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MTAN: A CRITICAL TARGET FOR ANTI QUORUM SENSING ATNTIBIOTICS

Cindy Chen1,4, Jake Yu2,4, Shanzhi Wang3,*, Fourth Author1

1Bronx High School of Science Bronx, US;2Montgomery High School, Skillman, US.

3Biochemistry Department, Albert Einstein College of Medicine, Bronx, USEmail: shanzhi.wang@einstein.yu.edu

4Contributed equally to the manuscript and listed alphabetically

Received Month Day, Year; Revised xxxx; Accepted xxxx

ABSTRACTThis review details the development and impact of drug resistant bacterial strains. Their rapid increase has prompted research into novel treatment methods that will compromise bacteria without killing them. One such method targets bacteria’s intercommunication process known as quorum sensing. Bacteria release sig-naling molecules called autoinducers. Once the autoinducers reach a critical level, or a quorum, all neigh-boring bacteria simultaneously switch gene expression and act in unison. This review shows that the enzyme 5' –Methylthioadelynosine/ S-adenosylhomocysteine nucleosidase (MTAN) is vital for autoinducer produc-tion. In addition, a recent study proposed a new menaquonone pathway in pathogenic bacteria H. pylori and C. jejuni in which MTAN plays a vital role. Thus, MTAN is a critical target for antibiotic drug design. Fu-ture studies will focus on how inhibition of MTAN will affect bacteria toxicity. This review furthers our un-derstanding of the inner workings of bacterial infections.

Keywords: MTAN, Quorum sensing, autoinducers, drug resistance, menaquonone

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AutoinducerPathogenic factors

Low Cell Density High Cell Density

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1. INTRODUCTIONAntibiotic resistant bacteria are a pressing problem across the world. Many disease causing bacteria such as Staphylococcus aureus, Mycobacterium tuberculosis, Es-cherichia coli, Vibrio cholera, Pseudomonas aeruginosa, and Streptococcus pneumonia have developed resistance against current antibiotics. To fight these ‘superbacteria’, current research focuses on novel antibiotics that limit bacteria’s toxicity without killing it, thereby reducing the chance of developing resistance. Disrupting quorum sensing to prevent biofilm formation and release of path-ogenic factors could be a viable strategy. The enzyme MTAN plays a vital role in quorum sensing pathways and thus is an important target for antibiotic drug design.

2. Antibiotic Resistant Strains2.1. Staphylococcus aurea Since the discovery of antibiotics, antibiotic resistant strains have significantly increased and become a major threat in post operative care departments. Each year ap-proximately two million hospitalizations result in noso-comial infections. A study in a large teaching hospital showed that illness due to nosocomial bacteremia in-creased intensive care unit stay by 8 days, hospital stay by 14 days, and the death rate by 35% among critically ill patients.1 The major cause of death in hospitals is an-tibiotic resistant microorganisms in post-operative care such as Staphylococcus aureus. Staphylococcus aureus causes a number of diseases as a result of infection of tis-sues in the body. Staphylococcal sepsis is the leading cause of shock and circulatory collapse. If left untreated, the mortality rate of S. aureus sepsis is higher than 80%2. When treated with β-lactam antibiotics, which have high efficacy against S. aureus, the mortality rate is still be-tween 20% and 40%. Recently, S. aureus has developed strains that are resistant to the drugs used to treat the bac-terium. These drug resistant strains of S. aureus are called Methicillin-resistant Staphylococcus aureus (MRSA). MRSA usually targets immunocompromised patients, in which the body cannot overcome and kill the strong bacteria colonies. 19,000 hospitalized American patients die from MRSA infections annually; this approaches the number of deaths due to AIDS, tuberculosis, and viral hepatitis combined . From 1994 to 2004, there was a 300% increase5 of staphylococcus infections caused by MRSA. MRSA is now endemic and epidemic in many hospitals, comprising nearly 30% of all S. aureus infections6.

