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Showcase College of Veterinary Medicine

The Sea as a Source of Novel Antiparasitic Compounds

The Sea as a Source of Novel Antiparasitic Compounds

Primary Author: Rachel Relat

Faculty Sponsor: Roberta O’Connor

 

Primary College/Unit: College of Veterinary Medicine

Category: Medical and Life Sciences

Campus: Pullman

 

Abstract:

 

PRINCIPLE TOPIC

Cryptosporidium, an intracellular parasite that causes severe diarrhea, is now recognized as a leading cause of waterborne disease worldwide, infecting both humans and valuable livestock.  Cryptosporidium infection is particularly devastating in children as it causes life-threatening diarrhea, along with developmental delays including growth stunting and cognitive impairment. Cryptosporidium is an especially important disease of immunocompromised individuals of all ages, leading to chronic, potentially fatal diarrhea. No effective treatment exists for many human or veterinary patients diagnosed with Cryptosporidium.

METHODS

To address this medical imperative, we designed a high throughput screen to test thousands of unique compounds, with the goal of discovering new, potent, anti-Cryptosporidium drugs and drug scaffolds. Our collaborators at the Harbor Branch Oceanographic Institute (HBOI) have amassed a library of over 125 natural compounds, and 6500 highly enriched fractions from extracts of marine organisms. We began screening this library as it contains compounds and fractions produced by sessile oceanic animals which frequently use chemical defenses.  We screened greater than 3,600 partially purified extracts from the HBOI library by infecting cells with Cryptosporidium parvum, allowing replication, and then treating with a specific highly enriched fractions or compounds, and then measuring parasite growth after 48 hours.

RESULTS

We identified 10 extracts derived from sessile marine organisms that effectively inhibit (>80% inhibition) Cryptosporidium without damaging the host cells in which these parasites live. This work will significantly contribute to the goal of discovering effective treatments against Cryptosporidium specifically, and further the search for novel anti-parasitic drugs.

 

Boromycin as a potential anti-toxoplasma and anti-cryptosporidium drug

Boromycin as a potential anti-toxoplasma and anti-cryptosporidium drug

Primary Author: Jaypee Abenoja

Faculty Sponsor: Roberta O’Connor

 

Primary College/Unit: College of Veterinary Medicine

Category: Medical and Life Sciences

Campus: Pullman

 

Abstract:

Toxoplasma gondii and Cryptosporidium parvum, members of the phylum Apicomplexa, are considered significant pathogens of both humans and animals worldwide. They are obligate intracellular parasites that cause serious conditions like neurological abnormalities, blindness and chronic diarrhea especially to immunocompromised individuals. Unfortunately, aside from significant toxicity on mammalian cells, current therapies against these parasites become ineffective through time because of drug resistance, making the discovery of new therapeutic drugs a priority. Here we described the activity of Boromycin (BM), a lipid-soluble antibiotic produced by Streptomyces antibioticus only known before as a drug for gram positive bacteria, against T. gondii and C. parvum.

BM’s in vitro activity against T. gondii and C. parvum was evaluated using various assays including proliferation inhibition, invasion assay and immunofluorescence to characterize the morphological changes of the parasites after being exposed to BM.

BM potently inhibits intracellular proliferation of T. gondii (EC50=2.13nM) and C. parvum (EC50=6.46nM) into their host cells. Irreversible inhibition on the ability of extracellular T. gondii to invade host cells was also observed after 2 hours of incubation with BM. Furthermore,

immunofluorescence of the parasites using anti-surface antigen glycoprotein-1 (SAG1) antibodies show detectable parasitophorous vacuoles (PV) but with randomly distributed surface antigens and complete loss of morphologically intact parasites within the vacuoles. We also determined after cytotoxicity assays that BM is very selective against parasites at the same time safe to mammalian host cells (Selectivity Index= of 3582.7). These promising results suggest BM as an exciting drug candidate for treating toxoplasmosis and cryptosporidiosis.

Identifying the mechanism of action of Tartrolon E, a broad-spectrum anti-apicomplexan compound

Identifying the mechanism of action of Tartrolon E, a broad-spectrum anti-apicomplexan compound

Primary Author: Gregory Bowden

Faculty Sponsor: Roberta O’Connor

 

Primary College/Unit: College of Veterinary Medicine

Category: Medical and Life Sciences

Campus: Pullman

 

Abstract:

 

PRINCIPAL TOPIC

The apicomplexan parasite Cryptosporidium is the cause of the severe diarrheal disease, cryptosporidiosis. Cryptosporidiosis is one of the most important diseases of children, immunocompromised individuals, and young ruminant livestock. Currently, there is no treatment for neonatal ruminants, and the only approved treatment for immunocompromised individuals is ineffective; a new treatment is needed.  Recently we identified Tartrolon E (TrtE), a compound from shipworm symbiotic bacteria, to have broad-spectrum anti-apicomplexan parasite activity, including activity against Cryptosporidium. The purpose of this work was to uncover the mechanism of action of TrtE against apicomplexan parasites by examining gene expression during treatment.

