PHYTOPHTHORA INFESTANS Eef3 FUNCTIONALLY COMPLEMENTS for LOSS of FUNCTION of SACCHAROMYCES
PHYTOPHTHORA INFESTANS eEF3 FUNCTIONALLY COMPLEMENTS FOR LOSS OF FUNCTION OF SACCHAROMYCES CEREVISIAE eEF3
Tanique Bennett, Kaitlyn Boyle, Emma Carlson, Arjun Gupta, Enoch Jiang, Ryan Jin, Aishwarya Kalyanaraman, Jeferson Mendoza, Pooja Nahar, Danielle Pergola, Siri Uppuluri, Sophia Velasquez
Advisor: Dr. Stephen Dunaway
Assistant: Jal Trivedi
ABSTRACT
The complex world of invasive mycoses has only recently emerged at the forefront of biomedical research as a serious threat to human health. Due to the increasing susceptibility of immunocompromised populations to invasive fungal infections (IFIs) and the ineffectiveness of current treatment methods, the field of medical mycology is in dire need of a novel therapeutic approach. In this paper, we focus on Eukaryotic Elongation Factor 3 (eEF3), one of three proteins in a family of translation factors known as eukaryotic elongation factors. eEF3 plays a critical role in polypeptide synthesis, aiding in the removal of deacetylated tRNAs from the ribosomal complex and recruiting aminoacetylated tRNAs from the cytosol to continue elongation. Curiously, it has been discovered that eEF3 is highly conserved in unicellular eukaryotes and is absent from, or at least functionally inactive in, more complex members of the domain. The functional exclusivity of eEF3 makes it a prime candidate for antifungal drug targeting, permitting the design of highly effective therapeutic compounds with minimal toxicity to host cells. However, the structural conservation of eEF3 varies among different species, and it is first necessary to determine the functional conservation of the protein as well as the conservation of integral functional domains. The current study presents an intuitive approach to answering these questions, employing the “plasmid shuffle” technique to transform P. infestans eEF3 into S. cerevisiae while forcing the latter species to forfeit its endogenous eEF3 gene. The results of the experiment indicated that, when placed under stringent environmental pressure, S. cerevisiae was able to survive on the P. infestans eEF3 homologue, suggesting that P. infestans eEF3 can functionally substitute for S. cerevisiae eEF3. This study provides a preliminary foundation for eEF3 targeting research and will hopefully have significant implications for antimicrobial drug development to combat fungal and other eukaryotic pathogens.
KEYWORDS
Eukaryotic elongation factor 3, eEF3, Phytophthora infestans, Saccharomyces cerevisiae, invasive fungal infections, antifungal drug targeting, Gibson Assembly Protocol, plasmid shuffle
INTRODUCTION
The contribution of fungal pathogens to the collective pool of human disease has been historically overlooked and has only recently captured the attention of the medical community, emerging in a variety of biomedical research avenues as an issue in need of addressing. Though the incidence of deaths related solely to invasive mycoses is comparably lower than that of other medical afflictions, the vast majority of these deaths occurs in patients with compromised immune systems due to pre-existing conditions, most notably Human Immunodeficiency Virus/Acquired Immunodeficiency Syndrome (HIV/AIDS) (1). This deadly combination of immunocompromisation and the resulting vulnerability to invasive fungal infections (IFIs) is responsible for over 50% of all AIDS-related deaths, earning a spotlight on the global medical stage (1, 2). The risk of contracting an IFI is also particularly heightened for transplant patients, preterm infants, and other individuals with underdeveloped/weakened immune systems or those undergoing immunosuppressive therapy (1,3). The net mortality rate due to both independently-acting invasive mycoses and IFIs is staggering; the seven most lethal fungal pathogens alone contribute to at least 1,600,000 recorded deaths every year (4).
The current state of affairs regarding the prophylaxis, diagnosis, and treatment of fungal infections is insufficient to combat the growing number of cases worldwide. The recognition and identification of a fungal infection is difficult and often results in a delayed diagnosis or no diagnosis at all (3). Our notable inability to distinguish among many existing fungal pathogens in microbiological cultures and the lack of simple serological tests further increases the threat that fungal pathogens pose to humans (3). Despite the availability of a vast array of antifungal therapeutics, an exponential increase in the administration of various antifungal drugs, and the expansion of the field of medical mycology, the susceptibility of human hosts to fungal pathogens continues to grow at an alarmingly rapid rate (1,5).
