential to virulence and viability of pathogenic species (Houben et al., 2013).

Three of the loci, ESX-1, ESX-3, and ESX-5, are involved in the secretion of proteins while

ESX-2 and ESX-4 are not known to be integral systems as neither have been proven to show

active secretion of substrates (Houben et al., 2013).

The first of the T7SS to be discovered was ESX-1 in Mycobacterium tuberculosis

(Houben et al., 2013).  When ESX-1 was discovered, it was shown to allow the secretion of two

small culture filtrate proteins (Houben et al., 2013). The proline-glutamic acid (PE) and proline-

proline glutamic acid (PPE) proteins, which have been consistently associated with the ESX

systems, are secreted by the ESX-1 and ESX-5 systems (Abdallah et al., 2007, Daleke et al.,

2012). The PE and PPE proteins are structurally similar to the ESX proteins containing the four-

helix bundle in antiparallel fashion and have a similar genetic organization (Houben et al., 2013).

The ESX-3 system is regulated by the availability of iron and zinc (Abdallah et al., 2007).

Currently, it is known that the ESX-3 locus encodes for a PE-PPE pair and a single Esp protein,

EspG; however, other substrates have yet to be identified (Stoop, Bitter, & van der Sar, 2012).

For the purposes of our study, we are focusing on ESX-1 and ESX-5 for their vital roles in the

virulence of Mtb. Attenuation of these two systems will result in an avirulent phenotype of Mtb.

The ESX-1 Type VII Secretion System of Mycobacteria

Genes for the T7SS ESX-1 are located on the Mtb genome in a locus known as region of

difference 1 (RD1) (Guinn et al., 2004). RD1 is imperative to the virulence of Mtb and several

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mutants lacking this region, including BCG, experience attenuation in vitro and in vivo (Guinn et

al., 2004). In addition to coding for the ESX-1 secretion system, RD1 codes for secreted factors

responsible for virulence (Guinn et al., 2004).

Currently, the exact functions of specific proteins coded for the ESX-1 are not well

defined, although certain products of RD1 fit known motifs for cellular activities (Simeone et al.,

2009). For example, Rv3869 is a protein that appears to be an AAA ATPase, and Rv3869,

Rv3870, and Rv3877 have structural features indicative of transmembrane domains (Simeone et

al., 2009). Rv3877 is confirmed to a transmembrane channel, which acts as a secretion channel

for proteins across the inner membrane (Fig. II-3). A sequence of Rv3871 implicates this protein

being needed for secretion of certain substrates of ESX-1 and it has also been identified that

Rv3871 interacts with Rv3870, which is a membrane protein (Fig. II-3) (Simeone et al., 2009).

Figure II-3. A diagram of current known protein-protein interactions in the ESX-1 secretion system (Abdallah et al., 2007).

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These results support evidence that Rv3871 interacts with substrates as well as membrane

proteins as a secretion mechanism (Simeone et al., 2009).

Wirth et al. (2012) give insights into the intracellular location of the ESX-1 system by

fluorescently tagging the protein Rv3871 and examining its location in the cell in M. smegmatis.

The protein was found to move to a distinct cell pole where there was a singular signal (Wirth et

al., 2012). The researchers used a different species of Mycobacteria, but their results do give us

insight into the possible location of the complex in Mtb, as well as a potential screening method

that could identify attenuation (Wirth et al., 2012). The six-kDa secretory antigenic target (EsxA)

protein encoded by RD1 is a known virulence factor whose exact mechanism is unknown (Stoop

et al., 2012). Similarly, 10-kDa culture filtrate protein (EsxB) is another protein secreted by

ESX-1, which also plays a role in virulence (Brodin et al., 2005). EsxA is a member of the

WXG100 family of proteins that are characterized by being approximately 100 amino acids in

length and having tryptophan-variable region-glycine as a conserved amino acid sequence

(Brodin et al., 2005). EsxA and EsxB form a tight 1:1 heterodimeric complex, which is then

secreted by Mtb through recognition of the C-terminus on EsxB by ESX-1 (Fig. II-3) (Brodin et

al., 2005). Secretion of this complex is also dependent on EspA, EspC, and EspD (Stoop et al.,

2012).  It should be noted that while other proteins known to be involved in the structure of

ESX-1 localize to the cell pole, EsxAB does not localize to the cell pole with structural

components (Wirth et al., 2012).

While the exact mechanism through EsxA acts is not precisely defined, Simeone et al.

(2012) show that Mtb lacking in RD1 are confined to the phagosome. Also, when transfected

with RD1 on a plasmid, BCG (BCG::RD1) is able to escape host phagosomes and move into the

cytosol (Simeone et al., 2012). This BCG mutant is successfully contained in host phagosomes

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when the esxA gene is knocked out of the bacterial chromosome, which indicates that EsxA is

vital for the bacteria to escape into the cytosol (Simeone et al., 2012). After gaining access to the

host cytosol, Mtb and BCG::RD1 can proliferate unhindered within the macrophage and cause

subsequent host cell necrosis (Simeone et al., 2012). Necrosis is not ideal for the host as it allows

for the rapid proliferation of virulent bacteria whereas programmed macrophage death does not

allow for the same levels of Mtb proliferation.

It has been demonstrated that ESX-1 is vital for the virulence of Mtb and strains, such as

BCG, that lack the presence of RD1 do not express the same virulence (Guinn et al., 2004).

Virulence is conferred by the EsxAB complex, which is targeted and secreted by ESX-1 (Brodin

et al., 2005). Secretion of EsxA allows for Mtb phagosome escape and cause subsequent host

cytotoxicity (Simeone et al., 2012). For these reasons, the ESX-1 secretion system and its

substrates are viable drug targets. In addition to ESX-1, the other T7SS we will be studying is

ESX-5.

The ESX-5 Type VII Secretion System of Mycobacteria.

In contrast to ESX-1, ESX-5 is most recently evolved and restricted to slow-growing

mycobacterium. This group of mycobacteria includes major pathogenic species Mycobacterium

tuberculosis, Mycobacterium leprae, Mycobacterium ulceran, and Mycobacterium marinum.

(Gey van Pittius et al., 2006). With a similar structure to other ESX clusters, the ESX locus

contains esx genes coding for the immunodominant vehicle EsxN. A stark difference arises in the

structure of ESX-5 when contrasting the roles the various genes play within the ESX-5 locus.

Upstream from the esxN gene, the ppe-pe19 genes named for their motifs near the N-terminus,

code for highly immunogenic associated proteins PE and PPE. The ESX ecc genes surrounding

the ppe-pe-esx genes encode for supporting membrane proteins involved in ATP-binding. (Di

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Luca et al., 2012). These genes are found to accompany adjacent PE and PPE proteins (Bottai et

al., 2012). Components of ecc, eccC5, eccD5, eccA5, and ppe27 were tested with mutants

knocking out factors individually, with the deletions of eccC5 and eccD5 relaying strong defects

in EsxN secretion. EccA5 and ppe27 show no significant difference from wild type secretion

levels. EsxN levels came back to norm only by the association of eccD5 with the ESX-5 gene

cluster, insinuating genes associated with multiple esx genes are intrinsic to the operon structures

within the cluster. EccD5 was found to be the component that modulates the antigenic range.