METHOD

We studied the mechanism of action of TrtE using the model apicomplexan parasite Toxoplasma gondii. Changes in gene expression during treatment were determined by RNA-sequencing and verified by RT-qPCR. We investigated the potential function of the responsive gene using bioinformatics and immunofluorescence assays. To examine the effect of this gene on parasite susceptibility to TrtE, we deleted the gene using CRISPR/Cas9.

RESULTS

RNA-sequencing data revealed that T. gondii responds to TrtE treatment by upregulating TGME49_272370 (TERG) in a rapid dose-dependent manner. TERG encodes for an unknown protein with multiple predicted transmembrane domains. Immunofluorescence assays located the TERG protein to punctate areas throughout the cytoplasm. The deletion of TERG resulted in a 22% increase in parasite susceptibility to TrtE. In uncovering the mechanism by which TrtE inhibits T. gondii parasites, we may identify a shared pathway critical to apicomplexan parasite survival and advance the search for a new treatment for cryptosporidiosis.

 

Highly Structured Nurse-to-Patient Interactions in an Acute Care Hospital ICU Reduces MRSA Transmission When Limited to Assigned Patient Groups: A Mathematical Model Approach

Highly Structured Nurse-to-Patient Interactions in an Acute Care Hospital ICU Reduces MRSA Transmission When Limited to Assigned Patient Groups: A Mathematical Model Approach

Primary Author: Matthew Mietchen

Faculty Sponsor: Eric Lofgen

 

Primary College/Unit: College of Veterinary Medicine

Category: Medical and Life Sciences

Campus: Pullman

 

Abstract:

PRINCIPLE TOPIC

The importance of nurse-to-patient contact in hospital settings for reducing healthcare acquired infections (HAIs) has been well documented. However, it is not well understood how specific interaction patterns effect pathogen transmission. Hospital staffing challenges are a reality, and in addition, are difficult and expensive to study. Using disease transmission models is a useful tool to explore the magnitude and the importance of patient care structure in an intensive care unit (ICU).

 

METHODS

A stochastic compartmental model of an 18-bed ICU was used to study how nurse interactions with patients affected the number of methicillin-resistant S. aureus (MRSA) acquisitions. A parameter, gamma, was introduced to represent the proportion of time a nurse spends within an assigned patient sub-group over a one year duration. The model was simulated 10,000 times with gamma being chosen randomly from a uniform distribution within the parameter range. A segmented Poisson regression model was fit to the MRSA acquisitions to detect any relationships or notable change points.

 

RESULTS

A non-linear relationship was observed, resulting in a decrease in acquisitions as the value of gamma increased. The regression model also detected a single change point in MRSA acquisitions at a gamma value of 0.40 (95% confidence interval: 0.37, 0.42). In conclusion, the proportion of time a nurse spends with an assigned group of patients may have large effects on MRSA transmission within an ICU. Highly structured nurse-to-patient contact with limited interactions outside of assigned patient sub-groups, may be important for hospital epidemiology prevention efforts.

 

Differential expression of the rhoptry associated protein -1a in Theileria equi; Implications for host cell invasion

Differential expression of the rhoptry associated protein -1a in Theileria equi; Implications for host cell invasion

Primary Author: Cynthia Onzere

Faculty Sponsor: Carlos Suarez

 

Primary College/Unit: College of Veterinary Medicine

Category: Medical and Life Sciences

Campus: Pullman

 

Abstract:

 

Principal Topic

Theileria equi (T. equi) is one of the causative agents of equine piroplasmosis, a severe tick-transmitted disease that affects all equid species. Presently, there is no vaccine for the control of the parasite. T. equi is an Apicomplexan organism characterized by the presence of secretory organelles, such as the rhoptries, on the anterior end that are essential for invasion and establishment of the parasite within the host cell. Rhoptry proteins have been shown to be important in the attachment of several apicomplexan parasites to the host cell during invasion. Despite their functional importance, the pattern of expression and potential of rhoptry proteins as vaccine targets in T. equi remain unexplored.

Method

We evaluated the expression of a putative T. equi rhoptry protein known as the rhoptry associated protein-1a (RAP-1a) in the parasite’s invasive stages in horses. RAP-1a was selected because it is widely conserved among the closely related Babesia and Theileria parasites.