2.2. Mycobacterium tuberculosisTuberculosis is a highly contagious airborne disease caused by Mycobacterium tuberculosis. If left untreated,

a person with active TB will infect between 10 and 15 people per year. In 2010, it was estimated that 8.8 mil-lion people were infected and 1.1 million people died from TB7. TB is resistant to almost any single drug treat-ment. TB is currently treated with a combination of three or four antibiotics with a treating period of six to nine months. However, two new strains of Tuberculosis, called MDR-TB (multi drug resistant) and XDR-TB (ex-tensively drug resistant) have developed. People infected with these strains have a high fidelity rate in spite of prompt antibiotic treatment.

2.3. Escherichia coliEscherichia coli is the most common bacterium that causes sepsis. This bacterium infects approximately 73,000 people in the United States every year 8. Around 500 people in the United States die every year due to E. coli related diseases. E. coli has developed strains that are resistant to penicillin and cephalosporin 9, drugs that are currently used to treat E. coli diseases.

2.4. Vibrio choleraCholera is an infectious disease of the intestine caused by Vibrio cholera. Every year, approximately three to five million people are infected by cholera, and 100,000 to 120,000 die from cholera10. The death rate due to cholera is about 1%, but if untreated, the mortality rate grows to 60% on average11. Doxycycline is one of the main antibi-otics used to treat cholera. However, strains of V. cholera have become drug resistant to these antibiotics. In 2009 50% of isolated strains of V. cholerae were tetracycline resistant 12.

2.5. Pseudomonas aeruginosaPseudomonas aeruginosa is a nosocomial pathogen that causes septic shock and pneumonia. The mortality rate in patients with septic shock is approximately 40% 13. Pneu-monia is one of the most common fatal nosocomial in-fections, leading to a yearly mortality rate of 33%14. P. aeruginosa infections are commonly treated with β -lac-tam antibiotics, such as first generation penicillin. How-ever, strains of P. aeruginosa quickly developed resis-tance to these drugs, prompting newer generation peni-cillin antibiotics such as Piperacillin sodium. The novel antibiotics took decades to produce, yet P. aeruginosa developed resistance in a matter of months.

2.6. Streptococcus pneumoniae

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Streptococcus pneumoniae is a bacterium that is the most common cause of otitis media, sepsis, pneumonia, and meningitis. Approximately 500,000 cases of pneumonia, 55,000 cases of bacteremia, and 6,000 cases of meningi-tis are caused by S. pneumoniae infections annually in the United States15.(source doesn’t check) In 2000, S. pneumoniae infections caused 6 million cases of otitis media, 100,000-135,000 hospitalizations for pneumonia, and 60,000 cases of invasive disease, including 3300 cases of meningitis16. After a vaccine was introduced in 2002, the rate of the invasive disease dropped from 21-33 cases per 100,000 population to 13 cases per 100,000 population16. Pneumonia is treated with antibiotics, such as macrolides, quinolones, and nafcillin. However, drug resistant S. pneumonia has increased. In 1992, 13,300 hospital patients died of bacterial infections that were re-sistant to antibiotic treatment. Today, 6.6 percent of pneumococcus (penicillin-resistant S. pneumoniae) strains are drug resistant17.

3. Quorum Sensing (QS)The rapid increase of antibiotic resistant pathogenic bac-teria calls for a new strategy that will fight bacteria with-out actually killing the bacteria itself. To develop such a method, we must first understand how single celled bac-teria can cause such widespread damage. Bacteria com-municate with each other and act in groups through the process of quorum sensing. By this mechanism, each in-dividual bacterium produces signaling molecules called autoinducers (AIs). When these autoinducers reach a cer-tain amount, each bacterium senses the autoinducers and recognizes that it is surrounded by other bacteria. Then, all the bacteria switch gene expression and act in unison. Thus, when cell density is low, bacterium lives as an in-dividual without communication; when cell density reaches a certain level, the quorum sensing mechanism is activated and bacteria exhibit synchronized behavior as shown in Fig. 1. Virulent bacteria cause damage when quorum sensing results in the release of pathogenic fac-tors including toxins, biofilm formation22-24, and tissue at-tachment factors . Disruption of quorum sensing systems has been shown to effectively compromise the infectivity of several pathogenic bacteria. In mice, mutant quorum sensing-deficient intranasal Streptococcus pneumonia in-fections are less capable of

Fig. 1. Bacterial QS system

spreading to the lungs and bloodstream29. In an infant rat infection model, a quorum sensing-deficient Neisseria meningitidis strain is unable to produce living bacteria in the blood30. Moreover, quorum sensing exists in bacteria and certain plants, but not in mammals. Therefore, anti-quorum sensing antibiotics would effectively target viru-lent bacteria without compromising human health 31. Be-cause such antibiotics would not kill bacteria, develop-ment of drug resistance is unlikely.