Furthermore, the secretion of EsxA was not affected (Sayes et al., 2012). Exact details of the

interactions are unknown, but their interdependence is made clear through resulting secretion

levels. (Houben et al., 2012)

To verify location of eccC5 and eccD5, antibodies were used to screen the cell envelope.

Immunoblot assays detected eccB5, eccC5, eccD5, eccE5, and mycP5 within the envelope

structure.  EccA5, EsxN, and EspG5 were discovered in the cytosolic fraction; furthermore,

ESX-5 was discovered to form a multimeric protein complex when analyzed by blue native-

polyacrylamide and immunoblot analysis. Four small complexes and a larger complex,

consisting of eccB5, eccC5, eccD5, and eccE5 of approximately 1500 kDa were formed. The

1500 kDa complex was detected to be the most crucial ESX structure present within the

mycobacterial cell envelope, while through BN-PAGE analysis, EccC5 molecules were found to

result from low amounts of protease digestion in the envelope when incubated with 10 or 50 mg

ml-1 trypsan.

Thus genes esxM, esxN, eccC5, eccD5, eccA5, and ppe27 constitute the ESX-5 operon

involved in secretion of the virulence proteins EsxMN, PE, and PPE. EsxM and EsxN dimerize

as immunodominant antigens, promoting T cell response. The exact roles of PE and PPE proteins

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are not known to date; however, a study has shown their interdependence in the secretion of

LipY in M. marinum (Daleke et al., 2011). PE and PPE proteins thus have a multitude of roles in

virulence, both structural and offensive. The immunogenicity of the PE and PPE proteins has

been found to relay back to EccD5 regulation. In a BLAST analysis of TubercuList database,

ESX-5 PPE proteins were found to play a distinct role in the MHC-1 pathway, though the

proteins’ part within the pathway is still unknown (Sayes et al., 2012). Further information and

experimentation is needed to pinpoint the exact mechanisms of these proteins. Aside from

secretion systems, another protein complex that we will further examine is the Nuo Operon of

Type 1 NADH Dehydrogenase.

The Nuo Operon of Type I NADH Dehydrogenase of Mtb

Type I NADH dehydrogenase is a vital part to cell respiration, of which the virulence

gene, NADH ubiquinone reductase G (NuoG), is a subunit. Type I NADH dehydrogenase

consists of enzymes that oxidize NADH into ubiquinone while transferring protons to use later as

proton motive force. This system consists of many various types of subunits including the Nuo

operon, which is made up of fourteen nuo genes coding for the fourteen subunits of the full

complex. Type I NADH dehydrogenase is favored over type II NADH dehydrogenase as type II

NADH dehydrogenase does not conserve the protons for later use. While type II NADH

dehydrogenase consists of only one subunit, it is not as efficient as much energy is lost with the

proton transport. The Nuo operon is a critical component of type I NADH dehydrogenase in

creating proton motive force (Archer & Elliott, 1995).

Previously, many experiments have been conducted within Mtb with the goal of seeing

the effect of attenuating the Nuo operon (Miller et al., 2010). In 2010, Velmurugan et al. found

that removing nuoG from the Nuo operon created an attenuated, Mtb mutant strain that resulted

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in a reduced bacterial load in mice. The decreased virulence was because of the mutant strain’s

inability to prevent apoptosis in host macrophages, with apoptosis acting as a primary defense

mechanism of the host (Velmurugan et al., 2010).

In 2010, Miller et al. attempted to better understand the mechanism by which the Nuo

operon, specifically NuoG, inhibits apoptosis. Phagocytic NADPH oxidase 2 (NOX2), is known

to be involved in microbicidal activity in phagocytes (Miller et al., 2010).

Previous experiments have shown that mice with lower levels of NOX2 were more

susceptible to infections (Bedard & Krause, 2007). However, there is no concrete evidence on

how NuoG affects levels of NOX2 derived ROS accumulation. One hypothesis on how NuoG

carries out this process is that it directly inhibits NOX2 activity by disturbing the attachment of

subunits to the phagosomes (Miller et al., 2010). Another possible method in which NuoG

inhibits apoptosis includes enzymatically detoxifying of NOX2 derived ROS (Miller et al.,

2010). Additionally, in a study conducted by Behar et al. (2011), it was found that stress caused

by ROS resulted in up regulation of pro-apoptotic proteins, Caspase 3, Caspase 8, Caspase 9,

Tumor Necrosis Factor (TNF)-α, and TNF Receptor-1. Archer & Elliott (1995) conducted an

experiment where electron acceptors were mutated in the type I NADH dehydrogenase system.

However, the expression of the nuo operon and type I NADH dehydrogenase were not reduced

as a result (Archer & Elliott, 1995). Currently, it is not known how the Nuo operon, specifically

NuoG, inhibits neutrophil apoptosis (Miller et al., 2010). The only evidence is attenuated Mtb

strains, which show that there is a link between nuoG and cell apoptosis (Velmurugan et al.,

2010). Additionally, interference with assembly of the Nuo complex would lead to an attenuated

phenotype, so assessing the interference of NuoG with any other subunit of the Nuo complex

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could be of great use. Because the mechanism is still unknown, potential targets and pathways

for the disruption of this operon remain elusive (Miller et al., 2010).

Conclusion

There still remains the question of how a potential drug could impact Mtb when it is

already inside of the cell without affecting healthy cells. Additionally, the delivery method for

the drug would have to be researched further in order to determine the most efficient way to

combat cases of MDR and XDR in tuberculosis. Thus, our team seeks to find novel treatments,

which will overcome the current problems related to expanding drug resistance of the bacteria.

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Chapter 3: Methodology

Background

After an analysis of the current state of Tuberculosis, the literature reveals two key

points. First, there is a global need to find additional treatments for Tuberculosis. Second, there

are three protein pathways identified as important components of Mtb’s virulence; they are

pathways that have not previously been targeted with drugs. These pathways are the interactions

between EsxA and EsxB for the ESX-1 system, EsxM and EsxN for the ESX-5 system, and

NuoF and NuoG for the type I NADH dehydrogenase complex. Thus, we have three protein

complexes, which we will target in order to identify a potential novel treatment for TB. In order

to find such treatments, we pose the question: what established drugs can be repurposed to

disrupt vital virulence pathways within Mycobacterium tuberculosis?

The first step in analyzing the efficacy of our drug panel on our model cell line involves

cloning our genes of interest, which code for dimer forming proteins, onto plasmids for use in

our M-PFC assay. These plasmids will be inserted into our model cell line; then, our M-PFC

assay will measure cell growth, which represents the level of protein-protein interaction within

the cell. This will be quantified by use of Alamar Blue. We also have the potential of using MTT

as well as an OD600 for measuring cell viability. Finally, any potential positive hits would go

through further validation studies to ensure that the drugs are truly affecting the protein-protein

interactions.