Fungal pathogens can also have devastating effects on non-human hosts such as crops, whose destruction often results in crippling economic implications and even widespread famine. P. infestans is an oomycete that causes potato late blight, a disease with the ability to wipe out countless potato crops, as well as other crops such as tomato (Solanum lycopersicum), pear melon (S. muricatum), and naranjilla (S. quitoense) (6). The horrific consequences of a P. infestans epidemic were epitomized by the Irish Potato Famine (also referred to as The Great Famine) in the mid-1840s, when the fungus destroyed over 75% of the potato harvest over the course of eight years. The potato crops were a major source of nutrition as well as a significant source of revenue for Irish farmers, and their loss resulted in nearly one million deaths, deep economic recession, and mass emigration (6). Even today, P. infestans remains an extraordinary threat to farmers around the world; in 1985, an outbreak in the state of Washington cost between $106.77 and $226.85 per acre affected, with a total management cost of nearly $30 million. The International Potato Center has estimated that in developing countries, P. infestans results in a minimum 15% harvest loss, or a total production loss of around $2.75 billion per year (6).
Currently, antifungal drugs catered to treat human diseases are typically sorted into one of two categories: systemic or topical (7). Topical drugs are applied directly to the surface of the skin, most commonly in the form of a cream or spray, whereas systemic drugs target the organism as a whole. The most common systemic antifungal drug used to treat life-threatening infections is amphotericin B; however, amphotericin B is linked to side effects including nausea, vomiting and fever (7). Researchers have developed lipid formulations of this drug in an attempt to decrease nephrotoxicity and other adverse effects. These lipid formulations, however, have not demonstrated much of an advantage, and are much more expensive than the typical amphotericin B. Due to the resounding structural similarities between fungi and mammalian cells, very few myco-specific agents exist as options for highly selective drug treatment (7). The majority of antifungal therapeutics target minute differences in cell membrane composition that distinguish some fungal species from non-fungal cells (8). However, this approach is limited in its application as it is not validated for all known fungal pathogens and is highly susceptible to the emergence of antifungal resistance strains.
The devastating effects of fungal pathogens on both human and non-human hosts have necessitated the search for novel diagnostic and therapeutic approaches against invasive mycoses and drug-resistant phenotypes. To address this concern, the present study focuses on intracellular translational machinery in an attempt to identify a highly conserved pathway in lower order eukaryotic organisms that may serve as a potential drug target. One of the driving forces behind this research is the search for such a molecule that is active and necessary for life in lower order eukaryotes, including pathogenic fungi, but is absent or functionally inactive in higher order eukaryotes - specifically the hosts of fungal infection. The answer lies in the central dogma of biology.
The central dogma delineates a very straightforward, albeit complex process that permits living organisms to produce proteins necessary for life using a universal genetic code. More commonly known as protein synthesis, the conversion of DNA to mRNA to polypeptide is comprised of two major steps: transcription and translation (9). Transcription is the process by which mRNA is synthesized by RNA polymerase using genomic DNA as a template. Assisted by additional transcription factors, RNA polymerase binds to a promoter region on the template strand and reads the nucleotide sequence in the 3’ to 5’ direction, synthesizing the mRNA strand in the 5’ to 3’ direction (9). Posttranscriptional modifications to the mRNA transcript include the addition of a 5’ guanosine cap and the polymerization of numerous adenines onto the 3’ tail, allowing the transcript to escape degradation by RNases while it is translated in the cytosol (9).
