The specific identification of a megalurous cercaria found in the snail Oxytrema plicifera was accomplished by completing the life cycle in the laboratory. This species is compared with the eastern Philophthalmus megalurus and P. gralli with which it was once confused. The eggs, miracidia, and rediae of the Oregon species were found to be similar to those of the above species. The body and organ sizes and sucker ratios for the cercariae and adult stages obviously indicate that the Oregon species is Philophthalmus megalurus.

Scrub typhus, caused by the obligate intracellular bacterium Orientia tsutsugamushi, afflicts one million people annually. Despite being a global health threat, little is known about O. tsutsugamushi pathogenesis. Here, we demonstrate that O. tsutsugamushi modulates the ER and ER-associated processes as mechanisms of nutritional virulence and immune evasion. To obtain amino acids to fuel replication, O. tsutsugamushi simultaneously induces ER stress and the unfolded protein response (UPR) while inhibiting ER-associated degradation (ERAD) during early infection time points. During exponential growth, the bacterium releases the ER bottleneck, resulting in generation of ERAD-derived amino acids that it parasitized for replication. The O. tsutsugamushi effector, Ank4, is linked to this process, as it impedes ERAD when ectopically expressed. O. tsutsugamushi expression of ank4 peaks during the ERAD inhibition window, but is absent when the pathway is restored. These data reveal a novel mechanism of nutritional virulence, whereby an obligate intracellular pathogen coordinates the modulation of multiple ER-associated processes. Like other intracellular pathogens, O. tsutsugamushi inhibits expression of MHC-I, but it does so in a novel manner by degrading the master regulator of MHC-I, NLRC5. This impedes production of the MHC-I components, human leukocyte antigen A and Beta-2 microglobulin. The NLRC5-reduction mechanism recapitulates across diverse cell types, but the degree and duration of inhibition is cell type-specific. NLRC5 modulation and MHC-I inhibition are linked to another O. tsutsugamushi Ank, Ank5. NLRC5 is a putative interacting partner of Ank5. Moreover, NLRC5 and MHC-I levels are reduced in cells ectopically expressing Ank5. To our knowledge, these are the first examples of a pathogen modulating NLRC5 to negatively regulate MHC-I expression and of a bacterial effector interacting with NLRC5. As we learn more about the bacterium’s ability to regulate its host cell, a unifying theme has emerged: modulation of the ER and ER-associated pathways. These projects reveal two novel mechanisms of O. tsutsugamushi pathogenesis, strategies to acquire the amino acids needed for replication and to decrease MHC-I antigen presentation by the host cell. These insights help in understanding how O. tsutsugamushi and potentially other related pathogens co-opt host cell processes to cause disease.

The growth of bacteriophage HK75 is inhibited by specific mutations in the zinc binding domain of the host RNA polymerase beta prime subunit. It shares this rare property with bacteriophage HK022 and other phages that use RNA mediated antitermination to promote early gene expression. Recent genomic analysis of HK75 and HK022 has confirmed the relatedness of these two phages and place HK75 in the lambdoid family of bacteriophages. Lambdoid phages are temperate and can adopt a lytic or lysogenic lifestyle upon infection of a suitable host. However, HK75 only forms clear plaques and thus appears to be defective in its ability to form lysogens. Based on published analyses of other lambdoid phages, a clear plaque phenotype is commonly due to a mutation in one of 5 phage genes: cI, cII, cIII, int, xis or the phage repressor DNA binding sites. To determine which mutation is responsible for the clear plaque phenotype of HK75, we cloned the cI and cIII genes and assayed their activities. The HK75 cI gene clone prevented super-infection by HK75. This result demonstrated repressor functionality and thus the clear plaque phenotype cannot be due to a mutation in the HK75 cI gene. Several amino acid differences were noted between the HK022 and HK75 CIII proteins. To determine if the clear plaque phenotype was due to mutations in the HK75 cIII gene, we cloned it into an expression vector. Only under conditions of cIII gene overexpression were lysogens of HK75 recovered. The phage CIII protein normally protects CII from proteolysis. Stabilization of CII by mutations in specific host proteases has been shown to suppress a clear plaque phenotype caused by mutations in the cIII gene. When HK75 was plated on a protease deficient strain of E. coli, turbid plaques were formed and lysogens were recovered. These results support the idea that the clear plaque phenotype of HK75 is due to a defect in the expression of the phage cIII gene.

