Sejal

=Design Project=



The design I created for this project is a synthetic genetic system in //E. coli// that can synergistically work with antibiotics currently used to combat MRSA, or methicillin-resistant //Staphylococcus aureus.// MRSA is a bacterium that has developed extreme antibiotic resistance and cannot be treated with the first-line antibiotics that usually cure //Staphylococcus// infections. At present, approximately 40%-50% of //S. aureus// isolates are resistant to newer semisynthetic antibiotics such as methicillin and vancomycin. These infections are rampant in hospitals because 1) MRSA normally infects immuno-compromised people (such as patients with cancer, requiring kidney dialysis, undergoing surgery, or with other underlying health conditions) and 2) because it resides primarily on the cutaneous and subcutaneous skin levels and is easily transmitted by touching contaminated inanimate objects (fomites). Additionally, because it is rarely eradicated due to its antibiotic resistance, those who are infected by MRSA relapse (symptoms of which include boils, skin abscesses, fever, rash, cough, chest pain, fatigue, and pneumonia) every few months following treatment. Due to the inability of physicians to use antibiotic drugs to treat MRSA infections, a method of debilitating resistance in the pathogen is necessary. This bacterium is designed to inhibit the various mechanisms of resistance towards β -lactam antibiotics in MRSA to promote efficacy of the drug and prevent periodic relapses.

Ironically, current technologies designed to overcome antibiotic resistance in these organisms fuel their prevalence in what is known as the “antibiotic paradox”. When bacteria are treated with antibiotics, a few bacteria usually exhibit resistance and survive, usually due to genetic mutations or lateral gene transfer. Through natural selection, these bacteria are the ones that survive to pass their genes down, increasing the allelic frequency of the antibiotic resistance genes in the gene pool. Too much antibiotic use selects more resistant mutants. For example, the prescription of va- ncomycin to treat MRSA resulted in the emergence of a new vancomycin-resistant strain, VRSA. This is why doctors advise against “unnecessary” use of antibiotics- overuse can result in a loss of drug potency. However, the only alternative is for the patient to simply rely on the body’s natural immune response and wait for the symptoms to subside. Relapse makes this problematic. My design solves for the antibiotic paradox because it increases the susceptibility of MRSA to existing β -lactam antibiotics, which lowers the MIC (minimum inhibitory concentration) needed to eradicate the bacteria and maintains drug efficacy. This removes the need for overuse of the drug or use of the strongest and most broad-spectrum drugs (which exacerbate the evolution of antibiotic-resistant strains) for effective treatment. This enables physicians to prescribe antibiotics without the fear of loss of drug potency, which means the patient doesn’t have to rely on the immune response each time the relapse kicks back in. The design is also uniquely beneficial in that it helps kill bacteria that would normally survive through natural selection by targeting antibiotic resistance genes, which reverses the existing evolutionary trend that promotes the horizontal and vertical transfer of these genes among strains.

MRSA has evolved to exhibit several mechanisms of β-lactam resistance, some through chance mutations and others through horizontal gene transfer. Typically, β-lactam antibiotics target penicillin-binding-proteins, or PBPs, enzymes anchored to the cytoplasmic membrane in bacteria that are involved in synthesizing peptidoglycan, a major component of cell walls. PBPs catalyze reactions that synthesize cross-linked peptidoglycan from lipid intermediates and remove D-alanine from the precursor of peptidoglycan. C-terminal domains of these proteins are sensitive to penicillin. Penicillin works by binding to the active sites of PBPs, which inhibits transpeptidase activity, disabling them from catalyzing the cross-linking step. This results in lesions of the cell wall and causes the cell to die. MRSA is able to survive in the presence of penicillins because it contains a horizontally acquired genomic island known as Staphyloccal cassette chromosome //mec//, or SCC//mec//. SCC//mec// contains an inducible gene known as //mecA//, which encodes for a protein known as PBP2a. //mecA// is constitutively repressed by a repressor known as MecI produced by the regulatory gene //mecI.// In the presence of β -lactam antibiotics, MecR1, a signal transducing transmembrane protein produced by regulatory gene // mecR1, // undergoes a signal transduction cascade that results in the proteolysis of MecI, inducing expression of // mecA. // PBP2a has a different structural conformation than standard PBPs, which gives it low affinity for the β-lactam ring in antibiotics. While penicillins inhibit all other PBPs, the inability for penicillin to bind to PBP2a enables the protein to continue cross-linking peptidoglycan and catalyze the formation of cell walls.