Origin of the Compound Library Source

NIH’s Clinical Collection will provide the drug library that will be used for this

experiment. This library includes plated arrays of 446 small molecules with known health

benefits. A large portion of these drugs were originally designed for other diseases, but may have

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untapped potential in disrupting mycobacterial biological pathways. (NIH Clinical Libraries,

n.d.). As these drugs have already passed clinical testing, they can be implemented more easily

than new compounds. The complete list of these compounds and their structures can be found on

the NIH Clinical Collection’s website (Appendix A).

Advantages of Using Mycobacterium smegmatis as a Model System

Mycobacterium smegmatis (Msm) was chosen to be the model organism for our research

project. Mtb and Msm both come from the Mycobacterium genus; therefore, they share many

important traits, such as various biosynthetic pathways and cell membrane type. However, Msm

also has several important differences from Mtb. Msm replicates much faster than Mtb and takes

up DNA more efficiently. Additionally, while a colony of Mtb requires about three weeks, a

colony of Msm requires only four days to grow. A shorter doubling time and quicker

transformation means these assays will take less time to complete. Msm is nonpathogenic in

humans and can therefore be safely handled in Biosafety Level (BSL)-2 labs, while Mtb must be

handled in a BSL-3 lab where greater safety precautions such as hazardous material body suits

must be exercised in order to prevent accidental infections (Singh et al., 2006). Overall, using

Msm as a model organism is beneficial because it is a safer and more efficient vehicle for

performing our assay.

Establishing a High-Throughput Liquid Screening Assay

Mycobacterial Protein Fragment Complementation Assay.

In order to measure drug interaction, we will need to use an assay that will give a clear

indication of protein-protein interaction inhibition. The M-PFC assay is specifically designed to

detect cytoplasmic and membrane-bound protein interactions within mycobacterium cells. In the

assay, two proteins with known interactions in vivo will be attached to mDHFR reporter

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fragments [F1,2] and [F3]. If [F1,2] and [F3] reassemble to a functional mDHFR, it will confer

resistance against the antibiotic trimethoprim (TRIM) by allowing the bacteria to digest the

antibiotic. [F1,2,3] refers to the polypeptide domains of the full enzyme mDHFR. There are

several advantages in using the M-PFC assay, including its ability to detect a diverse range of

protein-protein interactions. Previous tests have proved that M-PFC is successful in identifying

interactions among proteins originating from Mycobacterium tuberculosis (Tiwari et al., 2012;

Singh et al., 2006; Dziedzic et al., 2010). Although this system can detect a diverse range of

protein-protein interactions, it will only attain a positive result, or successful coupling, between

specific proteins that are known to have interactions (Singh et al., 2006).

Reconstitution of mDHFR due to protein-protein interactions can be simply monitored

via survival-based assay. The M-PFC provides in-depth analyses about mechanisms such as

protein modifications on protein-protein associations. Hence, one of our readouts for in-depth

analysis will be AB-TRIM, incorporating Alamar Blue. This compound has been used previously

to assess the viability of mycobacteria in the presence of antimycobacterial compounds in 96-

well formats. AB-TRIM is a colorimetric and fluorescent plate assay, as the color of AB will

transition from blue to a fluroescent yellow depending on the level of growth, which is heavily

dependent on the degree of reconstitution of mDHFR. Alamar Blue will be the primary method

of quantification of protein-protein interaction in Msm. Protein-protein interaction leads to the

reconstitution of the mDHFR, which leads to the digestion of TRIM that allows for cell

proliferation. The lack of protein-protein interaction means that the mDHFR will be non-

functional and the cells will die (Fig III-1) (Singh et al., 2006).

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Figure III-1. Diagram representing coupling of [F1,2]and [F3] mDHFR reporter fragments fused with interacting proteins in M-PFC assay (Singh et al., 2006).

Additional Methods to Quantify Protein-Protein Interaction

There are other means of assessing the level of growth in the M-PFC assay, which, in

turn, would also quantify the level of protein-protein interaction to allow for growth to occur.

Our second method of quantifying growth levels is using a spectrophotometer to assess the

optical density of Msm over time with a wavelength of 600 nm, which is commonly known as

OD600. This wavelength is a generally accepted standard for this assay (Peñuelas-Urquides,

2013). The values attained will depend on the concentration of the sample to be used for the

assessment, as well as the level of growth of the bacteria at the specified time period. Then, the

optical density of the sample of bacteria will be collected before and after the assigned growth

time periods to determine the differentials, if any are present. This value will be given in colony-

forming units (CFU). This particular read-out will only give us numerical values that quantify

the level of growth, but not an in-depth analysis of the protein-protein interaction.

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An alternative method for quantifying growth would be utilizing the 3-(4,5-

dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) dye to measure the growth of

mycobacteria by quantifying a change of color. MTT is naturally a yellow salt, but turns blue due

to the activity of the dehydrogenases of live cells. The amount of color change is proportional to

the amount of live cells, making this assay useful in measuring the level of drug interaction in the

Msm cells (Abate et al., 2004). The optical density is measured at 570 nm and would be

measured before and after the incubation period (Abate et al., 1998). The MTT assay is relatively

inexpensive and quick, which makes it a worthy alternative method.

Constructing Desired Fusion Proteins for the Mycobacterial Protein Fragment

Complementation System to Test for Protein-Protein Interaction

In order to perform the M-PFC assay, the pair of proteins that we suspect will interact

have to be expressed as a fusion protein with the mDHFR domains. This will require the genes of

the suspect proteins, or inserts, to be incorporated into plasmids that hold the genes for one of the

mDHFR domains. After each insert is incorporated into its corresponding plasmid, both plasmids

will be transformed into our Msm cell line. By doing this, the Msm cell line will produce our

desired proteins, which need to be present for the completion of our M-PFC assay.

To make these plasmids, we will employ the following cloning method. We will use M-

PFC plasmids that contain the mDHFR domain genes. The M-PFC plasmids will be digested

with a pair of restriction enzymes (RE) (Fig. III-2). They will create two unique sticky ends in

the multiple cloning site of the circular M-PFC plasmid to form a linear plasmid. The linear

plasmids will be treated with phosphatase to prevent self-ligations. Phosphatase activity will then

be terminated during clean up because inserts will be added to the linear plasmids following this

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step, and phosphate must be on the ends of inserts to retain the ability to pair up with the opened

plasmids (New England BioLab Inc., 2014).

To prepare the inserts, we will isolate them from their original plasmids (Fig. III-3). The

provided plasmids will be placed in a mixture with the pair of REs that were used before to

create the same unique sticky ends at each side of the insert. The inserts will be purified from

other DNA strands formed from the RE digestion with gel electrophoresis by selecting the band

that isolates the insert. The insert will be incorporated into the linear M-PFC plasmid by treating

the two parts with ligation (Fig. III-4) (New England BioLab Inc., 2014).

Figure III-2. Using restriction enzymes to form a linear plasmid from a circular plasmid (New England BioLab Inc. 2014).

Figure III-3. Using restriction enzymes to form an insert from a plasmid (New England BioLab Inc. 2014).

