Identification and prioritization of macrolide resistance genes with hypothetical annotation in Streptococcus pneumoniae
Blue Goad* and Laura Harris
Davenport University, Lansing MI, *Undergraduate researcher
Macrolide resistant Streptococcus pneumoniae infections have limited treatment options. While some resistance mechanisms are well established, ample understanding is limited by incomplete genome annotation (hypothetical genes). Some hypothetical genes encode a domain of unknown function (DUF), a conserved protein domain with uncharacterized function. Here, we identify and confirm macrolide resistance genes. We further explore DUFs from macrolide resistance hypothetical genes to prioritize them for experimental characterization.We found gene similarities between two macrolide resistance gene signatures from untreated and either erythromycin- or spiramycin-treated resistant Streptococcus pneumoniae. We confirmed the association of these gene sets with macrolide resistance through comparison to gene signatures from (i) second erythromycin resistant Streptococcus pneumoniae strain, and (ii) erythromycin-treated sensitive Streptococcus pneumoniae strain, both from non-overlapping data sets. Examination into which cellular processes these macrolide resistance genes belong found connections to known resistance mechanisms such as increased amino acid biosynthesis and efflux genes, and decreased ribonucleotide biosynthesis genes, highlighting the predictive ability of the method used. 22 genes had hypothetical annotation with 10 DUFs associated with macrolide resistance. DUF characterization could uncover novel co-therapies that restore macrolide efficacy across multiple macrolide resistant species. Application of the methods to other antibiotic resistances could revolutionize treatment of resistant infections.
Testing a Predicted Cytoskeleton Regulatory Gene for a Role in Filamentation
Hannah R. Kirshman* and Ian Cleary
Grand Valley State University, Allendale, MI, *Undergraduate researcher
Candida albicans is a member of the normal microbiota found within the body. However, for individuals with compromised immune systems, a C. albicans infection of their organ systems can be fatal. An important virulence factor is the ability to filament. The aim of our experiment was to examine the role of the uncharacterized gene orf19.2304 in C. albicans filamentous growth. We have constructed strains over-expressing this gene and are testing whether this leads to a changes in filamentous growth. Our results so far indicate that over-expressing this gene can affect cellular growth in some conditions, including an increase in flocculant growth.
Up-regulation of vitamin B pathways in antibiotic resistant Streptococcus pneumoniae
S. P. Harris1,2, W. DeHaven3,4 and Laura Harris4.5
1Bath Middle School, 2Lansing Community College GATE, 3Undergraduate 4Davenport University, Lansing MI, 5Science Faculty
Streptococcus pneumoniae (S. pneumoniae) infections can be life-threatening and are a major clinical concern. S. pneumoniae infections are usually treated with protein synthesis inhibitors (i.e. macrolides or aminoglycosides). Unfortunately, S. pneumoniae infections that cannot be treated with protein synthesis inhibitors exist and our understanding of resistance processes is incomplete, in part because of hypothetical genes. Prior work in Staphylococcus aureus showed detection of resistance processes improved by comparing pathway signatures (lists of pathways ranked by activity). In this project, we use pathway signatures created from comparing gene signatures from S. pneumoniae untreated and treated with protein synthesis inhibitors to 116 PATRIC pathways. We compared spiramycin and erythromycin pathway signatures to identify a set of 14 up-regulated pathways similar between the signatures (p=0.042). We found these 14 pathways also up-regulated in kanamycin treated S. pneumoniae cultures (p=0.021). Four of those pathways relate to vitamin B (folate biosynthesis, riboflavin metabolism, and 2 vitamin B6 metabolism pathways). From this we predicted that lowering vitamin B pathway activity reduces resistance to protein synthesis inhibitors. To test this, we examined minimum inhibitory concentration of a S. pneumoniae culture treated with streptomycin with or without vitamin B inhibitors and found treatment with trimethoprim to be effective, reducing concentration from 16 to 4mg/L. While validation is needed, this preliminary evidence indicates that co-therapy options with trimethoprim may overcome resistance to protein synthesis inhibitors in S. pneumoniae in patients.
Characterizing proteolysis of a Developmental Transcriptional Regulator in Myxococcus xanthus
Anna Gretzinger, Patrick T. McLaughlin, Penelope I. Higgs
Wayne State University, MI
Most bacteria live in multicellular communities in which cells are imbedded in an extracellular polymeric substance (i.e. biofilms). Many biofilms have detrimental effects on the environment and on human health due to their incredible resistance to various treatments. The mechanisms regulating biofilm formation need to be studied to design more effective treatments. Myxococcus xanthus is an environmental bacterium that forms a specialized biofilm. Upon nutrient starvation, M. xanthus cells undergo biofilm formation and differentiates into spatially distinct cell fates: 1) persister-like cells, 2) fruiting bodies with spores, 3) programmed cell death. We hypothesize cell fate segregation is largely governed by MrpC, a transcription factor that is differentially accumulated in the different cell types. MrpC is regulated transcriptionally and post-transcriptionally, but the mechanism that governs differential accumulation is currently unknown. One candidate that may dictate the differential accumulation of MrpC is the Esp signaling system, which stimulates an unknown protease to turnover MrpC during biofilm formation. While it is known that MrpC is subject to regulated proteolysis, the proteolytic recognition sequence in MrpC remains unknown. We designed a genetic screen to identify sequences within MrpC that play a role in proteolytic turnover. First, mrpC was subjected to random mutagenesis through error prone PCR to create a mutant library. The mutant mrpC library will then be transformed into M. xanthus and screened to identify clones that are resistant to proteolysis. We are currently optimizing the genetic screen to select for mutant defective in Esp-mediated proteolytic turnover of MrpC.
