The extensive and sometimes incorrect and noncompliant use of various types of antimicrobial agents has accelerated the development of antimicrobial resistance (AMR). In fact, AMR has become one of the greatest global threat to human health in this era. The broad-spectrum antibiotics aminoglycosides (AGs) display excellent potency against most Gram-negative bacteria, mycobacteria, and some Gram-positive bacteria, such as Staphylococcus aureus. The AG antibiotics amikacin, gentamicin, kanamycin, and tobramycin are still commonly prescribed in the U.S.A. for the treatment of serious infections. Unfortunately, bacteria evolve to acquire resistance to AGs via four different mechanisms: i) changing in membrane permeability to resist drugs from entering, ii) upregulating efflux pumps for active removal of intracellular AGs, iii) modifying the antimicrobial target(s) to prevent drugs binding to their targets, and iv) acquiring resistance enzymes to chemically inactivate the compounds. Amongst all, the acquisition of resistance enzymes, AG-modifying enzymes (AMEs), is the most common resistance mechanism identified. Depending on the chemistry each enzyme catalyzes, AMEs can be further divided into AG N-acetyltransferases (AACs), AG O-phosphotransferases (APHs), and AG O-nucleotidyltransferases. To overcome AME-related resistance, we need to better understand these resistance enzymes and further seek ways to either escape or inhibit their actions. In this dissertation, I summarized my efforts to characterize the AAC(6') domain and its mutant enzymes from a bifunctional AME, AAC(6')-Ie/APH(2")-Ia as well as another common AME, APH(3')-IIa. I also explained my attempt to inhibit the action of various AAC enzymes using metal salts. In an effort to explore the current resistance epidemic, I evaluated the resistance against carbapenem and AG antibiotics and the correlation between the resistance profiles and the AME genes in a collection of 122 Pseudomonas aeruginosa clinical isolates obtained from the University of Kentucky Hospital System. Besides tackling the resistance mechanisms in bacteria, I have also attempted to explore a new antifungal option by repurposing an existing antipsychotic drug, bromperidol, and a panel of its derivatives into a combination therapy with the azole antifungals against a variety of pathogenic yeasts and filamentous fungi.

Hydropathic molecular modeling is a computer-aided molecular design technique for obtaining, representing, and understanding the properties and interactions of biomacromolecular complexes in the biological environment. Hydropathic INTeraction (HINT) is a novel empirical force field to calculate the free energy of intermolecular interaction based on experimentally determined partition coefficients (log Po/w). It includes all the expected interactions between molecules such as hydrogen bonding, hydrophobic, electrostatic, acid-base, and Coulombic interactions, entropy, solvation and others. HINT tools were used to determine, evaluate, and analyze protein-ligand interactions in different research projects: 1) We used these tools to discover small molecule inhibitors of PsaA, a potential target for Streptococcus pneumoniae. We screened and scored potential molecules to obtain hits. After the growth conditions for both the wild type and PsaA mutant of S. pneumoniae were optimized, we then tested our hits. A few compounds passed through the three-stage assay protocol and confirmed the inhibition of PsaA with MICs between 125-250 μM. 2) The SAR of C-3 and C-5 pyrrole-based antitubulin agents at the colchicine-binding site with explicitly solvated models was performed. After docking with GOLD at the colchicine site, post-docking scoring and evaluation were performed with HINT. The total HINT score correlates with binding and activity; similarly, the significance of individual functional groups, protein residues and interactions amongst a collection of compounds can be quantitated. The possibility of water-mediated interactions in a way solvent accessible part of the pocket was considered by subjecting molecular models to MD simulations. Several water molecules were identified to be contributing to the binding and were confirmed by HINT scoring. Finally, using hydropathic molecular modeling tools helped us to understand, evaluate, analyze, and improve protein-ligand interactions in different biological systems.