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The specific M-PFC plasmids that will be used for this experiment include pUAB300 and

pUAB400. The pUAB300 plasmid includes [F1,2] of mDHFR while the pUAB400 plasmid

includes [F3] of mDHFR. pUAB300 and 400 will complement each other. The two proteins

suspected to interact will be tested by incorporating one of the inserts on pUAB300 and the other

insert on pUAB400.

After the desired plasmid vectors are formed, they will be transformed into Escherichia

coli, which will be grown on selectivity plates to ensure successful growth of only E. Coli that

hold the desired plasmids. The selectivity plate for E. coli containing pUAB300 will have the

antibiotic hygromycin, while the selectivity plate for E. coli containing pUAB400 will have the

antibiotic kanamycin. Each plasmid will have an antibiotic resistance gene for the corresponding

antibiotic in each selectivity plate; thus, growth in these plates will isolate E. coli that contain the

desired plasmids.

Figure III-4. Ligation of the linear plasmid and the insert to form the desired plasmid construct (assembled vector) (New England BioLab Inc. 2014).

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After an overnight growth of the selected colonies from the plates in liquid medium,

plasmid purification will be performed to isolate a large quantity of the desired plasmids.

Following that, the plasmids will be used in gel electrophoresis and sequencing to confirm the

success of the plasmid construct. Finally, M. smegmatis will be transformed by the plasmids into

the Msm mutants used in the M-PFC assays. To transform the desired plasmids into Msm, the

bacteria is electroporated and the plasmids are inserted (Velmurugan et al., 2010). The

transformed bacteria will be grown on hygromycin and kanamycin selectivity plates to isolate

Msm mutants with the desired plasmids (New England BioLab Inc., 2014).

The transformed Msm will contain both plasmids at once, each plasmid expressing a

specific protein fused to one half of mDHFR. If the proteins do interact, they will join,

combining the fragments of mDHFR. This will cause the bacteria to become resistant to

trimethoprim and will allow it to grow in the culture of media and antibiotic (7H11/TRIM plates)

(Fig. III-1) (Singh et al., 2006). Therefore, identifying protein-protein interaction in this assay is

determined by the growth of the Mycobacteria smegmatis in the culture.

These procedures are standard protocols for cloning with restriction enzymes. They are

widely used in experiments that require the synthesis of fusion proteins; thus, the procedures

have been proven to work successfully. For example, Clark et al. (1998) utilized the standard

restriction enzyme cloning method to investigate protein-protein interaction in the ethylene

pathway of plants. In 2010, Callahan et al. utilized this method of cloning to prepare plasmid

constructs to examine protein-protein interaction with the M-PFC system. Because this standard

cloning method does not require proprietary enzymes or vectors provided by a specific company,

we can simply apply it to our experimental needs. This standard cloning method is also the most

accessible because the required supplies are currently available at the laboratory provided for us.

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Additionally, compared to other cloning methods, such as Gibson Assembly or Gateway

Cloning, this is the least costly (New England BioLab Inc., 2014). For the procedures of

molecular cloning processes, refer to Appendix E.

Drug Screening to Determine Interference of Protein Interactions

Once our desired Msm mutants have been isolated, we can perform our M-PFC assay

with our drug panel. The M-PFC assay measures protein-protein interactions by correlating it to

cell growth in the presence of an antibiotic. The drug screen will use triplicate wells of a 96 well

plate, which are shown in Figure III-5, consisting of the Msm mutants and their fusion proteins at

the minimum effective concentration, the antibiotic, trimethoprim, growth media, and the

solvent, DMSO. If the drugs interact with the proteins, preventing the protein-protein interaction,

then the mDHFR will not digest TRIM, which would kill the cell (Mai, et al, 2011). Growth in

the presence of trimethoprim indicates that the protein-protein interactions were not inhibited and

that the drug had no effect. This means that the drug would be considered a negative result. No

growth is a positive result, which would express a blue color from the presence of Alamar Blue.

In the case where the drug does not inhibit the protein-protein interaction, the mDHFR complex

would form and express resistance from TRIM. This means that the cell would survive and

digest the Alamar Blue into a product that exhibits a fluorescent yellow color. The exact degree

to how blue or yellow the color is will be measured in OD by a spectrophotometer. Then, we will

continue onto further experiments to differentiate between true and false positives and negatives.

In addition to our experimental wells, we will have two sets of triplicate control wells.

The first set will be the negative control and will contain the mutant Msm, TRIM, and growth

media. In this control, there should be no inhibition of the protein-protein interaction because

there is no interfering drug and therefore should have full growth. This will confirm that the

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mechanisms behind the mDHFR work correctly. The other control will have the mutant Msm,

TRIM, growth media and DMSO. This control tests for any cytotoxicity from the drug solvent.

DMSO’s potential disruption is a confounding variable as we may incorrectly contribute cell

death or the lack of growth to the drug. Although we expect to see little disturbance from

DMSO, we will account for the difference, if significant, in our data collection. Our M-PFC

assay and the modified form for our drug screen will be performed in accordance with the

procedures located in Appendix E.

Figure III-5. The 96-well plate schematic will be numbered 1-12, left to right and lettered A-H from top to bottom (created by He Chun).

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Validation

False Positives.

Our M-PFC assay should indicate whether any of the drugs in our screening library have

an inhibitory effect towards EsxA and EsxB, EsxM and EsxN, or NuoG and NuoF interactions. It

is possible that a compound we test could have direct microbicidal activity and not have any

effect on EsxA-EsxB, EsxM-EsxN, or the assembly of the suspected NuoG-NuoF complex in the

type 1 NADH dehydrogenase. A drug with direct cytotoxic activity against Msm would yield a

positive test result because we will use a measure of bacterial viability as our indication of

protein interaction inhibition, which would be considered a false positive, as described in Figure

III-6. This presents a unique problem where we would be unable to distinguish between killing

activity attributed to TRIM or our drug of interest. In order to rule out direct cytotoxic activity

for positive signals, we will perform the proof-of concept screen similar to the one described by

Mai et al. (2011). This screen involves testing compounds with the M-PFC assay and no TRIM.

Drugs that are directly cytotoxic will show equal killing with and without TRIM.

The other possibility for false positives are drugs that interfere with the assembly of

mDHFR directly and have no effect on the interaction of the proteins being studied (Fig. III-6).

Our screen design should filter out these results. By testing inhibition of protein interaction for

two sets of proteins, results that show drug specificity toward the desired interaction should

affect one set of proteins and not the other. This also means that a drug, which affects both

systems, would be reported as a false positive. We can decrease the likelihood of losing a true

positive by screening our drug against another known set of interacting proteins.

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False Negatives.

A false negative (Fig. III-6) could arise when a cell grows in media containing TRIM, but

the drug actually interferes with the protein-protein interaction. If the negative result changes

after repeated trials, then the false negative will be identified. Another possibility for false

negatives is well to well contamination when pipetting our drugs onto our plates. We will have

each drug be repeated in triplicate on each plate and each plate will be repeated twice so that

each drug has a total of nine trials. Additionally, we will take extra caution when performing this

step of the protocol and will always follow lab standards set up to avoid human error. All false

negatives will be identified by the repetition of our experiment.