Elucidating the Mechanism of Host Fatty Acid Tolerance in Metabolically Altered Staphylococcus aureus
Phillip C. Delekta* and Neal D. Hammer
Michigan State University, East Lansing, MI, 48824
Antibiotic-resistant Staphylococcus aureus is the leading cause of healthcare-associated infections. An expansive tissue tropism and ability to withstand antimicrobial therapies make S. aureus a significant clinical challenge. S. aureus proliferates in diverse host tissues using a versatile metabolism that fluctuates between aerobic respiration and fermentation. Aerobic respiration represents another layer of metabolic flexibility as a branched respiratory chain composed of two terminal oxidases, QoxABCD and CydAB, catalyze the terminal reduction of oxygen to water. CydAB and QoxABCD are required to colonize the heart and liver, respectively, underscoring the importance of aerobic respiration to pathogenesis. These facts support the hypothesis that the terminal oxidases have unique biophysical properties that allow them to function in distinct environments. To define these properties, we seek to identity host-derived factors that influence the function of each terminal oxidase, filling considerable gaps in our knowledge of staphylococcal pathogenesis. In this study, we found that genes within the exogenous fatty acid incorporation pathway are significantly downregulated in a qoxA mutant compared to wildtype. Exogenous fatty acid incorporation is utilized by S. aureus to synthesize phospholipids and has been proposed to play a role in detoxifying the membrane from harmful concentrations of free fatty acids. We observe that saturated fatty acids, which are innocuous to aerobically respiring cells, severely limit the proliferation of respiration-arrested cells. Our data and the fact that the liver is a significant host reservoir of free fatty acids, support the hypothesis that host-derived fatty acids represent an extrinsic challenge for respiration-impaired S. aureus during infection. Specifically, we reason that the aberrant accumulation of free fatty acids leads to toxicity in metabolically-restricted S. aureus. This research is the first to link antimicrobial activities of fatty acids to the metabolic status of a bacterial pathogen and establishes respiration as a fatty acid-resistance determinant.
Effect of Serotype and CRISPR Arrays on Streptococcus mutans Biofilm Formation
Hanaa Saleh*, Rita Salim*, Joshua J. Thomson
University of Detroit Mercy, MI
Dental caries, also referred to as tooth decay or cavities, is caused by erosion of enamel on the tooth surface and without intervention can eventually lead to tooth loss. One of the main factors leading to caries is the accumulation of bacterial biofilm, also called plaque. The gram positive bacterium, Streptococcus mutans, is highly correlated with the initiation of dental caries due to its ability to produce a sticky matrix of extracellular polysaccharides (EPS) while growing in the presence of sugars. Additionally, S. mutans can metabolize dietary sugars results in the production of lactic acid which can directly degrade enamel and lead to tooth decay. Four serotypes of S. mutans exist based on extracellular characteristics: c, e, f, and k. Almost 80% of S. mutans found in the oral cavity are serotype c, while serotypes e and f are not as prevalent, yet still contribute towards dental disease. Moreover, serotype k is commonly associated with cardiovascular disease. The objective of this study is to determine if serotype influences the ability of S. mutans to form biofilm in vitro. Additionally, the CRISPR-Cas system of bacterial defense against foreign DNA elements has recently been shown to correlate with increased virulence in S. mutans. Therefore, we propose to determine the prevalence of CRISPR arrays in different serotypes of S. mutans and the correlation with biofilm formation. These studies will enhance our understanding of the impact of S. mutans serotype and CRISPR presence on disease-causing ability persistence/prevalence in the population, as well as the possible resistance to foreign genetic elements.
Maximizing Efficiency of Training Undergraduate Students in a Molecular Biology Research Lab Using Polymerase Chain Reaction
Payal M. Patel*, Sarah C. Plecha, and Joshua J. Thomson
University of Detroit Mercy, MI
Common barriers to productively facilitating scientific research with a full-time undergraduate student may include lack of time for the student to consistently conduct research and variations in scientific principle comprehension. Therefore, maximizing the efficiency of laboratory training is essential for the process of developing a student’s ability to conduct benchtop science while learning to effectively and critically analyze a research project. The success of the training period is of high importance to foster a mutually beneficial partnership between faculty and student. The goal of this project was to have an experienced undergraduate lab member develop a series of regimented experiments specifically designed to efficiently train new undergraduate research students in the foundational principles and techniques of molecular biology regardless of prior lab experience. The project focused on determining the most efficient sequence of instruction and experimentation. The teaching design was based on one fundamental molecular biology experiment: Polymerase Chain Reaction (PCR). Using this technique as the basis of introduction into the research setting, the new undergraduate members were taught how to appropriately write in a lab notebook, make solutions, learn equations and unit conversions, all while being trained in the use of basic lab equipment. Through this process we aspired to determine the most appropriate sequence of instruction, experimental techniques, and reinforcement needed for an incoming student to successfully investigate research questions, regardless of their previous research experience or year of study in an undergraduate program.
Homology driven interaction between viral protein ICP0 and host restrictive factor PML I in HSV-1 infection
Yi Zheng, Behdokht JanFada*, Ayette Dourra, and Haidong Gu
Department of Biological Science, Wayne State University
Infected cell protein 0 (ICP0) is one of the immediate early proteins of herpes simplex virus-1 (HSV-1) that is mainly responsible for counteracting host antiviral defense mechanisms. Upon HSV-1 infection, several cellular antiviral factors converge at the incoming viral genome, attempting to silence viral genes. Simultaneously, ICP0, which is an E3 ubiquitin ligase, targets some of these host restrictive factors for proteasomal degradation to alleviate host defense and enhance viral gene expression. Promyelocytic leukemia protein (PML) is an important cellular protein involved in several cellular pathways including antiviral defense that gets degraded by ICP0 in this process. Previously, we reported that ICP0 degrades PML isoforms differentially and a bipartite domain located upstream and downstream of the RING-type E3 ubiquitin ligase domain of ICP0 mediates its interaction with PML I. In the current study, we report that (i) either one arm of the ICP0 bipartite PML I-interacting domain is sufficient for PML I interaction, ubiquitination and degradation, whereas deletion of both arms of the bipartite domain hinders binding, ubiquitination and consequently the degradation of PMLI. (ii) The two arms of bipartite PML I-interacting domain in ICP0 share sequence similarity, which is necessary and sufficient for PML I interaction and ubiquitination. (iii) The homologous sequences in the two arms of bipartite PML I-interacting domain also share sequence similarity to the C-terminus of PML I. (iv) Deletion of PML I C-terminus inhibits the ICP0-PML I interaction. These results suggest that ICP0 may have evolved to partially mimic PML I sequence. This possible adaptation enables ICP0 to target PMLI specifically and regulate its ubiquitin-mediated degradation.