Results/implications

Our findings indicate that RAP-1a is expressed in the merozoite stage of development that invades red blood cells (erythrocytes) but it’s not expressed in the sporozoite stage that invades peripheral blood mononuclear cells. This indicates that T. equi RAP-1a could be important for invasion of erythrocytes and could therefore be a marker for pathogenicity because the symptomatic stage of T. equi infection is associated with the intra-erythrocytic stage of the parasite’s development. Future work will be focused on defining the functional relevance of RAP-1a in T. equi and its role in eliciting protective immune responses.

 

Modeling the Impact of Staff to Patient Ratios on MRSA Acquisition in an Intensive Care Unit

Modeling the Impact of Staff to Patient Ratios on MRSA Acquisition in an Intensive Care Unit

Primary Author: Stephanie Sikavitsas Johnson

Faculty Sponsor: Eric Lofgren

 

Primary College/Unit: College of Veterinary Medicine

Category: Medical and Life Sciences

Campus: Pullman

 

Abstract:

 

Staff-to-Patient ratios in hospitals impact several hospital acquired infections (HAIs). Intuitively, increasing the number of nurses and doctors in an intensive care unit (ICU) should impact rates of HAI transmission by reducing the number of patients per provider, allowing less patient-patient interaction. Designing a study to determine how different staffing ratios would affect HAIs is difficult to in person because of lots of different factors that make each hospital and unit unique. Mathematical modeling can be used instead to simulate how staffing ratios affect methicillin-resistant S. aureus (MRSA) being acquired in a 15-bed ICU.

 

We varied the number of doctors in the ICU from one to three. For each doctor scenario, the nurse -patient ratio was 1:1, 1:2.5, 1:3. And 1:5. We ran each model 1000 times, with each time simulating 1 year in the ICU. The outcome was the mean number of MRSA acquisitions in one year for each of the 12 models. Our baseline model we compared everything to was the single doctor, 1:3 nurse-patient ratio model.

 

Adding more doctors slightly decreased the number of acquisitions, with the steeper decline going from one doctor to two. Going from a 1:3 nursing ratio to 1:1 ratio decreased MRSA acquisition by 64%. Increasing to a 1:5 ratio raised acquisitions 21.5%. These results suggest reduction in provider contact may have an impact on HAI rates. Appropriate staffing should be considered in infection control guidelines, and the cost of staffing be weighed against its impact on infection prevention.

 

Naturally derived peptides enhance killing efficiency of colistin against colistin-resistant bacteria

Naturally derived peptides enhance killing efficiency of colistin against colistin-resistant bacteria

Primary author: Kaitlin Witherell
Faculty sponsor: Dr. Douglas Call

Primary college/unit: College of Veterinary Medicine
Campus: Pullman

Abstract:

Colistin is a “last-line” antibiotic that is used to treat multidrug-resistant, Gram-negative bacteria, but colistin resistance has emerged. In susceptible bacteria, colistin binds lipid A and destroys the bacterial membrane. MCR (encoded by mcr-1 and others) is a phosphoethanolamine transferase that modifies lipid A resulting in decreased affinity for colistin. We are investigating two cysteine-dense peptides (CDPs), “B03” and “B11” that appear to kill bacteria by interacting with the bacterial membrane. We propose that using CDPs in combination with colistin we can kill colistin- and multidrug-resistant bacteria at lower concentrations and more quickly compared to colistin alone. We have shown B03 and B11 are able to reduce the colistin resistance profile of mcr-1 harboring and multidrug-resistant Escherichia coli AR Bank #0346. By adding 100 µg/ml of peptide B03 we are able to significantly reduce the minimum inhibitory concentration (MIC) of colistin from 1.25 µg/ml to 0.063 µg/ml. When 50 µg/ml of peptide B11 is added the MIC of colistin is reduced to less than 0.0005 µg/ml. We also found that by adding either peptide, 346 is killed faster and at lower concentrations compared to using colistin alone. We are determining the mechanism of action of these peptides by performing membrane potential assays and employing scanning electron microscopy.

Compared to C57BL/6J Mice, C57BL/6N Mice Exhibit Reduced Ethanol Consumption That Is Not Due to a Discrepancy in Sweet Taste Perception

Compared to C57BL/6J Mice, C57BL/6N Mice Exhibit Reduced Ethanol Consumption That Is Not Due to a Discrepancy in Sweet Taste Perception

Primary author: Talia Thuet
Co-author(s): Kevin Douglas

Primary college/unit: College of Veterinary Medicine
Campus: Pullman

Abstract:

DBA/2J (D2) and C57BL/6J (B6J) mice are widely used as models for low and high ethanol (EtOH) consumption genotypes respectively. However, a key determinant of low EtOH consumption in D2 mice is taste-aversion to EtOH, which precludes voluntary consumption to neurologically active concentrations of EtOH. This phenomenon is explained in part by D2 mice having an allele of the sac locus which encodes a sweet taste receptor with reduced responsivity to sweet compounds like saccharin, sucrose, and EtOH. Furthermore, vast genetic differences between B6J and D2 mice make it difficult to identify key underlying neuro-molecular-genetic factors that contribute to their respective drinking profiles. To address these complications, we studied EtOH and sucrose reinforcement in C57BL/6N mice (B6N), a substrain of the original C57BL/6J line, distinguished by only five small nucleotide polymorphisms, that exhibits significantly reduced EtOH consumption. To determine whether taste-aversion to EtOH is a primary determinant of low EtOH consumption by B6N mice, we subjected animals to a modified sucrose fade procedure using a drinking in the dark, two-bottle-choice paradigm. We determined that; 1) B6Ns consume the same amount of a 10% sucrose solution as B6Js, 2) B6Js consume significantly more of a 10% EtOH/10% sucrose mix than B6Ns, but both achieve physiologically relevant blood alcohol concentrations (15-219 mg%), and 3) B6Js consume significantly more 10% EtOH than B6Ns upon removal of sucrose. Taken together, these results suggest that genetic differences in neurological response to EtOH contribute to differences in EtOH consumption between genetically similar B6J and B6N mice.

Predicted Dermacentor andersoni iron metabolism genes and their response to iron reduction in cultured tick cells

Predicted Dermacentor andersoni iron metabolism genes and their response to iron reduction in cultured tick cellsPredicted Dermacentor andersoni iron metabolism genes and their response to iron reduction in cultured tick cells

Primary author: Muna Solyman
Faculty sponsor: Susan M. Noh

Primary college/unit: College of Veterinary Medicine
Campus: Pullman

Abstract:

For most organisms, iron is an essential nutrient due to its role in many cellular processes. Consequently, insufficient iron levels cause cell damage and death while high concentrations of iron are toxic due to the formation of oxidative radicals, which damage cellular components. As obligate hematophagous parasites, ticks have access to large amounts of iron. Unlike all other hematophagous invertebrates, blood meal digestion within ticks occurs intracellularly rather than in the gut lumen. Due to their unique physiology, little is known about iron uptake and regulation in ticks. Some genes have been identified that are hypothesized to be involved in iron metabolism based on the presence of conserved domains and some limited experimental evidence. In this project, thirteen likely iron metabolism genes were chosen based on the literature and bioinformatics. Using, Dermacentor andersoni ticks, which do not have a published genome, we identified and sequenced these genes. Next, we developed a method to reduce iron in cultured tick cells using an iron chelator 2,2′-bipyridyl and measured the transcriptional response of the D. andersoni iron metabolism genes to iron reduction. Overall, the response of tick cells to iron reduction was less marked than in mammalian cells. The iron transport gene, ferritin 2, and the mitochondrial iron transporters ferrochelatase and mitoferrin were down regulated, while iron regulator protein 1 was up-regulated in response to reduced iron levels. This work lays a foundation for an improved understanding of iron metabolism in ticks and the potential identification of anti-tick targets.

Transcriptional R-loops in Archaea

Transcriptional R-loops in Archaea

Primary author: Marguerite Smith
Co-author(s): Michael Rolfsmeier; Cynthia Haseltine
Faculty sponsor: Cynthia Haseltine

Primary college/unit: College of Veterinary Medicine
Campus: Pullman

Abstract:

To maintain genome integrity, cells must contend with multiple DNA structures that arise during replication, transcription, and repair. Transcriptionally active regions of the genome are particularly susceptible to DNA damage through the production of R-loop regions, consisting of an RNA:DNA hybrid along with a displaced ssDNA non-template strand, which is particularly susceptible to damage. Failure to dissociate RNA:DNA hybrids can lead to extensive ssDNA regions, stalled transcription machinery, or persistent protein:DNA complexes, all of which are potentially deleterious to the cell. Thus, all organisms must have robust mechanisms to contend with RNA:DNA hybrids within actively transcribed regions of the genome. While transcriptionally associated R-loops have been detected in bacteria and eukaryotes, there have been no reports of their occurrence in archaea. We used the hyperthermophilic crenarchaeon Sulfolobus solfataricus as a model archaeal species to investigate the formation of RNA:DNA hybrids. We established methodology for immunoprecipitation of RNA:DNA hybrids from S. solfataricus and evaluated abundance and persistence of these hybrids for both transcriptionally active and inactive regions of the genome using multiple cell backgrounds. In parallel, we examined topological alterations in the supercoiling state of a plasmid carrying an inducible gene using an in vitro transcription approach. Our molecular characterization of RNA:DNA hybrids in S. solfataricus indicates they are strongly associated with transcriptionally active regions and likely represent R-loop structures. Additionally, variation of production and persistence of these R-loops in strain backgrounds with differential DNA damage responses implicates DSB repair processes in maintaining genome stability during transcriptional activities in S. solfataricus.