Figure III-6. For the purposes of our research, positive signifies the desired result of drug interference of the protein interaction. Because mDHFR dimerization causes TRIM resistance, an interfered interaction will result in non-TRIM resistant bacteria. Negative will be deemed as the undesired result of no drug interference of proteins. Growth signifies this negative result because

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Human Cell Cytotoxicity.

Another follow up test for our positive results will be a cytotoxicity screen against

HEPG2 and HEK293 human cell lines for liver and kidney cells, respectively. These cell lines

would be provided by Dr. Briken and would be screened using a different assay than our M-PFC

assay. It is important to test cytotoxicity toward human cells because if a drug is effective against

our protein interaction but is toxic toward human cells, it is not a viable candidate for future

clinical testing. We will be using liver and kidney cell lines because those organs are responsible

for filtering toxins from the blood, which means that they will be exposed to our drugs in the

highest concentrations in the body.

Alternative Methods

Compound Structure Analysis.

Based on the outcome of our M-PFC assay there are alternative methods that we could

take that are dependent on our results. For any drugs in our 446 drug compound which test

positive for protein inhibition by our M-PFC assay and our proof-of-concept assay, there are a

few possible directions for additional methods on these drugs. Any drugs which result in a

positive hit or exhibit toxicity toward human cells should be analyzed on a structural basis to

determine if there are any other compounds that exist in the same family. A drug family is

categorized by a group of compounds that share many similar structural characteristics. If any

compounds like this can be identified, we can look into possibly requesting to have those sent to

us by NIH or synthesized for us by an organic chemist. By further analyzing drug families

similar to molecules already found to be effective, we would hope to find a plethora of possible

Figure III-6. For the purposes of our research, positive signifies the desired result of drug interference of the protein interaction. Because mDHFR dimerization causes TRIM resistance, an interfered interaction will result in non-TRIM resistant bacteria. Negative will be deemed as the undesired result of no drug interference of proteins. Growth signifies this negative result because

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drugs based on structural similarities to already defined positive hits. Additionally, by further

analyzing the drug families from drugs that exhibit toxicity, we could determine underlying

structural characteristics which make these compounds less toxic to our human cells without

losing their ability to inhibit our desired protein interaction.

Dose-Response Relationship.

After our cytotoxicity assay against our human cell lines, we would hope to move onto

further tests with our drugs. The drugs of our screen will be sent to us in unknown

concentrations, which means that we may have varying amounts of any drug that gives a positive

result. If a compound yields a positive result and we have enough of it for further testing after

our cytotoxicity assay, we could try and develop a dose-response relationship. By making serial

dilutions of our drug in media, we will be able to observe different levels of protein inhibition at

different concentrations of the drug. We can then plot percent inhibition of protein interaction

against the base ten logarithm of our drug concentration and generate a curve to model this

relationship.

Compounds are Inert Against Protein Interaction.

Conversely, if our M-PFC assay returns all negative results for our initial drug panel

there is also information to be gained and studies that could be performed. If we get no positive

hits from our initial drug panel, we will have effectively eliminated all of the drugs in our panel

as possibilities to knock out our specific protein systems. This information will provide us with

insights into what types of compounds are ineffective at disrupting Mtb and will help us to

identify which drug families will not be good matches. From these insights we could look into

identifying other families that might be more effective based on their differences from the

families that we identified as ineffective. Additionally, if our initial panel of 446 drugs is

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ineffective at identifying possible drugs, we could test another panel of similar size. The new

panel could incorporate the different families that we think might have a greater chance of

disruption by drawing from information of the original drug panel. Through this method, we

would have a better chance of identifying compounds that will disrupt the integral interactions in

Mtb and thus decrease its virulence.

Future Research

In addition to these alternate methods, there are further studies that could be pursued

based on our results from our M-PFC assay. These could further our research and would be

beneficial to complete. Positive hits from our M-PFC assay will allow us to pursue further

studies. These studies include testing in virulent Mtb to ensure that the drugs are effective in the

true disease causing bacteria. Additionally, tests in vivo could account for how the drug behaves

in a model animal system. Tests in virulent TB require special training and lab space, and an

animal model necessitates Institutional Animal Care and Use Committee (IACUC) approval.

However, under our methodology it is extremely unlikely that we would have the time or

resources to complete either of these studies. The ultimate goal for future research on effective

drugs would be to moving to clinical trials with patients in varying stages of TB. These studies

are integral components to confirming the effectiveness of positive hits. While we will not have

the time to complete them, we would recommend that further directions include these studies.

Additionally, there are also future studies that could be performed if we have zero

positive hits. In alternative methods, we discussed testing a second panel of similar size to our

initial panel if we are unable to identify any drugs from the first panel. However, a panel of 446

compounds is relatively small and moving forward, we could test a different, larger panel of

compounds that incorporates many distinct compounds and families. Such a panel can

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incorporate the insights gained from the original drug panel. Unfortunately, a panel such as this

would be too large for the scope of the Gemstone experience.

Logistics

To begin the project, three teams will focus on cloning plasmids which correspond to

ESX-1, ESX-5, and type I NADH dehydrogenase. A fourth team will establish a high-throughput

liquid screening assay by testing the M-PFC system with proteins of known interactions. After

the liquid screen is established, the members of that group will be distributed into the other three

groups and these groups will begin testing the mutant cells in the M-PFC system. Once

interactions are established in any of the three systems, the results will be validated with other

studies. Each group will be assigned a lab liaison that will be responsible for lab work

accountability and reproducibility. If this method encounters problems, we will modify in order

to produce maximum efficiency. Our team will follow the general timeline shown in Appendix

B. We will receive funding from the Gemstone Program and grants, which will be spent in

accordance with the budget (Appendix C).

Conclusion

Overall, the M-PFC assay is useful in quantifying the levels of protein-protein

interactions by using cloned plasmids in our Msm cell line. This is an integral part of finding

potential drugs for to target the actions of our identified systems. After successful drugs have

been identified, possible secondary studies that we can perform on our positive results include

cytotoxicity assays on both our Msm and human cell lines as well as identifying a dose-response

relationship. We have also identified future directions that we can pursue if we find no positive

hits. In summary, we hope to find, confirm, and analyze positive results from our drug panel to

identify which drugs can be used to treat Tuberculosis. If unsuccessful in finding hits, we hope to

36TEAM MTB PROPOSAL

identify trends or clues in the structure of drugs that did not work to suggest drugs that will not

be effective at treating TB and suggest structures that may lead to effective TB treatment.

Appendix A: List of Terms

Cloning: The process of creating recombinant DNA and replicating it in a host organism.

Compound: Chemical substance

Culture: A growth of cells in medium.

DMSO: Dimethyl sulfoxide, a common polar, aprotic organic solvent for drug solutions.

Dimer: A complex consisting of two molecules linked together, often resulting from protein-

protein interaction.

Electroporation: A method of increasing permeability of cell membrane by applying electricity

so that DNA can be introduced to the organism.

ESX-1: Type VII secretory system within mycobacterium.

ESX-5: Type VII secretory system within mycobacterium.

Fusion protein: The protein product of two genes that are joined together.