A second antibiotic-resistance mechanism is the chromosomal gene //blaZ//, homologous to //mecA. blaZ// produces the enzyme β-lactamase, which hydrolyzes the four-atom β-lactam ring in antibiotics. Like //mecA, blaZ// is constitutively repressed by repressor protein BlaI and is regulated by //blaI// and //blaR1//. The extracellular domain of BlaR1 (homologous to MecR1) is acylated by a β-lactam ring. This results in signal transduction to the intracellular zinc protease domain of BlaR1, which activates and then proteolyzes BlaI, resulting in expression of //blaZ//. Because //mecA// and //blaZ// are so homologous, there is a lot of crosstalk between repressor proteins MecI and BlaI, which means that both regulatory networks need to be inhibited simultaneously in order to render MRSA susceptible to β-lactams.

A third mechanism used by MRSA is that of MDR (multi-drug resistant) efflux pumps. Efflux pumps are in the cytoplasmic membrane and use active transport to confer resistance to toxic host substances by essentially “pumping” them out of the cell. //S. aureus// utilizes broad-spectrum efflux pumps such as NorA, effluxing a range of antibiotics, and specific pumps such as TetK, effluxing only tetracycline. Inhibition of efflux can prevent growth and colonization, debilitating the cell.

The genetic system I’ve designed takes into account these major methods of antibiotic resistance and inhibits them simultaneously to weaken the pathogen. In order to sense the presence of //S. aureus//, the system employs the //agr// (accessory gene regulator) quorum sensing system, which is used in //S. aureus// to decrease expression of cell surface proteins and increase expression of secreted virulence factors while cell growth transitions into stationary phase. The //agr// locus is activated by the P2 and P3 promoters. The P2operon, through RNA II, encodes for the transcription of four proteins: agrA, agrB, agrC, and agrD. I modified this operon by removing agrC and agrD, which encode for proteins that synthesize the auto-inducing octapeptide signaling molecules. agrA and agrC encode for proteins that synthesize the transmembrane protein receptor, AgrC (histidine kinase). This receptor senses auto-inducing octapeptides produced by //S. aureus// and, upon binding with an AIP, modulates AgrA, which is then phosphorylated. This subsequently activates P2 and P3. In //S. aureus//, the increased transcription of the P3 operon usually increases production of RNAIII, which encodes the toxin δ -hemolysin and other virulence factors. However, I used this promoter to activate the sequence of genes that would produce various inhibitors. This way, the production of inhibiting enzymes and proteins would be regulated directly by the presence of MRSA as communicated through quorum sensing.

The quorum sensing device activates both the P2 operon (which regulates production of histidine kinase AgrC) and the P3 operon. The P3 operon contains four genes that encode for specific inhibitors, each preceded by a ribosome binding site (RBS), indicated in the diagram as an orange circle. Each mRNA transcript contains a place at the RBS for the ribosome to bind in order to produce the protein in question. Each operon is terminated by Terminator 1 and Terminator 2 (the two red octagons at the end of the gene sequence).

The first open reading frame of the P3 operon is ALO1, which produces an enzyme called D-Arabinono-1, 4-Lactone Oxidase, or ALO. ALO, which is not naturally produced in //E. coli//, catalyzes the terminal step in the biosynthesis of D -erythroascorbic acid (EASC), a “cousin” of Vitamin C, in //Saccharomyces cerevisiae.// //Because// //E. coli// //naturally already produces// d -arabinono-1,4-lactone, another necessary component for this enzymatic pathway, adding a gene to the operon that produces ALO will allow for the synthetic production of EASC. Ascorbate then acts as an induction inhibitor for β-lactamase.

The second open reading frame is from a cyslabdan synthase gene from //Streptomyces// K04-1044. Cyslabdan is a diterpene, or protein, that inhibits transpeptidase activity by inhibiting the //femA// gene by inducing the repressor protein FemA, which causes cyslabdan to accumulate nonglycyl and monoglycyl monomers in the peptidoglycan layer of the cell wall. This prevents MRSA from forming cell walls despite being equipped with PBP2a.