Examining the Growth of C. albicans Filamentous Mutants In Embedded Conditions
Curtis M. Mack*, Ian A. Cleary, Grand Valley State University, Allendale MI
Candida albicans is an opportunistic fugal pathogen and a member of the normal human microbiota. Various environmental conditions stimulate changes in cellular morphology between round yeast cells and filamentous hyphae and pseudohyphae. One such stimulus is growth embedded in solid media. Examination of scientific literature revealed that several mutant strains known to grow filamentously under yeast conditions had not been tested in embedded growth. One example is a strain over-expressing the transcription factor SFL2. Our initial goal was to close this gap in our knowledge of these strains. Interestingly, our experiments revealed that over-expression of SFL2 was also able to overcome the repression of filament induction that results from over-expression of the repressor NRG1. This effect appears to be specific to embedded conditions, since in several liquid media tested simultaneous over-expression of SFL2 and NRG1 produces only elongated yeast cells. We are continuing to examine this question by growing our over-expression strain in other conditions known to induce filamentous growth.
Characterization of Putative Salmonella enterica Serovar Typhimurium Virulence Genes
Luke Rozema and Aaron Baxter
*Best undergrad poster*
Salmonella enterica serovar Typhimurium is a Gram-negative pathogen responsible for a large percentage of food-borne illnesses in the US each year. Upon entry, S. Typhimurium invades the epithelial cells and Peyer’s patches of the small intestine via a type III secretion system. Invasion requires a region of the genome known as Salmonella Pathogenicity Island 1 (SPI-1), and intracellular survival requiring Salmonella Pathogenicity Island 2 (SPI-2). Central to the upregulation of SPI-1 is the gene hilA, which is both positively and negatively regulated via a series of proteins that senses the bacteria’s current environment. One of the major negative regulators of hilA is the gene hilE. Previously, it was found that the genomic area surrounding hilE carries key characteristics commonly seen in pathogenicity islands. The purpose of this research is to characterize the effects two polar mutations, Δ4501 and Δ4502, located in this region, have on virulence as part of a continuing study of this pathogenicity-island-like region. Current work was done by measuring hilA expression under inducing and non-inducing conditions via beta-galactosidase assays in mutant and wild type strains. Further, motility and twitching assays were performed on both mutant and wild type cells. Results indicate no significant change in expression of hilA in the absence of Δ4501 and Δ4502 in both inducing and non-inducing conditions. Results from the twitching and motility assays are currently in progress. Thus far, the results indicate that Δ4501 and Δ4502 have insignificant effects on S. Typhimurium virulence. Future work will focus on the effects Δ4501 and Δ4502 have on invasion through invasion assays, the characterization of expression of other known regulators, macrophage survival and adherence.
Evaluating Bacterial Microcompartment Shell Proteins as Building Blocks for Self-Assembly of Predictable Nano-scaffolds
Wright Jacob K.1,2*, Young Eric J.1,2, Ducat Daniel C.1,2
1Michigan State University, East Lansing, Michigan 48823
2MSU-DOE Plant Research Lab, East Lansing, Michigan 48823
*Best undergrad poster*
At the nanoscale, cellular pathways are frequently organized upon scaffolding complexes that physically co-localize related components. Natural Nano-scaffolds act to increase pathway flux and fidelity, while also insulating the pathway from unwanted side-reactions and reducing accumulation of pathway intermediates. An outstanding goal for biological engineers is to recapitulate the advantages of natural scaffolds to improve the performance of heterologously installed pathways. Our approach is to use a naturally-occurring protein that self-assembles into defined intracellular structures (bacterial microcompartment shell proteins) as “building blocks” for the assembly of new, user-defined Nano-structures. Here, we show that heterologous expression of these self-assembling building blocks can be used to form macromolecular protein assemblies both in vitro and in vivo. We describe our efforts to attach functionalizing adaptor domains to these protein building blocks in order to allow for programmable protein-protein interactions on the surface of these Nano-scaffolds. Within the context of this broader goal, my project seeks to understand how attaching adaptor domains influences the self-assembly characteristics of shell proteins under a range of different physiological temperatures and pH ranges. By analyzing their ability to continue to form higher order structures, even with the addition of these functional adaptor domains, we aim to characterize a promising biomaterial that could be used as a tool in future bioengineering purposes.
Testing the Role of an Uncharacterized Gene in Candida albicans Filamentation
Eric Vaikevicius and Ian Cleary
Grand Valley State University
The fungus Candida albicans is in most human intestines and mucous membranes, and it typically does not cause disease in healthy individuals. However, with changes in the host, such as in immune compromised individuals, disease can arise due to C. albicans ability to change cellular shape between round yeast and elongated filaments. In C. albicans cellular shape is regulated by a genetic program. The function of many genes associated with filamentation is unknown. Our goal is to understand the particular contribution to this process of one gene, FGR16. FGR16 overexpression caused increased biofilm growth in some media, and increased clumping in some broth cultures. Changes in plate growth included reduced filamentous growth, as well as increased invasion. These results confirm that FGR16 overexpression affects cell shape. We are testing how well this gene’s function is maintained between organisms; by overexpressing homologs in C. albicans and looking for effects on cell shape.
Nutrient Shifts Affect Adherence Ability and Cell-Surface Properties of Lactobacillus rhamnosus GG
Rielinger, Amanda L* and Clemans, Daniel
Eastern Michigan University
*Best undergrad poster*
Lactobacillus rhamnosus GG (LGG) is one of the best studied probiotic organisms. The ability of probiotics to adhere to other microorganisms and the intestinal epithelium is thought to play a major role in their protective functions. Coaggregation is thought to be an important mechanism for biofilm formation by microorganisms. Recent studies from our lab suggest that there is extensive coaggregation between human gut microbes. It is hypothesized that nutritional variation may affect the ability of LGG to coaggregate and form biofilms, and thus affect its probiotic characteristics and ability to colonize the gastrointestinal tract. The goals of this study were to examine the ways in which nutrient variation affects coaggregation between LGG and other intestinal species; biofilm formation between LGG and B. thetaiotaomicron wild-type or B. thetaiotaomicron capsule-deficient mutant in vitro; and LGG cell-surface properties and structures. Lactobacillus rhamnosus GG was cultured anaerobically in different formulations of tryptone, yeast extract, and glucose (TYG) medium in order to simulate nutritional shifts Coaggregation ability between LGG and 22 Bacteroides spp. and Parabacteroides spp. and 8 mutant capsular types of Bacteroides thetaiotaomicron were tested. There was a significant difference (p<0.001) in coaggregation when compared by media type. Twenty-four-hour static biofilm assays were conducted in plastic 24-well microplates, and the optical density of the stained biofilms was determined in order to quantify the level of adherence. Biofilm formation of LGG with either the B. thetaiotaomicron wild-type strain or the B. thetaiotaomicron capsule-deficient mutant strain was observed to significantly differ by media type (p<0.001). Hydrophobicity values of LGG grown under differing nutrient conditions were also examined, and were found to significantly differ by media type (p<0.001). These results suggest that different nutrient conditions may enhance the ability of LGG to act as a successful probiotic.