Gel Electrophoresis: A process by which DNA or proteins are separated on the basis of size by

the speed at which they move through a gel (normally agarose based).

Granuloma- An inflammation formed from groups of immune cells that the immune system

can’t eradicate

Immunodominant: refers to the restricted peptide specificity of T cells that are considered

detectable after a primary immune response

in vitro: Process performed outside of living organism, often in a test tube or culture dish.

in vivo: Process performed within a living organism.

Ligation: Combining two nucleotides via the formation of a phosphodiester bond.

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Lysosome: An organelle in the cytoplasm of eukaryotic cells containing degradative enzymes

murine dihydrofolate reductase (mDHFR): A protein complex that digests trimethoprim.

MDR-TB: Multidrug-resistant Tuberculosis

Media: A substance, often an agar or liquid, which provides the nutrition through which cultures

of bacteria may grow.

Microbicidal: Ability to destruct microbes.

Multiple Cloning Site: A region on a plasmid that includes many restriction sites. It is where the

insert will eventually be incorporated.

mycobacterial protein fragment complementation (M-PFC) assay: An assay that tests for

protein-protein interaction by exploiting the mDHFR protein

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT): Naturally a yellow salt,

but turns blue due to the activity of the dehydrogenases of live cells

Nicotinamide adenine dinucleotide (NADH) dehydrogenase: Enzyme complex used in cellular

metabolism.

Necrosis: The death of most or all cells in a tissue or organ due mainly to the failure of blood

supply, disease, and injury.

Optical Density: A measurement of light transmittance through an object, often used as an

indicator of turbidity or concentration.

Phagosome: A vacuole, or vesicle in the cytoplasm of the cell

Phosphatase: An enzyme that removes a phosphate group.

Plasmid: A small, often circular piece DNA that can replicate independent of chromosomes. It is

often used in the laboratory to manipulate genetic framework. It is a specific type of vector.

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Protein-protein interaction: An interaction between two proteins, which are often particular to

certain biological systems.

Restriction Enzyme: An enzyme able to cut out a desired portion of DNA.

Selectivity Plates: Plates with medium that allow the growth of cell culture. Because the vector

that is transformed into the cells has an antibiotic resistance gene, the plates included have the

corresponding antibiotic to prevent the growth of any cells that do not have the vector.

Ultimately it promotes the growth of cells with a desired vector successfully transformed into

said cells.

Sticky Ends: a single-stranded end of DNA or RNA nucleotides which complement another

single strand end via base pairing, allowing for the two strands to be fused. This is a useful

technique for engineering purposes.

Synergistic- Two drugs acting jointly to increase the overall effectiveness

XDR-TB - Extensively drug-resistant tuberculosis

Virulence: The degree of pathogenicity within a group or species

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Appendix B: Timeline

Fall

2014

Complete research proposalPrepare for first semester of laboratory research

Spring 2015

Attain required lab trainingPresent research proposal to Gemstone and a panel of expertsEstablish M-PFC system

Fall

2015

Finish drug screenIdentify and apply for potential conferences and grantsPresent findings at Junior Colloquia

Spring 2016

Further testing for specific hitsPresent findings at Undergraduate Research DayBegin draft of final thesis

Fall

2016

Finish data collectionContinue drafting thesis and preparing for Thesis Conference

Spring 2017

Present and defend thesis at Team Thesis ConferenceFind collaboration to continue studiesWrite Publications

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Appendix C: Budget

Item Unit Cost Quantity Subtotal Details

GeneralGrowth medium (7H10 Agar Base) (500g)

$155 1 $155 Sigma-Aldrich

Growth medium (7H9 Broth Base) (500g)

$162 1 $162 Sigma-Aldrich

microcentrifuge test tubes (Pack of 500 tubes, 1.0-2.0) mL

$100 3 $300 Sigma-Aldrich

micropipette tips (1 uL) $75 1 $75 Fischer Scientificmicropipette tips (960 tips, 200 uL)

$75 1 $75 Sigma-Aldrich

micropipette tips (20 uL) $75 1 $75 Sigma-AldrichInk Bottles $2 300 $600 Sigma-Aldrich

Flat-bottom 96 well plates (case of 100)

$313 1 $313 Sigma-Aldrich

Round Bottom Test Tube (12 mL) (~1000)

$100 1 $100 Fischer Scientific

M. smegmatis cells 0 0 Provided by PILab notebook $20 5 $100 AmazonDyes: Alamar Blue (100 mL)

$414 1 $414 Fischer Scientific

Cloning

miniprep kits $2 150 $300 Sigma-Aldrich

restriction enzymes $50 12 $600 Sigma-Aldrichbacterial growth plates $2 100 $200 Sigma-Aldrich

inoculation loops (pack of 100)

$50 4 $200 Sigma-Aldrich

Primers $15 12 $180 Sigma-Aldrich

PCR kits $200 2 $400 Sigma-Aldrich

DNA Sequencing $8 48 $384 Sigma-Aldrich

Screening

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Drug Panel from NIH $800 2 1600 NIH

Total: $6,233

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Appendix D: NIH Clinical Collection

The NIH Clinical Collection is a plated array of 446 drugs that have a history of use in

clinical trials. These Food and Drug Administration approved drugs are rich sources of

undiscovered bioactivity and therapeutic potential, with known safety profiles. The detailed

characteristics of each drug can be found here:

http://www.nihclinicalcollection.com/NCC_SDFILE_NCC-104_Update2.pdf

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Appendix E: Protocols

Restriction Enzyme Digestion Protocol:

Original protocol can be found in: Promega (2011). The full citation can be found in the

references.

Objective: To create linear DNA molecules (restriction enzyme digests) that will have sticky

ends on both terminals of the DNA sequence.

1. In a 1.5 mL microcentrifuge tube, add these components in the following order.

Component Volume (μL)

Sterilized, deionized water 16.3

Restriction enzyme 10x buffer 2.0

Acetylated BSA (10 μg/μL) 0.2

DNA (1 μg/μL) 1.0

2. Mix by pipetting.

3. Add to the 1.5 mL microcentrifuge tube.

Restriction Enzyme (10 μg/μL) 0.5 μL

Final Volume 20 μL

4. Mix gently by pipetting.

5. Microcentrifuge the tube for a few seconds.

6. Incubate at enzyme’s optimal temperature for 1-4 hours.

7. Incubate reaction at 70 oC for 15 minutes to stop restriction enzyme activity.

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Dephosphorylation of Restriction Enzyme Digests with Antarctic Phosphatase Protocol:

Original protocol can be found in: New England BioLabs Inc. (n.d.) with the title “Protocol for

Dephosphorylation of 5'-ends of DNA using AnP (M0289).” The full citation can be found in the

references.

Objective: To prevent self-ligation of linear DNA molecules.

1. Add 10X Antarctic Phosphatase Reaction Buffer to 1-5 μg of restriction enzyme digest so

that the buffer will be diluted to 1X in the mixture (e.g. if 9 μL of 0.5 μg/μL restriction

enzyme digest was used, 1 μL of buffer will be added).