Pseudomonas aeruginosa is a dreaded opportunistic pathogen that causes severe and often intractable infections in immunocompromised and critically ill patients. This bacterium is also the primary cause of fatal lung infections in patients with cystic fibrosis and a leading nosocomial pathogen responsible for nearly 10% of all hospital-acquired infections. P. aeruginosa is intrinsically recalcitrant to most classes of antibiotics and has the ability to acquire additional resistance during treatment. In particular, resistance to the widely used β-lactam antibiotics is frequently mediated by the expression of AmpC, a chromosomally encoded β-lactamase that is ubiquitously found in P. aeruginosa strains. This dissertation delved into the role of a recently reported chromosomal β-lactamase in P. aeruginosa called PoxB. To date, no detailed studies have addressed the regulation of poxB expression and its contribution to β-lactam resistance in P. aeruginosa. In an effort to better understand the role of this β-lactamase, poxB was deleted from the chromosome and expressed in trans from an IPTG-inducible promoter. The loss of poxB did not affect susceptibility. However, expression in trans in the absence of ampC rendered strains more resistant to the carbapenem β-lactams. The carbapenem-hydrolyzing phenotype was enhanced, reaching intermediate and resistant clinical breakpoints, in the absence of the carbapenem-specific outer membrane porin OprD. As observed for most class D β-lactamases, PoxB was only weakly inhibited by the currently available β-lactamase inhibitors. Moreover, poxB was shown to form an operon with the upstream located poxA, whose expression in trans decreased pox promoter (Ppox) activity suggesting autoregulation. The transcriptional regulator AmpR negatively controlled Ppox activity, however no direct interaction could be demonstrated. A mariner transposon library identified genes involved in the transport of polyamines as potential regulators of pox expression. Unexpectedly, polyamines themselves were able induce resistance to carbapenems. In summary, P. aeruginosa carries a chromosomal-encoded β-lactamase PoxB that can provide resistance against the clinically relevant carbapenems despite its narrow spectrum of hydrolysis and whose activity in vivo may be regulated by polyamines.

Fish of the genus Cottus were found infected with heterophyid metacercariae. Laboratory animals were infected with the metacercariae, and adult heterophid trematodes were recovered. These flukes were found to represent an undescribed species of the genus Euryhelmis. Various streams in the Willamette Valley and coastal areas were sampled for infected Cottus sp. Snails of the genera Oxytrema and Fluminicola were collected. Several possible definitive hosts were examined. A partial review of the subfamily Apophallinae and a complete review of the genus Euryhelmis are presented. Euryhelmis cotti n. sp. is placed in the subfamily Apophallinae, and its life cycle is partially described.

Salmonella enterica serovar Typhimurium is one of the most prevalent food-borne pathogens, affecting millions around the world every year, making it a threat to global health. Salmonella possesses the ability to survive the normally lethal condition of desiccation, however, discovery of the genes and mechanisms behind this phenomenon are still ongoing. Using a transposon mutagenesis approach to construct a broad transposon library, this study aimed to uncover genes that may be contributing to changes in Salmonella’s survivability under desiccation, particularly when exposed to the antimicrobial molecule salicylic acid. Building on previous findings showing salicylic acid can alter cell viability through differential gene regulation, transposon mutants were exposed to salicylic acid and subsequently desiccated to screen for mutants that displayed an alteration in survival phenotypes. This work identified a transposon mutant with an interruption of the porin-coding gene ompC that displayed an augmented survivability phenotype under these conditions, leading to further exploration into the origin of this phenomenon.

Polymicrobial communities of bacterial and fungal species are present on the skin and mucosal surfaces of the body. Invasive infections caused by Candida species are commonly seen in immunocompromised individuals (HIV, transplants, cancer) and ranks as the third leading cause of infection in hospitalized patients. C. albicans is a polymorphic opportunistic fungus that infects critically ill patients and has the ability to change its morphology from yeast to hyphal form.The morphogenesis of C. albicans is a major aspect of its virulence and is regulated by quorum sensing (QS) molecules they produce, as well as the presence of neighboring microbes.In this study, we examined two transporter mutants, cdr4 and ssu1, for their ability to form biofilms in the presence of cyclic-di-GMP and 3-oxo-12-homoserine lactone. To quantify biomass, wild type and mutant cells were grown overnight at 30˚C in YPD. The cells were washed, counted and diluted to a desired density of 106 cells/ml in medium 199, pH7.5. Cells were added to 96-well plates pre-incubated with 5% fetal bovine serum at densities of 105, 104, and 103 cells/well and allowed to adhere at 37˚C for one hour. The wells were then covered with fresh M199 media containing the QS molecule and monitored for 48 hours at 37˚C. After this time, the media and planktonic cells were removed. The biofilms were fixed with methanol, dried, then stained with 0.05% crystal violet. Bulk biomass was assessed by spectrophotometry. We did observe a difference in biofilm density when incubated in the presence of cyclic-diGMP. We noted that for the wild type and ssu1 strain their biofilms biomass increased by as much as 10% at 104 and 103 cell densities when compared with the control. While the cdr4 strain had a slight reduction in biofilm density when cyclic-diGMP was present. This result also indicates a potentially positive role in which cyclic-diGMP can help C. albicans develop denser biofilms, potentially in the presence of bacteria like P. aeruginsa, which secrete cyclic di-GMP, but kill hyphal forms of C. albicans. Additionally, it has been shown that C. albicans mutants lacking the hybrid histidine kinase, Chk1p, are refractory to the effects of farnesol, a QS molecule that inhibits morphogenesis.Given that mutations in CDR4 and SSU1 impact the QS response in C. albicans, we investigated whether these genes were regulated through two-component signaling by Chk1. To assess CDR4 and SSU1 expression, wild type and mutant strains were grown overnight in YPD media at 30˚C. Cells were then harvested and RNA was obtained by acid phenol extraction. Using RT-PCR, we determined both CDR4 and SSU1 expression is reduced or highly repressed in the chk1, ypd1, and skn7 null strains. These results suggest the two genes are downstream targets in a pathway regulated by Chk1p. The finding that QS proceeds through a two-component pathway can be exploited in antifungal drug development. Given that two-component signaling is absent in mammalian cells, development of novel compounds that interfere with this pathway may be a useful alternative for treating patients with candidiasis.