Carbohydrate-recognizing Regulators from Clostridium thermocellum
Bohr*, Margaret, C. and Blumer-Schuette, Sara, E.
Orthogonal regulation is important for the field of synthetic biology in order to reduce cross-talk between endogenous and recombinant regulators in engineered microbes. My goal for this project was to determine the feasibility of using a carbohydrate sensing regulatory system from the thermophilic bacterium Clostridium thermocellum in Bacillus subtilis. Both Firmicutes evolved from a common ancestor; however, this regulatory system evolved in C. thermocellum and not in B. subtilis, making it a candidate for orthogonal regulation. If successful, this system will be used to construct a biosensor microorganism, using B. subtilis, that will respond to plant-derived carbohydrates such as cellulose, xylan, and pectin, by producing green fluorescent protein. C. thermocellum anti-sigma and sigma factors will be cloned into a replicating B. subtilis plasmid using isothermal assembly, while the cognate promoter will be cloned in front of gfp on a non-replicating plasmid using Golden Gate assembly. We modified an existing non-replicating plasmid for use in Golden Gate cloning, including replacing the strong lactose-inducible promoter with a lacZ cassette flanked by BsaI sites and using site directed mutagenesis to remove a BsaI recognition site from the β-lactamase gene. Both plasmids were transformed into supercompetent B. subtilis SCK6. Using a spectrofluorimeter, we quantified levels of non-target expression from the C. thermocellum promoter in B. subtilis. Constructed plasmids and engineered B. subtilis strains will ultimately be used to construct biosensors for use in directed evolution of carbohydrate recognizing proteins.
Effects of Orf19.2302 on Growth Under Various Metal and pH Conditions in Candida albicans
Caiden J. Walter
Grand Valley State University
The gene orf19.2302 is upregulated during filamentous growth in the fungal pathogen Candida albicans. This gene encodes a protein of unknown function and cellular localization. Based on sequence similarities to proteins in other fungi, the protein is a predicted to be a permease and is thought to be a possible transporter of cations between the endoplasmic reticulum and the cytoplasm. To test its function in cation transport we have examined the growth of a deletion strain in the presence of metal stressors. In the presence of the divalent cation chelator EDTA, deletion or over-expression of this gene had no effect. Growth on media containing iron or magnesium at neutral or acidic pH was also unaffected. However, growth in the presence of zinc or copper at acidic pH was altered. In acidic copper medium the deletion strain was more resistant to metal stress, whereas in acidic zinc medium an over-expression strain was more sensitive. These results suggest that this protein is involved in metal ion transport in C. albicans and is required for appropriate stress responses in some growth conditions.
Fungal Pathogen Candida Albicans GAL10 Gene is Imperative to Biofilm Formation
Diana McMahon, Angelina Antonyan, Alex Jackman, Marcellio Shammami, Nikol Shllaku, Jonathan S. Finkel Ph.D.
Department of Biology, University of Detroit Mercy
Infections by the opportunistic fungal pathogen Candida albicans is a critical health issue with a ~30% mortality rate and few available antifungal drugs for treatment. While C. albicans is commensal in the human gut, it can become virulent and form biofilms in the body on implanted devices such as artificial joints, catheters, and pacemakers, especially in immunocompromised individuals. The cell wall of C. albicans is the outermost component of a fungal cell and is comprised of cell wall proteins along with chitin and glucans. Cell wall proteins are of great importance to the survival of the C. albicans as they are the first to encounter the cell’s environment and report the external environmental condition including whether the location is an appropriate site for cellular adherence. Once the yeast cells have adhered, a biofilm can develop. In this study we identified from a screen of cell wall insertion mutant that the gene GAL10 is essential for biofilm formation. Here we report the results of study of two mutant isolates of gal10 for their ability to form biofilms in anaerobic conditions and aerobic conditions, and their sensitivities to different cell wall perturbing agents in an attempt to identify the cause of the biofilm defect. Our results show that GAL10 has an essential role in the ability of C. albicans to form a biofilm, and that one possible role in biofilm formation is GAL10 important function of galactose metabolism.
Antibiotic Resistant Pseudomonas aeruginosa and Phage on Blood and in Serum in Undergraduate Research
James F. Graves* (F/S) and Cameron M. Johns (UG)
Department of Biology, University of Detroit Mercy
Pseudomonas aeruginosa is an antibiotic resistant bacterium that is known to cause infections of blood. This study examined growth of P. aeruginosa ATCC 13388 with a phage isolated from stream sediment in presence of blood and blood serum. An antibiogram made on Luria-Bertani (LB) agar revealed that the host strain of P. aeruginosa was able to grow up to the edge of 3 out of 5 different antibiotic discs. The Enterotube II/EnteroPluri-Test, which is a multiple biochemical test system made of 12 test chambers in a series, indicated that the host strain when inoculated in presence or absence of phage was nonfermentative. With prolonged incubation positive reactions for arabinose or glucose were occasionally produced. In the routine test dilution (RTD) assay, to discover the highest titer of phage to give complete lysis, carried out on trypticase soy agar (TSA) with sheep blood, clear zones on lawns of cells on agar for dilutions up through 10 raised to the power of -6 were apparent. This RTD result was the same as observed for the phage with the assay performed on brain heart infusion (BHI) agar without blood. The effect of phage on growth of P. aeruginosa in blood serum was assessed by measurement of culture optical density (OD) with a Klett-Summerson colorimeter. Inclusion of phage at a multiplicity of infection (MOI) of approximately 1.0 resulted in a bacterial culture with OD 35, in contrast to a culture without phage with OD 160 after 6 days. In BHI, in absence of blood factors at an MOI of approximately 100, a culture containing phage with OD 38 in contrast to a culture without phage with OD 138 resulted in 24 h. However, when an MOI of approximately .01 was used the culture with phage was only slightly inhibited. Streak plate cultures after experiments produced survivor colonies that were green from pyocyanin pigment. Development of bacterial biofilm on blood was inhibited by the phage. Growth of the bacteria in blood serum was slow and phage further decreased growth.