2. Add 1 μL of Antarctic Phosphatase and mix.

3. At 37 oC, incubate for 15 minutes for 5’ ends or 60 minutes for 3’ ends.

4. Heat inactivate phosphatase for 5 minutes at 70 oC.

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Agarose Gel Electrophoresis Protocol:

Protocol for cloning can be found in: Addgene (n.d.). The full citation can be found in the

references.

Objective: To identify DNA molecules by their size in units of base pairs. To isolate a specific

DNA sequence.

Recipe for a stock of 50X TAE (for 1 L):

Component Amount

Tris-base 242g

Acetate (100% acetic acid) 57.1 mL

0.5M sodium EDTA 100 mL

Deionized H2O Add to make total volume 1 L

Dilute to 1X TAE (for 1 L):

Add 20 mL of 50X TAE stock to 980 mL of deionized water.

Recipe for 1% Agarose Gel:

1. Add 1 g of agarose into a 500 mL Erlenmeyer flask.

2. Add 100 mL of 1X TAE into flask.

3. Microwave until agarose is completely dissolved and light boiling (usually 1-3 minutes).

4. Let agarose solution cool for 5 minutes.

5. Add 0.2-0.5 μg/mL ethidium bromide to the agarose solution.

6. Pour the solution into a gel tray with well combs.

7. Let the gel sit for 20-30 minutes at room temperature to completely solidify it.

46TEAM MTB PROPOSAL

Loading DNA samples and running the gel:

1. Add loading buffer to each of the restriction enzyme digest samples.

2. Place the solid agarose gel into a gel box.

3. Add enough 1X TAE into the gel box for the gel to be covered.

4. Load a standard DNA ladder into the first lane of the gel with a micropipette.

5. Load each digest sample into separate wells on the gel.

6. Operate the gel at 80-150 V.

7. Run the gel until the dye of the loading buffer travels about 75% of the distance down the

gel.

8. Turn off the power source and remove the gel from the gel box.

9. Observe the gel under UV light to illuminate the DNA bands.

10. Compare the location of the band on the gel with the location of the standard DNA ladder

bands to identify the size of the digest sample bands.

11. Cut out the bands that match the size of the DNA molecules needed for the next steps

with a razor.

47TEAM MTB PROPOSAL

Ligation with T4 DNA Ligase Protocol:

Original protocol can be found in: New England Biolabs Inc. (n.d.) with the title “Ligation

Protocol with T4 DNA Ligase (M0202).” The full citation can be found in the references.

Objective: To incorporate the digested insert into the digested M-PFC plasmid vector, which

ultimately forms the desired plasmid construct that will be transformed into cells and used in the

M-PFC assay.

1. Add the following into a 1.5 mL microcentrifuge tube:

Component Amount

10X T4 DNA Ligase Buffer 2 μL

Vector DNA (4kb) 50 ng

Insert DNA (1kb) 37.5 ng

Sterilized, deionized water Add to make total volume 20 μL

T4 DNA Ligase* 1 μL

*Add ligase last, but include the 1 μL into the calculation for the volume of water to add.

2. Mix by pipetting.

3. Microcentrifuge for a few seconds.

4. Incubate at 16 oC overnight or at room temperature for 10 minutes.

5. Heat inactivate ligase at 65 oC for 10 minutes.

48TEAM MTB PROPOSAL

Transformation into Bacteria by Electroporation:

Objective: To introduce the M-PFC plasmid constructs that contain the insert into a bacterium’s

genome, which ultimately creates mutant bacteria.

Transformation into E. coli is used for isolating a large quantity of the M-PFC plasmid

constructs.

Transformation into Msm is used for the M-PFC assay.

E. coli cells used for electroporation will be commercially prepared.

Prepare Msm cells for electroporation:

Original protocol can be found in: Goude et al (2008). The full citation can be found in the

references.

1. Obtain Msm colonies grown on 7H11 agar plates that contain Tween-80.

2. Inoculate single colonies into 7H9 liquid broth containing Tween-80 that is contained in a

culture tube.

3. Vortex to homogenize the suspension of cells in the cell culture tubes.

4. Incubate the liquid cell cultures at 37 oC and shaking at 100 RPM in a growth chamber

overnight.

5. Inoculate the overnight culture into 100 mL of 7H9 broth in a 250 mL E. Flask. Add a

volume of the overnight culture to maintain a 1:100 dilution of overnight culture to broth.

6. Incubate the cell culture at 37 oC with shaking for about 16-24 hours.

7. Check OD to equal 0.8-1 which is the optimal point of growth.

8. Incubate cells on ice for 1.5 hours to increase transformation efficiency (do not incubate

any longer).

9. Pour culture into large centrifugation tubes.

49TEAM MTB PROPOSAL

10. Centrifuge the tubes at 300 G for 10 minutes.

11. Wash cells with ice cold 10% glycerol 3 times. Use decreased volumes of glycerol each

time: for a culture volume of 100 mL, add up to 20 mL of glycerol for the first wash, add

up to 10 mL for the second wash, add up to 5 mL for the last wash.

12. Resuspend cells in 1:10 to 1:100 of the original volume of ice cold 10% glycerol.

13. Transfer aliquots of cells to 1.5 mL microcentrifuge tubes to be stored at -70 0C.

14. Prior to use: thaw the frozen cells, centrifuge the cells, and resuspend in fresh 10%

glyrcerol

Electroporation (can be use with both electrocompetent Msm or E. coli):

Original protocol can be found in: Gonzales et al (2013). The full citation can be found in the

references.

1. Add up to 1 μg of plasmid DNA (in up to 1 μl water or Tris-EDTA buffer) to the 40 μl

bacterial suspension and transfer this mixture into a pre-chilled, sterile 0.2 cm

gap cuvette The salt concentration in the DNA sample must be lowa, as it will contribute

to arcing of the pulse in the next step.

2. Insert the cuvette into the electroporation chamber of the pulse control module,

and electroporate at 1.8 kV, 25 μF. The time constant should be ~5.0 msec, and no arcing

should occur.

3. Quickly recover the cell suspension by resuspending into 1 ml LB broth and transfer into

previously autoclaved borosilicate glass test tube.

4. Allow the cells to recover by incubating under aerated growth conditions (in a roller

drum) at 37 °C for 30 min without antibiotic selection.

50TEAM MTB PROPOSAL

5. Plate the bacteria onto the previously prepared LB agar plates in the presence of the

appropriate selective agent (antibiotic), and incubate at 37 °C O/N. If bacteria are

transformed with a high concentration of purified vector (0.1-1 μg supercoiled plasmid),

deliver 10 μl of bacterial culture onto the edge of the LB agar plate and with a sterile

inoculating loop streak for isolated colonies using the quadrant streak method. If a

ligation mixture was transformed, deliver 100 μl of bacterial culture onto each agar plate

and evenly spread it with the sterilized glass cell spreader.

M-PFC Assay Protocol:

Protocol for M-PFC assay can be found in: Singh et al (2006). The full citation can be found in

the references.

Objective: To determine protein-protein interactions of mycobacterial proteins.

Host strain:

M. smegmatis strain mc2155: A high frequency transformation derivative of M. smegmatis

mc26.