4. MTAN’s role in autoinducer production5’ –Methylthioadelynosine/ S-adenosylhomocysteine nu-cleosidase (MTAN) is a dual substrate enzyme in bacte-ria that catalyzes the hydrolytic reactions of 5’ –Methylthioadenosine (MTA) and S-adenosylhomocys-teine nucleosidase (SAH). S-adenosylmethionine (SAM) pathways lead to polyamine synthesis which yields MTA, and methyltransferase reactions which yield SAH. Polyamines are critical for bacterial cell growth and methyltransferase reactions are crucial for cell survival. By inhibiting MTAN, excess MTA and SAH would ac-cumulate and inhibit their respective pathways. Further-more, MTAN plays a key role in the production of au-toinducers. AHL synthase transfers the amino acid moi-ety of SAM to an acyl acceptor to yield acylhomoserine lactones (AI-1), and produces MTA as a byproduct 32. MTAN catalyzes the hydrolytic reaction of SAH to form S-ribosylhomocysteine (SRH), which is the precursor to AI-2. MTAN

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Fig. 2. MTAN’s role in autoinducer production

inhibition would accumulate MTA which would inhibit AI-1 production, and directly block the formation of SRH, a precursor to AI-235. The blockage of both AI-1 and AI-2 would disrupt quorum sensing.

4.1 MTAN as a possible targetAs of today, MTAN is only found in prokaryotic cells and some plants, while mammalian cells utilize another enzyme, 5’ –Methylthioadelynosine phosphorylase (MTAP), to consume MTA. MTAP utilizes inorganic phosphate instead of water to cleave the glycosidic bond of MTA, yielding adenine and methylthioribose phos-phate. In contrast to MTAN, it does not use S-adenosyl-homocysteine as a substrate. The crystal structure of hu-man MTAP is available and it is structurally similar to prokaryotic MTANs . However, MTAP has a smaller binding pocket for the 5’ region of the substrate, discrim-inating the binding of S-adenosylhomocysteine40. Thus, inhibitors with a large 5’ substitution would only bind to MTAN, without inhibiting human MTAP. In theory, in-hibitors specific for SaMTAN could be a potential antibi-otic with little or no interaction with human enzymes.

A new menaquinone pathway was suggested in H.pylori and C.jejuni recently 41-43. MTAN was shown to be es-sential in the pathway. In contrast to the inhibition of quorum sensing in other bacteria 44, inhibition of MTAN of H.pylori using tight inhibitor leads to growth arrest 45. Because this new pathway exists in only few bacteria, not in human or normal flora, MTAN specific inhibitors are expected to exhibit few side effects. In addition, low

drug resistance is also expected due to the rarity of the pathway.

Fig. 3. MTAN’s critical role in the menaquonone pathway

5. CONCLUSIONMTAN’s critical role in bacterial pathways makes it a possible target for future antibiotics. Inhibiting MTAN should reduce bacteria toxicity and not lead to drug

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REFERENCES (Heading 6)Use the author/date system of references. In the text refer to the authors’ name (without initials) and year of publi-cation. All publications cited in the text should be presented in a list of references following the text of the manuscript.

1. Examples for a single authorPeterson (1993) has shown that ……This is in agreement with the results obtained by several authors (Kramer, 1994; Smith, 1995; Brown, 1999)2. Examples for two authorsSmith and White (1999) reported that…….This was later found to be incorrect (Amir and Ahmed, 2000)”.

3. Examples for three or more authors

Moore et al. (1990) stated that …..Similar results were reported recently (Smith et al., 2003).

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