Determining the role of mRNA secondary structure on non-Shine-Dalgarno translation initiation using DMS-seq
Nisansala Muthunayake*, Mohammed Bharmal, Jared M. Schrader
Department of Biological Science, Wayne State University
Translation initiation is an essential process in which ribosomes engage the mRNA at the start codon. In E. coli, translation initiation at the start codon is facilitated by base pairing of the ribosome and a Shine-Dalgarno (SD) site in the mRNA. Surprisingly, recent genome surveys revealed that only half of bacterial genes contain SD sequences, with some bacterial species having as few as 8% of their genes encoded with upstream SD sequences. To understand the mechanism(s) of non-SD translation initiation, we utilize Caulobacter crescentus, an α-proteobacterium that is highly adapted to non-SD initiation. We hypothesize that mRNA folding plays a major role in non-SD mRNA translation initiation. To test this hypothesis, we used computational analysis of the mRNA folding stability in C. crescentus translation initiation regions which revealed that the mRNA structures are typically less stable around the start codon. However, RNA secondary structure prediction is subject to biases, therefore we will use experimental genome-wide secondary structure probing approaches to verify the low secondary structure content at start codons. Dimethyl sulfate (DMS) rapidly and specifically modifies unpaired adenines and cytosines which blocks reverse transcriptase at the site of modification. The combination of DMS modifications with next generation sequencing methods (DMS-seq) will be used to map genome wide mRNA structure in C. crescentus. The findings of our DMS-seq experiments will provide further evidences for the existence of mRNA structure dependent translation initiation mechanism in C. crescentus. In our preliminary experiments we used the DMS probing technique to validate the secondary structure of C. crescentus 5S ribosomal RNA and will present progress on adaptation of DMS-seq to C. crescentus mRNAs.
Role of mRNA Folding in non-Shine-Dalgarno Translation Initiation
Mohammed-Husain M Bharmal, Jared M. Schrader
Department of Biological Science, Wayne State University
Translation initiation is an essential process in which ribosomes engage the mRNA at the start codon. In E. coli, translation initiation at the start codon is facilitated by base pairing of the ribosome and a Shine-Dalgarno (SD) site in the mRNA. Surprisingly, recent genome surveys revealed that only half of bacterial genes contain SD sequences, with some bacterial species having as few as 8% of their genes encoded with upstream SD sequences. To understand the mechanism(s) of non-SD translation initiation, we utilize Caulobacter crescentus, an α-proteobacterium that is highly adapted to non-SD initiation. We hypothesize that mRNA folding plays a major role in non-SD mRNA translation initiation. To test this hypothesis, we used computational analysis of the mRNA folding stability in C. crescentus translation initiation regions which revealed that the mRNA structures are typically less stable around the start codon than at AUG codons within the coding sequence. To test if the start codon region’s structural stability is functionally relevant to initiation we made different mutations in the 5’ UTR’s of SD and non-SD mRNAs that alter the mRNA’s structural stability and assayed their translation using YFP. Mutations destabilizing secondary structures surrounding the start codon increase translation, while mutations stabilizing the secondary structure surrounding the start codon lower translation. These data support a model in which the C. crescentus ribosome initiates preferentially on single stranded AUG codons. Interestingly, the leaderless mRNAs which completely lack a 5’ UTR, show a high amount of YFP production as compared to SD or non-SD mRNAs, suggesting that C. crescentus is also highly adapted for leaderless mRNA translation.
α proteobacterial degradosomes assemble liquid-liquid phase separated RNP bodies
Nadra Al-Husini*1, Dylan T. Tomares2, W. Seth Childers2, and Jared M. Schrader1
1 Wayne State University, Detroit, MI,2University of Pittsburgh, Pittsburgh, PA
Bacteria have distinct challenges to organize their cellular pathways as they generally lack membrane-bound organelles. In eukaryotes, membraneless organelles called biomolecular condensates provide distinct liquid-liquid phase separated structures that organize cellular components. We discovered that RNase E, the rate-limiting enzyme controlling mRNA decay in bacteria, assembles biomolecular condensates termed Bacterial RNP-bodies (BR-bodies) with similar properties to eukaryotic P-bodies and stress granules. RNase E requires RNA to assemble a BR-body, and disassembly requires RNA cleavage, suggesting BR-bodies provide localized sites of RNA degradation. The intrinsically disordered C-terminal domain of RNase E is necessary and sufficient to assemble the core of the BR-body and other alpha-proteobacterial RNase E proteins also assemble BR-bodies. Ability to form a condensate stimulates the initial endonucleolytic cleavage of mRNA, while recruitment of exoribonucleases into the BR-body stimulates subsequent decay of cleaved mRNA fragments. BR-bodies therefore provide an effective strategy for the subcellular organization of biochemical pathways in bacterial cells without the use of membrane-bound compartments.
Absolute Measurements of mRNA Translation in C. crescentus Reveal Important Fitness Costs of Vitamin B12 Scavenging
James R. Aretakis*, Alisa Gega, Jared M. Schrader
Department of Biological Science, Wayne State University
Caulobacter crescentus is a model for the bacterial cell cycle which culminates in asymmetric cell division, yet little is known about the absolute levels of protein synthesis of the cellular parts needed to complete the cell cycle. Here we utilize ribosome profiling to provide absolute measurements of mRNA translation of the C. crescentus genome, providing an important resource for the complete elucidation of the cell cycle gene-regulatory program. Analysis of protein synthesis rates revealed ~4.5% of cellular protein synthesis are for genes related to vitamin B12 import (btuB) and B12 independent methionine biosynthesis (metE) when grown in common growth media lacking B12. While its facultative B12 lifestyle provides a fitness advantage in the absence of B12, we find that it provides lower fitness of the cells in the presence of B12, potentially explaining why many Caulobacter species have lost the metE gene and become obligates for B12.