Syphilis, a systemic sexually transmitted disease, is on the rise in the US, with infection rates the highest recorded since 1994 according to the CDC. Useful laboratory testing is an important diagnostic tool for determining individual syphilis infection and preventing community-wide disease spread. The purpose of this study was to determine the usefulness of a specific automated treponemal test method, the CaptiaTM Syphilis IgG EIA, and the syphilis reverse algorithm interpretation for detecting syphilis infection among patients seeking care in a public health clinic. The study employed a retrospective, nonexperimental descriptive correlational design with data collected between 2012-2013 from 4,077 public health clinic patients with 21% of the patients diagnosed with syphilis infection. There was a statistically significant difference between the CaptiaTM Syphilis IgG and the Fujirebio Serodia TP-PA test results; between the CaptiaTM Syphilis IgG Signal to Cutoff (S/CO) and the MacroVue RPR titer continuous variables; and between the reverse and traditional syphilis interpretation algorithms. The reverse algorithm using the CaptiaTM Syphilis IgG test method provided more useful performance measures with a sensitivity of 82%; specificity of 99%; accuracy of 95%; positive likelihood ratio of 63.06 and negative likelihood of 0.18 than the traditional algorithm using the MacroVue RPR test method. Statistical comparison of the area under the curve (AUC) for the continuous variables, CaptiaTM Syphilis IgG S/CO and RPR titer, concluded that the Syphilis IgG AUC (0.9500) was higher than the RPR titer (0.8155) indicating greater accuracy for detecting syphilis infection. This was the first study to determine that the CaptiaTM Syphilis IgG, the S/CO value, and reverse algorithm are useful diagnostic predictors of syphilis infection among public health clinic patients. The data from this study can be utilized by future researchers and scientists who are developing or improving syphilis detection methods.

The invasive pathways, InlA- and InB-mediated uptake and M cell transcytosis, that Listeria monocytogenes uses to invade the intestine have mainly been studied using infection models that do not truly replicate what occurs during a natural infection. Recently, our lab has developed an oral infection model that is more physiolocally relevant to what occurs during food borne listeriosis. We have sought to evaluate the relative roles of the previously defined invasive pathways, in our oral model of infection. We have done this by utilizing an InlAmCG Lm strain that is able to bind murine E-cadherin, knockout Lm strains, ΔinlA Lm, and ΔinlAΔinlB Lm. We also took advantage of a knockout mice strain CD137-/-that has M cells that are deficient in M cell transcytosis. We were able to show that these invasive pathways are relevant in our oral infection model, that M cell transcytosis is a compensatory pathway for InlA-mediated uptake, and that there might be another mechanism that L. monocytogenes uses to invade the intestines. To confirm this, it is necessary though that the M cell transcytosis deficiency be confirmed in the CD137-/- mice.

The sterol biosynthetic pathway is essential for growth of all eukaryotic cells and the main target of antifungal agents. The emergence of resistance to these antifungals in an already ill patient population indicates a need to develop drugs that have a broad spectrum of activity among pathogenic fungi and have minimal patient toxicity. Squalene synthase is the first committed step in the sterol pathway and has been studied intensively for development of antifungal agents. While the overall architecture of this enzyme is identical throughout eukaryotes, it was shown that plant and animal genes cannot complement a squalene synthase knockout mutation in yeast unless the carboxy-terminal domain is swapped for one of fungal origin. This implies that there is a component of the fungal carboxy-terminal domain that is responsible for the complementation phenotype and that is unique to the fungal kingdom of life. To determine the role of the carboxy-terminal domain of squalene synthase in the sterol pathway, we used the yeast Saccharomyces cerevisiae with a squalene synthase knockout mutation and expressed squalene synthases originating from fungi, plants, and animals. In contrast to previous observations, all enzymes tested could partially complement the knockout mutation when the genes were weakly expressed. When induced, non-fungal squalene synthases could not complement the knockout mutation and instead led to the accumulation of carboxysterol intermediates, suggesting an interaction between squalene synthase and the downstream sterol C4-decarboxylase. Overexpression of a sterol C4-decarboxylase from any kingdom of life both decreased the accumulation of carboxysterol intermediates and allowed non-fungal squalene synthases to complement the squalene synthase knockout mutation. Using chimeric squalene synthases from each kingdom of life, the motif in the C-terminal domain responsible for preventing this toxicity was mapped to a kingdom-specific 26-amino acid hinge motif adjacent to the catalytic domain. Furthermore, over-expression of the carboxy-terminal domain alone containing a hinge motif from fungi, not from animals or plants, led to growth inhibition of wild-type yeast. Since this hinge region is unique to and highly conserved within each kingdom of life, this data provides evidence for the development of an antifungal therapeutic as well as for tools to develop an understanding of triterpene catalytic activity and identify similar motifs in other biosynthetic pathways.