The translation stage of eukaryotic protein synthesis involves three major steps: initiation, elongation, and termination. During initiation, a eukaryotic ribosome dissociates into two subunits denoted 40S and 60S (10). Initiation Factor 3 (IF3) then binds to an initiator tRNA carrying a methionine and the anticodon loop sequence UAC, creating a complex which binds to the 40S ribosome subunit. Initiation Factor 4 (IF4) then binds to the 5’ end of mRNA, guiding the 40S subunit with the initiator RNA and associated IF3 to the mRNA. The complex travels along the mRNA until it reaches the AUG start codon, and upon annealing of the anticodon to the codon, the 60S subunit is signalled to localize to the transcript-subunit complex and start elongation (10). Elongation begins when an acetylated tRNA molecule, through recognition of the complementary mRNA codon, binds to the A site (aminoacyl site) of the ribosome complex (11). The tRNA in the A site carries an amino acid which it then attaches to the polypeptide chain carried in the P site (peptidyl site). Elongation factors assist the tRNA in the A site to move to the P site, where it will adopt the growing polypeptide chain (11). The tRNA molecule in the P site then releases its polypeptide chain and moves to the E site (exit site), where the deacetylated tRNA will be released from the ribosome complex into the cytosol, allowing it to restart the process with a new amino acid. Termination of translation occurs when the ribosome reaches a stop codon such as UAG, termination proteins bind to the ribosome, and the polypeptide chain is released (11).
A critical component of translation in eukaryotes is the involvement of various eukaryotic elongation factors, which assist the tRNA molecules in moving between the ribosomal binding sites (Fig. 1). The roles of different elongation factors, however, are not equally conserved among different eukaryotic species, making them a particularly interesting object of study. One such elongation factor, eukaryotic elongation factor 3 (eEF3), is present and active exclusively in lower order eukaryotic cells (12). eEF3 is responsible for transporting deacetylated tRNA out of the E site, allowing the subsequent tRNA molecule to move from the P site to the E site. This action is pivotal to the continuation of the elongation phase of translation as it continuously frees the E site, and thus the P site and the A site, once the tRNA is ready to exit the ribosome complex, allowing new, charged tRNAs to bind the complex and continue synthesis of the polypeptide (12).
Figure 1 | Various roles of Eukaryotic Elongation Factors in translation. The process of translation in eukaryotes is facilitated by a class of proteins known as eukaryotic elongation factors (eEFs). eEF3 hydrolyzes ATP to remove deacylated tRNA from the E site of the ribosome-mRNA complex, allowing subsequent tRNAs to move into their appropriate locations and continue the synthesis of the polypeptide product (12, 13). It has been further suggested that eEF3 plays a direct role in recruiting aminoacetylated tRNAs to the A site of the ribosome, allowing elongation to continue (12, 13).
The structure and function of eukaryotic elongation factor 3 (eEF3) in S. cerevisiae has been studied in great depth, and prior research has established a sound foundation on which the present study is based. The interaction of eEF3 with deacetylated and aminoacylated tRNAs is critical to the proper progression of elongation (12). eEF3 not only removes deacylated tRNAs from the ribosomal E site, but also recruits charged tRNAs localized near the site of translation to the A site, where they are annealed to complementary mRNA codons. This multifaceted activity of eEF3 is mediated by various conformational changes made possible by ATP hydrolysis, exposing different functional domains and allowing eEF3 to interact with a host of targets (12). Recognizing the complexity of the protein, prior researchers have presented a crystal structure of eEF3 in S. cerevisiae and have mapped the various motifs to their respective amino acid regions in the original, pre-functional polypeptide (Fig. 2). This information gives significant insight into the activity of the translation factor in vivo and its various mechanisms of action (12).
Figure 2 | Structures of S. cerevisiae eEF3 amino acid sequence and tertiary protein (12). a, Schematic representation of functional domains mapped to regions on primary amino acid sequence (12). b, Crystal structure of eEF3 bound to ADP in S. cerevisiae (12). Colored regions from Fig. 2a corresponding colored domains in tertiary structure. It is important to note that ABC1 and ABC2 are ATP binding cassettes, which allow eEF3 to hydrolyze ATP and make conformational changes. The HEAT, 4HB, and Chromo motifs interact with rRNA and tRNAs to facilitate elongation.
eEF3 was first observed in S. cerevisiae and has since been discovered as an active component of various other species (14). Under cursory observation of eEF3’s role in lower order eukaryotes, it would seem logical to conclude that the function of the protein is so critical to eukaryotic translation and, by proxy, to the existence of eukaryotic life forms that it must be conserved ubiquitously throughout the domain. However, simple protein analysis has revealed the startling reality that eEF3 is only present in single-celled eukaryotes (15). The absence of eEF3 from higher order eukaryotes contributes immensely to its potential to serve as a drug target for potent, broad-spectrum antifungal agents. This is primarily because administering an eEF3 inhibitor via a systemic drug delivery system would interfere with a fundamental life process in single-celled fungal pathogens with predictably lethal consequences without affecting translational activities in the cells of the host.