Vitamin B Inhibitors Co-therapy Restores Streptomycin’s Efficacy in Streptococcus agalactiae and Streptococcus pyogenes
Cynthia Konan* and Laura Harris
Davenport University, Lansing MI, *Undergraduate researcher
BACKGROUND: Streptococcus are a serious threat to public health. They can many diseases ranging from minor infections to life threatening conditions. Examples of streptococcus species are: Streptococcus pneumonia (pneumonia), Streptococcus agalactiae (group B), and Streptococcus pyogenes (group A). Macrolides and aminoglycosides inhibit protein synthesis and are common treatments, but resistances exist. Here, we identify the similarities and differences in macrolides and aminoglycoside resistance mechanisms across streptococcal species focusing on Streptococcus agalactiae (group B), and Streptococcus pyogenes. RESULTS: We present a review of Streptococcus agalactiae (group B), and Streptococcus pyogenes resistance to macrolides, aminoglycosides, and trimethoprim-sulfamethoxazole (TMP-SMX). Overall the Streptococcus agalactiae strain used for this experiment is four times more resistant to streptomycin than Streptococcus pyogenes strain. When streptomycin is co-treated with trimethoprim-sulfamethoxazole, streptococcal species’ resistance to streptomycin decreased immensely. CONCLUSION: Effective treatment of resistant S. agalactiae and pyogenes is a growing concern. New classes of drugs, newer formulations of older drugs, combination antibiotic therapy, better oversight of antibiotic usage, and enhanced preventive measures hold promise. Vitamin inhibitors can decrease S. agalactiae and S. pyogenes resistances to macrolides and aminoglycosides and restores drug efficacy. Our macrolide resistance pathway panel could be a useful clinical tool for diagnosing resistant Streptococcus agalactiae (group B), and Streptococcus pyogenes (group A) genes pathways involved in the resistance of these macrolides and aminoglycosides. Moreover, it can provide ideas of therapies to change already resistant strains of bacteria to be susceptible to the same antibiotics.
Thermostable type IV pilus: a 'helping hand' for Caldicellulosiruptor bescii in plant polysaccharide degradation
Khan*, Asma. and Blumer-Schuette, Sara, E.
At the upper thermal limits for biological crystalline cellulose hydrolysis, only one bacterial genus, the Caldicellulosiruptor thrive. Among these, Caldicellulosiruptor bescii exhibits an extraordinary cellulolytic ability which is generally attributed to its modular, multi-functional carbohydrate acting enzymes. Additionally, non-catalytic mechanisms like adherence to lignocellulosic biomass and polysaccharides via proteins like the tāpirins, S-layer proteins, substrate binding proteins or type IV pili (T4P) are likely to increase its cellulolytic efficiency. We identified a putative T4P operon in C. bescii, located close to an upstream glucan degradation locus (GDL), using genome annotation. Interestingly, the T4P from highly cellulolytic Caldicellulosiruptor are evolutionarily divergent from those of weakly cellulolytic species. Based on pilin-like protein domains, transcriptomics and proteomics analysis, we annotated Athe_1880 (PilA), as the major pilin and produced a soluble, recombinant form: rPilA. Using epifluorescence, we showed that PilA is expressed in C. bescii cells. We also quantified PilA expression using immuno dot blots. Maximum PilA expression was seen during growth on xylan. Cell adherence inhibition assays confirmed that PilA inhibits C. bescii cells from attaching to insoluble polysaccharides. However, we saw no direct interaction of PilA with these polysaccharides. Based on the proximity of the T4P operon to the GDL and data from PilA cell adherence inhibition assays we propose that the major C. bescii pilin PilA, and by extension it’s T4P, facilitate cellulose degradation by attachment.
Investigating the Coaggregation of Potential Probiotics Isolated from Fermented Foods
Colvard, Brittney M*, Chan, Lily Z, and Clemans, Daniel
Eastern Michigan University
Probiotic microbes propagate within the human gastrointestinal tract through the formation of biofilms by means of coaggregation with genetically distinct organisms. The aim of this study was to observe the coaggregative abilities of microorganisms isolated from fermented foods with one another and with a panel of representative known probiotic strains and native gut microbes. Twelve unique bacterial and fungal species, including Lactobacillus and Kazachstania, were isolated from fermented foods such as sauerkraut and kimchi and identified using molecular techniques. Using standard coaggregation assays, the interactions of these various microorganisms was investigated. Our results concluded that extensive adhesion among the fermentation isolates occurs, with the yeast species showing the strongest patterns of coaggregation. These results will allow us to further understand how coaggregation plays a role in gut biofilm formation between probiotics from food and normal microbiota.
An Antimicrobial Agent Found in Over-The-Counter Nasal Sprays
Lammers, Carolyn J*; Campbell, Ashley; Klausing, Sydney; Shetron-Rama, Lynne
Eastern Michigan University
*Best undergrad poster*
Over-The-Counter moisturizing nasal sprays were tested to investigate antimicrobial properties. The active ingredient of one spray was tested against various microorganisms commonly found in the nose, including Streptococcus pneumoniae and Methicillin-resistant Staphylococcus aureus (MRSA), were measured by 5 hour time kill, disc diffusion, and biofilm assays. The ingredient shows antimicrobial activity against MRSA, Klebsiella pneumoniae, and S. pneumoniae in 5-hour time kill assays. It also appears to have anti-biofilm implications against S. aureus. These results signify this ingredient as a promising research target against drug-resistant pathogens such as MRSA, which present a growing threat to human health.
An N-Terminal TTSS Motif on a Crp/Fnr Homolog, MrpC, is Essential for the Developmental Program of Myxococcus xanthus
Brooke Feeley*1, Vidhi Bhardwaj2, Patrick McLaughlin1, Penelope I. Higgs1
1Wayne State University, Detroit, MI, 48202
2Max Planck Institute for Terrestrial Microbiology, Marburg, Germany, 35043
Transcriptional regulators of the CRP/Fnr family are generally controlled by binding a small-molecule ligand. MrpC is a CRP homolog found in Myxococcus xanthus, and controls the complex developmental program this soil bacterium undergoes in response to starvation, in which multicellular, spore-filled fruiting bodies are formed. MrpC is a negative autoregulator, activates transcription of target genes needed for development to proceed, and is a key output of a large signaling network in M. xanthus. MrpC is not known to be controlled by binding a ligand, instead, it’s been proposed that it’s regulated in a novel way—by phosphorylation within its unique N-terminus by the Ser/Thr kinase, Pkn14. The N-terminus contains a TTSS putative phosphorylation motif that is essential for MrpC’s function, as a TTSS to AAAA quadruple substitution mutant (mrpCAAAA) fails to develop. Various single or consecutive double alanine substitutions within the TTSS motif allow development to proceed normally, while triple or non-consecutive double substitution mutants develop with variable timing. These data suggest that while only one residue within the TTSS must remain intact to promote development, significant perturbation of the motif leads to a loss of developmental robustness. We have shown that an intact TTSS motif is required for full phosphorylation of MrpC by Pkn14 in vitro. However, in vivo genetic analyses suggest that Pkn14 is not the only MrpC kinase, since a pkn14 kinase-inactive mutant develops normally. We have also shown that mrpCAAAA shows a small reduction in negative autoregulation and fails to properly activate target genes, likely due to ineffective binding to the promoters of mrpC and of target genes, respectively. However, mrpCΔ1-25, which expresses an MrpC isoform lacking the amino 25 residues, including the TTSS motif, is abolished in negative autoregulation but binds DNA with wildtype affinity. We are currently attempting to identify whether the N-terminus of MrpC is needed for protein interactions, the in vivo phosphorylation status of MrpC, and additional MrpC kinases likely belonging to the previously identified signaling network.