The purpose of this study was to determine the prevalence of Lyme Disease (LD) bacteria in adult Ixodes pacificus ticks collected from the mountains of San Bernardino County in Southern California. Seven hundred fifty four I. pacificus adults were collected from the Pacific Crest Trail and adjacent areas. The Polymerase Chain Reaction (PCR) was used to screen ticks for Borrelia burgdorferi infection by targeting two different DNA loci. Oligonucleotide primers targeting both the ospA and fla genes were used in the assay. Ticks were processed in pools of three, and genomic DNA from the ticks was extracted with a commercial mini-kit utilizing silica matrix spin-columns. All ticks tested negative for B. burgdorferi infection regardless of primer pair used. In addition, ticks were negative following examination by dark-field microscopy. This study confirms previous reports that the prevalence of LD in Southern California is quite low.

Chlamydia is the most common bacterial sexually transmitted infection in the United States and worldwide. It often goes unnoticed due to lack of symptoms and left untreated it can ascend the female genital tract to cause sequelae like pelvic inflammatory disease and irreversible tubal infertility. In reproductive-aged women, female sex hormones estrogen (E2) and progesterone (P4) concentrations fluctuate during the menstrual cycle and are influenced by hormonal contraceptives and hormone replacement therapy. E2 and P4 influence genital Chlamydia infection in women and mice, but these multifactorial interactions are not entirely mapped out. The complex interplay of E2 and P4 with Chlamydia and the host response demand further study to determine the effect of hormonal environment and host susceptibility to Chlamydia. E2 primarily signals through estrogen receptors (ER) ERα and ERβ. We used ERα or ERβ knockout (KO) mice to study the role of E2 and ERs in chlamydial progression and examined the host immune response at day 9 post-infection, when we expected the immune response to be the most robust. ERαKO, but not ERβKO mice had significant differences in the progression of Chlamydia and the host immune response. Future studies should test the immune response at additional timepoints, and a model should be utilized wherein ERα and ERβ are simultaneously silenced by chemical knockdown of ERβ in ERα knockout mice using ER agonist ICI 182, 680. 3 Mice are widely used in Chlamydia research, but due to its short estrus cycle, infection cannot be established naturally before infected cells are shed. To overcome this, mice are pretreated with depot medroxyprogesterone acetate (DMPA), an exogenous progesterone that halts the estrus cycle. However, a mouse model not reliant on DMPA pretreatment is needed because 1.) DMPA can affect the immune response and 2.) the hormonal environment in women is not static. Our model uses mice that are ovariectomized to stop the production of endogenous E2 and P4, then treated with physiologically relevant levels of E2 and P4 via implantation of a hormone-filled capsule. We observed that E2 protected mice from Chlamydia, making our model a good alternative for in vivo Chlamydia studies.

Tuberculosis (TB), a transmissible disease caused by Mycobacterium tuberculosis (Mtb), is a global health threat. To design an effective vaccine, we need to better understand how different elements of our immune system collaborate to fight against Mtb. CD4 T cells are crucial in protective immunity to Mtb because they produce cytokines including interferon-γ. In contrast, CD8 T cells are thought to play a modest role. Whether CD4 T cells act as “helper” cells to promote optimal CD8 T cell responses during TB is unknown. We argue CD8 T cells’ role are likely underestimated because CD8 T cell functions are compromised without CD4 T cells. Here, using two independent models, I show that CD4 T cell help promotes CD8 T cell effector functions and prevents CD8 T cell exhaustion. I demonstrate CD4 and CD8 T cells synergistically enhance the survival of infected mice. Purified helped, but not helpless, CD8 T cells effectively restrict intracellular Mtb growth. Thus, CD4 T cell help is indispensable for generating protective CD8 T cell responses. In addition, I investigate the mechanisms of CD4 T cell help. Signals from CD4 T cells, and signals relayed by antigen presenting cells collectively shape CD8 T cell responses. We infer that vaccines aimed for eliciting both CD4 and CD8 T cells, in which CD8 T cells are properly helped by CD4 T cells, are more likely to be successful.