The third open reading frame is a corilagin synthase gene from // Arctostaphylos uva-ursi //. Corilagin is a diterpenoid that potentiates methicillin by inhibiting PBP2a cross-linking activity, increasing lesions and cell wall damage and lowering the MIC of methicillin. Because // mecA // and // blaZ // have crosstalk, coraligin has also been shown to markedly inhibit production of β-lactamase.

The fourth gene is the Columbus gene, which encodes the HMG-CoA that synthesizes geranylgeranyl pyrophosphate. This undergoes a diterpene metabolic pathway that results in the formation of totarol. Totarol is an efflux pump inhibitor (EPI) that inhibits NorA efflux pump activity in //S. aureus//.

The final operon in the system is an ACL5 or ACAULIS5 operon. This operon functions to make the bacteria resistant to antibiotics, as it is used synergistically with β-lactams and must be used simultaneously as the activity it inhibits is only induced in the presence of β-lactams. The ACL5 promoter constitutively expresses the ACL5 gene, a spermine synthase, which results in the formation of the polyamine spermine. Spermine will decrease permeability of the cell membrane by binding to one of two binding sites in the porins of the outer membrane. In gram negative bacteria, β-lactams pass through chemically selective porin channels to reach their targets. The inhibition of this transport through the use of spermines will ensure that the cell will not die in the presence of β-lactams.

When the cell is working perfectly, the presence of the MRSA auto-inducing octapeptides should induce production of AgrC histidine kinase, EASC, cyslabdan, corilagin, and totarol. The ACL5 gene should be constitutively expressed.

Truth Table (Perfectly Functioning)
 * AIP || AgrC || EASC || Cyslabdan || Corilagin || Totarol || Spermine ||
 * 1 || 1 || 1 || 1 || 1 || 1 || 1 ||
 * 0 || 0 || 0 || 0 || 0 || 0 || 1 ||

The cell will be working at an imperfect but acceptable level as long as EASC and either cyslabdan or corilagin are being produced in response to the AIP input through a functioning AgrC signal transducer, because they will work to inhibit the //mecA-blaZ// coregulators of PBP2a and β-lactamase, the main mechanism of methicillin resistance. In this case, spermine would still be a necessary component to maintain antibiotic resistance of the cell (otherwise, the presence of β-lactams would kill the bacteria).

Truth Table (Acceptably Functioning)
 * AIP || AgrC || EASC || Cyslabdan || Corilagin || Totarol || Spermine ||
 * 1 || 1 || 1 || 1 || 0 || 0 || 1 ||
 * 1 || 1 || 1 || 0 || 1 || 0 || 1 ||
 * 0 || 0 || 0 || 0 || 0 || 0 || 1 ||

The system does pose several potential problems that need to be explored through developmental research. First, the exact genomic sequences producing cyslabdan and corilagin have yet to be isolated, and the BioBricks for each part of the system developed. Second, researchers will need to determine how much EASC needs to be produced to ionize to such an extent that MIC for ascorbate is reached. Additionally, researchers will need to find a way to make spermine bind to the specific porins that transport β-lactams and ensure that the molecules do not inhibit transport in other necessary porins used to transport sugars and other molecules. In addition, researchers will need to make sure the //agr// locus specifically targets AIPs released from MRSA- the wrong //agr// locus could be induced by octapeptides from other Staphylococci like //S. epidermis,// which exist naturally on human skin. The AIPs produced by MRSA can already be classified as AIP I, AIP II, AIP III, or AIP IV based on the symptoms exhibited. Researchers may be able to develop specific //agr// loci for each of these classifications. These are a few of the many issues that must be addressed and troubleshot when developing this bacterium.

Several different experiments would need to be conducted in order to fully test the efficacy of this system. One way is to use a nitrocefin assay to measure β-lactamase activity of MRSA in the presence of β-lactams alone and β-lactams with the //E. coli// inhibiting system. Nitrocefin is a chromogenic cephalosporin susbtrate that contains a β-lactam ring. Once its ring is hydrolyzed by β-lactamase, it changes color from yellow to red. This would allow for a quantitative and qualitative measure of the degree of change in β-lactamase activity in response to the insertion of the system.

There is an obvious need for further endeavors in the research and development of this resistance-inhibiting genetic system. However, it holds a great deal of promise in its ability to effectively treat MRSA relapse, mitigate the loss of drug potency, and reverse the natural selection of horizontally and vertically conferred antibiotic resistance genes. The development of this technology will help combat one of the most powerful and prevalent nosocomial infections in the world.

PowerPoint presentation file:

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