SinKM: A Signal Integration System Controlling the Myxococcus xanthus Developmental Program
Glaser, Maike M., Lall, Dave*, and Higgs, Penelope I.
Wayne State University, Detroit, MI, USA
His-Asp phosphorelay (a.k.a. two-component signal transduction) proteins are the predominant mechanism used in most bacteria to control behavior in response to changing environmental conditions. In addition to systems consisting of a simple two-component system utilizing an isolated histidine kinase / response regulator pair, some bacteria are enriched in histidine kinases that serve as signal integration proteins; these kinases are usually characterized by non-canonical domain architecture and the responses that they regulate may be difficult to identify. The environmental bacterium, Myxococcus xanthus, is highly enriched in these non-canonical histidine kinases. M. xanthus is renowned for a starvation-induced multicellular developmental program in which some cells are induced to aggregate into fruiting bodies and then differentiate into environmentally resistant spores. Here, we characterize the M. xanthus orphan hybrid histidine kinase, SinK, which consists of a histidine kinase transmitter followed by two receiver domains. Non-phosphorylatable sinK mutants were analyzed under two distinct developmental conditions and a new high resolution developmental assay. These data suggest SinK impacts onset of aggregation, mobility of aggregates, and sporulation efficiency. SinK activity is controlled by a genus-specific hypothetical protein (SinM) and the two SinK receiver domains. We are currently working to understand how SinM modulates SinK activity.
Discovery & Analysis of Genes Regulating Biofilm Formation of Candida albicans
Nikol Shllaku, Angelina Antonyan, Alex Jackman, Diana McMahon, Marcelio Shammami, Jonathan S. Finkel Ph.D.
Biology Department, University of Detroit Mercy
Microorganisms can have a profound effect on their surrounding environment. The only conditions that must be met are slight moisture and organic matter to trigger growth. Through the slight buildup of microorganisms on the surface a biofilm can be created. Biofilm formation can cause many health related issues in its host. Candida albicans infections are commonly associated with a wide variety of implanted medical devices. They are the primary source of systemic infection in a hospital environment. In order to identify molecular determinants of biofilm formation an insertion mutant library of cell wall genes was screened for its ability to form a biofilm. Our studies identified that mutations in orf19.212 have lead us to believe that having a biofilm defect allows for the host cell the ability to respond in such a way to kill the yeast cells. With a mutation in that specific gene the chance that hyphal growth being affected is almost certain as well as complications in development. Nonetheless, without the ability to form hyphae the extracellular matrix cannot be developed, which is important in developing resistance against antifungals. Our results stipulate that without certain genes the formation of an extracellular matrix is not possible and results in little to no resistance against the host cells.
The TodK histidine kinase and its role in cell fate segregation during the Myxococcus xanthus developmental program.
Chris Mataczynski*+, Maike Glaser+, and Penelope I. Higgs
Dept. of Biological Sciences, Wayne State University, Detroit, MI 48202
+Authors contributed equally to this work
Myxococcus xanthus is a model organism for bacterial signaling complexity. Under nutrient limiting conditions, these bacteria enter a multicellular developmental program wherein cells follow different fates: aggregation into mounds (fruiting bodies) followed by differentiation into environmentally resistant spores, differentiation into a persister-like state termed peripheral rods, or programmed cell death. MrpC, a developmental transcriptional regulator, appears to play an important role in cell fate segregation. MrpC accumulates heterogeneously in the developing population and mis-accumulation of MrpC interferes in appropriate cell fate segregation. TodK is a histidine kinase that appears to be necessary for regulation of MrpC accumulation. Though we show that TodK cannot autophosphorylate in vitro, kinase activity is required in vivo for proper MrpC accumulation. We demonstrate that a todK mutant over-accumulates MrpC and sporulates more quickly than the wild type, and expression of todK from a constitutive promoter leads to an extremely delayed developmental phenotype and reduced MrpC accumulation. Using an mrpC transcriptional reporter, we have determined that mrpC expression starts earlier in a todK mutant, suggesting that TodK represses mrpC transcription. We propose TodK either represses MrpB (the transcriptional activator of mrpC) or enhances the negative autoregulation of MrpC. By qPCR and using a transcriptional reporter, we determined that TodK does not affect the expression of mrpB. We are currently testing how TodK accumulates in developing populations using fluorescent microscopy.
The TcpPH Complex and Regulation of Virulence in Vibrio cholerae
Demey Lucas M*, LeVeque Rhiannon and DiRita Victor J.
Department of Microbiology & Molecular Genetics, Michigan State University
Vibrio cholerae is a bacterial gastrointestinal pathogen that causes the life-threatening diarrheal disease cholera. To cause disease and remain competitive in endemic marine environments, V. cholerae must tightly regulate the production of virulence factors, such as cholera toxin (CtxAB) and the toxin co-regulated pilus (TCP). Expression of these major virulence factors, ctxAB and tcpA-F, is regulated by ToxT and indirectly by ToxR and TcpP, two membrane localized transcription regulators that modulate expression of toxT. Levels of TcpP are regulated by a two-step proteolytic process, termed Regulated Intramembrane Proteolysis. Under conditions not suitable for virulence factor production, TcpP undergoes proteolysis first by Tail-specific protease (Tsp; site-1 protease), cleaving within TcpP’s periplasmic domain, and secondly by a transmembrane metalloprotease (YaeL; site-2 protease), cleaving within the transmembrane domain of TcpP, thereby inactivating TcpP. Under conditions suitable for virulence factor gene expression, TcpP is protected by TcpH, a single pass transmembrane protein. In the absence of TcpH, TcpP undergoes constitutive degradation leading to a colonization defect in vivo. Currently, the cues that promote protection of TcpP in vivo are not understood. We are investigating localization determinants for TcpH that control its ability to interact with TcpP in the membrane. We replaced the natural transmembrane (T/M) domain of TcpH with T/M or periplasmic localization domains of other V. cholerae proteins including EpsM, ToxS, and CtxB. In growth experiments carried out in vitro, the TcpH T/M chimeras protected TcpP from degradation and led to expression of cholera toxin. In contrast, the TcpH T/M chimeras had a significant colonization defect compared to wild type, approximately ten-fold less than wild type. This decreased level of colonization by the TcpH T/M chimeras is equivalent to that observed with a tcpH null mutant. We hypothesize that the native TcpH transmembrane domain responds to alterations in the cytoplasmic membrane that occur in vivo, induced perhaps by bile or fatty acids, thereby promoting protection of TcpP.