Vibrio parahaemolyticus is a food-borne pathogen commonly associated with the consumption of raw or undercooked seafood resulting in primary infections of the human gastrointestinal tract. It is estimated to cause about 4500 illnesses each year in the United States. However, infection from this food-borne pathogen can be avoided if this organism is detected in the implicated food, prior to consumption. Current standard methods of detecting this organism are dependent on the culturability of the bacteria. Detection based on an organism’s culturability may be problematic as V. parahaemolyticus has been known to exist in a viable but nonculturable (VBNC) state. Bacteria in the VBNC state are characterized by low levels of metabolic activity and the inability to be cultured by standard laboratory practices. When bacteria enter the VBNC state, their gene expression profile may be different than the culturable counterpart. We were interested in comparing the expression of two virulence-associated genes between VBNC and culturable cells of V. parahaemolyticus. V. parahaemolyticus RIMD2210633 was incubated at 4°C in modified Morita mineral salt solution supplemented with 0.5% NaCl (MMS) or trypticase soy broth supplemented with 2% NaCl (TSBS), which represented nutrient poor and rich conditions, respectively. The number of VBNC and culturable cells were determined by standard plate count and fluorescence microscopy. The expression levels of virulence-associated genes tdh2 and escU, were measured relative to the housekeeping gene, pvsA, by qRT-PCR. Nutrient availability and temperatures exerted variable effects on the virulence gene expression. It is possible that VBNC V. parahaemolyticus cells may retain their pathogenicity potential.

<p>Department not listed. Molecular Biology</p> / <p>The yeast S. cerevisiae Is able to use several different metabolic pathways for the generation of metabolic energy, and is able to vary the synthesis of these multlenzyme systems in response to changes in the external environment. The responses of the yeast to various changes in the environment have been studied and correlated with changes in the rate of synthesis of various enzymes. Two of these pathways are found in the mitochondrion, a sub-cellular organelle, the structural integrity of which is necessary for the functioning of these integrated systems. The experiments described thus provide information about the control of structure and function in cellular organelles, as well as about the general changes in energy metabolism.</p> / Doctor of Philosophy (PhD)

Periodontitis is a chronic inflammatory disease caused by pathogenic bacteria residing in a complex biofilm within a susceptible host. Amixicile is a non-toxic, readily bioavailable novel antimicrobial that targets strict anaerobes through inhibition of the activity of Pyruvate Ferredoxin Oxidoreductase (PFOR), a major enzyme mediating oxidative decarboxylation of pyruvate. Our study aimed to evaluate the efficacy of amixicile, when compared to metronidazole, in inhibiting the growth of bacteria present in a microbiome harvested from patients with chronic periodontitis. Plaque samples were harvested from patients with severe chronic periodontitis and cultured under anaerobic conditions. The microbiomes were grown in the presence of amixicile and metronidazole and the growth was compared to that of bacteria grown in the absence of the antimicrobials. Following 24 hour growth the bacterial DNA was analyzed using quantitative PCR (qPCR) using primers specific for 12 bacterial species: P. gingivalis (Pg), P. intermedia (Pi), F.nucleatum (Fn), S.gordonii (Sg), S. anginosus (Sa), V. atypical (Va), L. acidophilus (La), A.actinomycetemcomitans (Aa), T.denticola (Td), S.mutans (Sm), and S.sanguis (Ss). Both drug treatment groups yielded a statistical significant reduction for several anaerobic bacteria: Pi (P