Quorum sensing molecules have become a recent focus of study to learn if and how they can be used, both on their own and in conjecture with current antimicrobial methods, as a means of bacterial control. One such quorum sensing molecule is the sesquiterpene alcohol, Farnesol, which is synthesized and released by the fungus, Candida albicans. In most in-vivo cases, our laboratory has shown that Alcaligenes faecalis overtakes C. albicans, preventing its growth. However, as a way to counteract this inhibitory effect, Farnesol may be one way that Candida has found to fight back. In this study, we focused on the inhibitory properties of Farnesol for growth and motility of A. faecalis, as well as, the molecule’s ability to prevent Alcaligenes from creating biofilms and/or degrading them once they have already been established. Our experiments show evidence that Farnesol is able to inhibit both the growth and motility of A. faecalis, and determination of the specific concentrations of Farnesol needed to see the largest effects on A. faecalis biofilms. Our hope is that in future studies, we will be able to add varying concentrations of the Farnesol to known and widely used antibiotics in order to increase the effectiveness of antibiotics against bacterial strains, both in the Alcaligenes genus and in other genus, that have previously been considered “antibiotic resistant”.

In 2008 the Nobel Prize in Physiology or Medicine was awarded to the co-discoverers of the Human Immunodeficiency Virus Type 1 (HIV-1), the causative agent of Acquired Immunodeficiency Syndrome (AIDS). This award acknowledged the enormous worldwide impact of the HIV-1/AIDS pandemic and the importance of research aimed at halting its spread. Since the syndrome was first recognized, 25 million people have succumbed to AIDS and over 33 million are currently infected with HIV-1 (www.unaids.org). The most effective strategy for ending the pandemic is the creation of a prophylactic vaccine. Yet, to date, all efforts at HIV-1 vaccine design have met with very limited success. The consistent failures of vaccine candidates stem in large part from the unprecedented diversity of HIV-1. Among the novel theories of vaccine design put forward to address this diversity is the targeted vaccine approach. This proposal is based on the finding that mucosal transmission of HIV-1, the most prevalent form, occurs across a selective bottleneck such that typically only a single (or a few) variants of the viral swarm present in a donor are passed to the recipient. While the mechanisms controlling the selection are largely unknown, the targeted vaccine approach postulates that once they are identified, we can utilize this understanding to design vaccines specifically targeted to the characteristics shared by the rare, mucosally transmissible HIV-1 variants. The studies described in this work were conducted to improve our understanding of the factors influencing viral variant selection during mother-to-child-transmission of HIV-1, a route of mucosal transmission which has globally become the leading cause of child infection. A unique panel was generated, consisting of nearly 300 HIV-1 envelope genes cloned from infected mother-infant pairs. Extensive characterization of the genotypes, phenotypes and phylogeny of these clones was then done to identify attributes differentiating early infant from maternal variants. Low genetic diversity of HIV-1 envelope variants was detected in early infant samples, suggesting a bottleneck and active selection of variants for transmission. Transmitted variants did not differ from non-transmitted variants in CD4 and CCR5 use. Infant isolates replicated poorly in macrophages; a cell subtype hypothesized to be important in the establishment of infection. The sensitivity of infant envelope variants to neutralization by a panel of monoclonal antibodies, heterologous and autologous plasmas and HIV-1 entry inhibitors varied. Most intriguingly, envelopes cloned from infants infected during delivery exhibited a faster entry phenotype than maternal isolates. Together, these findings provide further insight into viral variant selection during mother-to-child transmission. Identification of properties shared by mucosally transmitted viral variants may allow them to be selectively targeted, resulting in improved methods for preventing HIV-1 transmission.