Structural Insights Lead to the Identification of Multiple ToxR-binding Sites along the toxT Promoter in Vibrio cholerae
Jamal A. Alhabeil1, Nour El Yaman*1, Sarah C. Plecha1, Jonathan S. Zora1, Joshua J. Thomson1, Albert Canals2,3, Simone Pieretti2,3, Rosa Pérez2,3, Miquel Coll2,3, and Eric S. Krukonis1
1University of Detroit Mercy School of Dentistry, Detroit, MI 48208; 2Institute for Research in Biomedicine, Barcelona Science Park, Barcelona, Spain; 3Institut de Biologia Molecular de Barcelona (CSIC), Barcelona Science Park, Barcelona, Spain; *firstname.lastname@example.org; *email@example.com Institute for Research in Biomedicine, Barcelona Science Park, Barcelona, Spain
Cholera, an acute diarrheal disease caused by Vibrio cholerae, is estimated to cause over 100,000 deaths each year. Cholera toxin and toxin co-regulated pilus, two key virulence factors, are directly regulated by ToxT, while the toxT promoter is activated by ToxR in conjunction with TcpP. ToxR also directly regulates ompU, encoding the outer membrane porin OmpU, without TcpP. We previously defined two ToxR-binding sites within the toxT promoter, but recent crystallographic studies of ToxR bound to the toxT promoter identified three other ToxR-binding sites. Based on these structural findings, ToxR mutants were generated based on their predicted impact on DNA binding activity. ToxR residues W64, D72, R84, T99, K102, and Y105 when mutated to alanine (or proline for D72) resulted in ToxR molecules unable to activate either of the two ToxR-dependent promoters, toxT or ompU. To assess DNA-binding activity, the DNA-binding and transactivation domains of these ToxR mutants were purified and used in electrophoretic mobility shift assays (EMSAs). ToxR-W64A, ToxR-R84A, ToxR-T99A, ToxR-K102A, and ToxR-Y105A were completely defective for DNA binding. ToxR-D72P bound the toxT and ompU promoters similar to wild-type ToxR despite failing to activate both promoters. This suggests the D72P substitution alters presentation of the transactivation loop for interaction with RNA polymerase. Two mutants affecting the DNA-recognition helix, ToxR-Q78A and ToxR-S81A, had differential effects on toxT and ompU activation which was reflected in their binding affinities to those specific promoters. Finally, EMSA analysis supports the structurally-based identification of additional promoter-proximal ToxR-binding sites on the toxT promoter from -75 to -41 and confirmed ToxR binding to three regions of the ompU promoter: -187 to -148, -130 to -91, and -53 to -24. Future studies will more specifically define the DNA sequences required for ToxR binding and regulation of V. cholerae virulence.
Anti-Staphylococcal Mechanism of the 3,4-Dihydroquinazolines
Malcolm, Celina I., Davies, Steven B., *Hutchens, Martha A., Mosey, R. Adam
All authors are affiliated with Lake Superior State University, Sault Sainte Marie, MI, 49783
Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that causes of a wide variety of serious infections such as surgical site infections, and bloodstream infections. Infections can often be difficult to treat due to MRSA’s resistance to penicillin-based antibiotics. With the increasing problem of bacterial resistance, there is a need for new antibiotics. One possible source of these could be the 3,4-Dihydroquinazolines. These compounds are effective MRSA growth inhibitors, however, their mechanism of anti-microbial action is unknown. The objective of this study is to determine the mechanism of action of the 3,4-Dihydroquinazolines. S. aureus was cultured in high concentrations of the most potent 3,4-Dihydroquinazoline compound (SD-1-123) to generate a mutant strain. Five commercial antibiotics, each representing one of five known mechanisms of action, were then tested against the mutant S. aureus. The mutant strain of S. aureus was found to be resistant to SD-1-123, and Daptomycin. These results suggest that the compound SD-1-123 may use the same mode of entry or mechanism as the commercial drug Daptomycin. However, the mechanism of the other 3,4-Dihydroquinazoline compounds remains a mystery. Synergistic assays are underway, to determine if the other compounds work through one of the five known pathways or through an undiscovered pathway. Further research with these compounds may be able to give a potential solution to the ever-growing antibiotic resistance problem in healthcare.
Determining Drug Targets in Candida albicans Biofilm Formation
Marcelio Shammami, Angela Antonyan, Alex Jackman, Diana McMahon, Nikol Shllaku, Jonathan S. Finkel Ph.D.
Candida albicans is a naturally occurring yeast in the gut microflora that can become deadly through the formation of biofilms on medical implants in immunosuppressed patients. Biofilms form in four steps, adherence, where yeast cells adhere to a medical implant or device, initiation, where the yeast cells begin to clump and create larger colonies as well as begin to filament, maturation, where filamentation continues and an extra cellular matrix forms causing the biofilm to become virulent, and dispersal, where yeast cells break off from the hyphal cells and begin the cycle again. Our research is focused on finding specific cell wall mutations as possible targets for drugs, by testing an assay library of insertion mutants of C. albicans. To determine if there is a defect we run adherence assays, drop tests on different compounds that would identify defects, including Congo Red and NaCl, and look at death rates in Galleria mellonella (wax worms), an organism that only has a primary immune system, after injection of each strain to test initial response to infection. As a lab we have confirmed four mutants with more being retested to confirm a defect. One of the insertion mutants we found a defect in is in gene VPS28, the known mutations in VPS28’s genome include a defect in pH response, a filamentation defect, a pathogenesis defect, and a protein transport defect in the vacuoles. The next step for our lab would be to run a CRISPR/Cas9 system to confirm that gene VPS28 is causing the mutation, and then determining possible drug solutions that attack this defect in the cell walls.