However, the eEF3 gene varies greatly between unicellular eukaryotic species, even those within the same genus (15). This raises an alarming question regarding the viability of eEF3 as a potential drug target for broad spectrum antifungal agents: are the specific roles of eEF3 in translation conserved throughout pathogenic fungi, and if so, is the structural conservation of eEF3 sufficient to allow researchers to design a drug targeting a functional motif that, if blocked, will render eEF3 nonfunctional in all pathogenic species?
The purpose of this study is to provide relevant, preliminary insights and to open the door to an avenue of research that we hope will one day provide the answer to that question. As representative models of the evolution of eEF3 in fungi and fungi-like species, the P. infestans eEF3 gene and the S. cerevisiae eEF3 gene are being compared. A quick alignment of their respective amino acid sequences using the NCBI Basic Local Alignment Search Tool (BLAST) revealed a 47% identity conservation and a 63% positive (physicochemical property) conservation. Furthermore, it identified the specific regions in the amino acid sequence which were most closely maintained between the two species. Applying this information in conjunction with the mapping of the amino acid sequence of S. cerevisiae eEF3 to its ultimate motifs in the tertiary structure (Fig. 2) permits us to make an educated guess regarding the conservation of functional domains between P. infestans eEF3 and S. cerevisiae eEF3.
Taking into account the high accuracy alignment, especially in regions encoding for critical functional domains like the ATP binding site, it can be hypothesized that the function of eEF3 is conserved between P. infestans and S. cerevisiae, despite significant structural differences. Results supporting our hypothesis would indicate more specifically the most necessary motifs within the eEF3 structure that contribute to maintenance of its activity in vitro. Our hope is that the results of this study provide significant implications for the appropriation of eEF3 as a viable drug target for antifungal agents with minimal toxicity to the host.
EXPERIMENTAL THEORY
The synthesis of recombinant DNA is essential to this study’s analysis of eEF3 functionality in both P. infestans and S. cerevisiae. In order to determine whether or not eEF3 holds the potential to become a target for antifungal infection drugs, a molecularly engineered plasmid must be introduced into S. cerevisiae. The basic procedure used to engineer this vector is to amplify an empty plasmid marked with the LEU2 gene by introducing it into Escherichia coli. This transformation is more formally known as the Gibson Assembly process. In this particular experiment, this process synthesizes DNA plasmids necessary to transform empty gel purified plasmids into a vector that incorporates P. infestans’ eEF3 gene, resulting in the desired vector. The plasmid will be extracted and will undergo electrophoresis to confirm that the preferred vector has indeed been amplified. This process is then going to be repeated, with the added step of bacterial transformation. This transformation results in a plasmid engineered to contain P. infestans’ eEF3 gene with a LEU2 marker (16).
Gel electrophoresis is a useful method in analyzing the plasmid deoxyribonucleic acid essential to this experiment. The procedure allows scientists to detect and preliminarily characterize DNA. This process begins with the insertion of the dyed plasmids of interest into a porous 1% agarose gel. When an electric field is applied to this gel, the fragments of DNA migrate at a rate related inversely to their molecular weights (17). In this experiment, gel electrophoresis will be used to confirm that the plasmid has been isolated from E. coli, utilizing the method’s ability to separate DNA from other components. It will then be used again to determine if the plasmid does, as intended, take up the P. infestans eEF3 gene by comparing the results to an expected model.
Bacterial transformation is a process involved in the horizontal transfer of genetic material, and is integral to this experiment, for it allows the experimenters to amplify, alter, and mark genetic material (18). Transformation refers to a cell’s ability to uptake exogenous DNA and incorporate that DNA into its own genetic material (18). In this experiment, E. coli will be transformed with the recombinant plasmid containing a P. infestans eEF3 gene in order to promote amplification. After amplification, S. cerevisiae cells will be transformed with the recombinant plasmid.