Growth of Mycobacteria

20% v/v Tween-80: dissolve 20 ml of Tween-80 in 80 ml of deionized water. Heat at 56

OC and bring into solution with thorough mixing on magnetic stirrer. Filter sterilize

through 0.2 µm filter. Store at 4 OC.

50% glycerol: mix 250 ml glycerol with 250 ml deionized water and stir thoroughly until in

solution. Sterilize by autoclaving. Store at 4 OC.

50% Glucose: dissolve 50 gm of glucose with 60 ml of deionized water and stir

thoroughly. Make up the volume to 100 ml. Filter sterilize through 0.2 µm filter. Store at 4

OC.

51TEAM MTB PROPOSAL

Kanamycin sulfate (Sigma): Prepare a 50 mg/ml stock solution. Filter sterilize and store at

-20 OC.

Hygromycin B: Purchased as a stock solution of 50 mg/ml.

Trimethoprim (Sigma, Cat # T7883): Prepare a 50 mg/ml stock solution in 100% DMSO.

Filter sterilize and store at -20 OC.

Middlebrook (MB) 7H9 broth (Difco): Dissolve 4.7 g of MB 7H9 powder in 900 ml

deionized water, mix thoroughly and autoclave. Add 0.5% glycerol, 0.5% glucose and

0.2% Tween-80.

Middlebrook (MB) 7H11 agar (Difco): 21 g of MB7H11 agar to 970 ml of water and

autoclave. Cool and add to a final of 0.5% glycerol, 0.5% glucose and 0.2% Tween 80.

When necessary add 25 µg/ml of kanamycin, 50 µg/ml of hygromycin and 30-50 µg/ml

of trimethoprim

M-PFC Vectors:

M-PFC vectors were transformed into E. coli DH10B and grown in LB supplemented with

Kan (25 µg/ml) or Hyg (150 µg/ml). All DNA manipulations were performed using standard

protocols.

pUAB100: Episomal E. coli–Mycobacterial shuttle plasmid harboring GCN4 homo-

dimerization domain fused to N-terminus of mDHFR fragment F[1,2] and glycine

linker. Gene of interest can be cloned in this vector by replacing GCN4 domain using

MscI/ClaI or BamHI/ClaI. This plasmid contains the hygromycin resistance marker.

pUAB200: Integrative E. coli–Mycobacterial shuttle plasmid harboring the GCN4 homo-

dimerization domain fused to N-terminus of mDHFR fragment F[3] and glycine linker.

Gene of interest can be cloned in this vector by replacing GCN4 domain using MunI/ClaI.

52TEAM MTB PROPOSAL

This plasmid contains the kanamycin resistance marker.

pUAB300: Episomal E. coli–Mycobacterial shuttle plasmid harboring mDHFR fragment

F[1,2] and glycine linker. Gene of interest can be cloned in this vector at multiple

cloning sites to generate in frame fusion with the C-terminus of F[1,2]. This plasmid

contains the hygromycin resistance marker.

pUAB400: Integrative E. coli–Mycobacterial shuttle plasmid harboring mDHFR fragment

F[3] and glycine linker. Gene of interest can be cloned in this vector at multiple cloning sites

to generate in frame fusion with the C-terminus of F[3]. This plasmid contains the

kanamycin resistance marker.

The preferred plasmid choice for making DNA libraries is pUAB300.

The M-PFC assay:

Clone genes of interest in the M-PFC vectors (e.g Gene A in pUAB100 or pUAB300 and

Gene B in pUAB200 or pUAB400). Care should be taken to clone genes in-frame with the

complementary mDHFR fragments. Constructs should be verified by sequencing. Prepare

high quality DNA for transformation into M. smegmatis. M. smegmatis electrocompetent cells

can be prepared by using standard protocol. Check the efficiency of the cells (104-105

transformants/µg of DNA). Electroporate M-PFC clones into M. smegmatis using standard

protocol. Allow transformants to recover for 3h at 37 OC in 1 ml of MB 7H9 and plate entire

transformation mix on MB 7H11 Kan/Hyg plates. Incubate MB 7H11 plates at 37 OC for 3

days. Protein-protein interaction between the gene products of A and B can be analyzed by

subculturing Kan/Hyg transformants on MB 7H11 plates containing Kan/Hyg and

Trimethoprim (30-50 µg/ml). Subculturing on trimethoprim plates can also be performed by

making a suspension of few colonies in 100 ul of sterile 1xPBS followed by streaking of 10

53TEAM MTB PROPOSAL

ul of this suspension on the plates. Incubate MB 7H11 Kan/Hyg/Trim plates at 37 OC for 4-

5 days for growth. Time and robustness of growth can be influenced by the strength of

interaction; stronger interactions will emerge earlier than weaker interactions.

Growth on trimethoprim is indicative of protein-protein interaction. The specificity of

the interaction can be determined by analyzing interaction with an unrelated protein (negative

control). pUAB 100 and pUAB 200 can be used as a positive control (both of these construct

contain GCN4 homo-dimerization domains from Saccharomyces cerevisiae) and associate

strongly. Also, any one of these clones can be used as negative control with your clone of

interest.

Notes:

➢ Fusions should be carefully verified by sequencing.

➢ Do not replace 7H11 with 7H10 or 7H9 + agar for the assay.

➢ Do not modify concentrations of antibiotics (Kan, Hyg and Trim) in the assay.

➢ To check for the interactions in the mycobacterial membrane use pUAB100 and

pUAB200.

➢ Some proteins may require a free N- or C- terminus for interactions. It will be

useful to check parallel-antiparallel interactions as well as parallel-parallel

orientations.

➢ The strength of interactions can be measured by performing the AB-TRIM assay as

described (Singh et al., 2006).

54TEAM MTB PROPOSAL

Drug Screen

Protocol for M-PFC assay can be found in: Singh et al (2006). The full citation can be found in

the references. For our drug screen, we will make a variation of the 96-well plate procedure

described in the above reference.

Drug stocks will be first diluted in DMSO from original stock. Diluted drug

concentrations will be re-dissolved in 7H11 media. Transformed Msm cell line will be at a final

concentration of 105 cells/ ml in each well. Trimethoprim will be kept at a concentration of 30-50

µg/ml in each well. For our initial drug screen, our drugs will be plated at a final concentration of

25 µm in each drug well. Our negative control well will contain transformed Msm, and

Trimethoprim dissolved in media at the same concentrations of the experimental wells. Vehicle

control will contain transformed Msm, Trimethoprim, and DMSO at its concentration in our

experimental wells. All trials will be performed in triplicate, and DMSO will never exceed a

concentration of 0.2% in each well. Refer to figure III-5 for the well layout. Plates will be

incubated at 37 OC for 4-5 days of growth. After incubation is complete, the AB-TRIM assay

will be performed as described in Singh et al. (2006). We will obtain statistics by measuring

absorbance of wells by using a plate spectrophotometer at 650 nm as provided by Dr. Briken.

Validation Studies

We have yet to establish our exact methods for validation. They will be based off of the papers by

Singh et al. (2006), and Mai et al., (2011). The full citations are contained in the references.

55TEAM MTB PROPOSAL

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