Doctor of Philosophy, Case Western Reserve University, 2024, Molecular Biology and Microbiology

Antimicrobial resistance is one of the greatest threats to human health. The β-Lactams (including penicillins, cephalosporins, carbapenems, and monobactams) are safe, effective, widely-used antibiotics. Mimicking an essential peptide bond and inhibiting enzymes involved in peptidoglycan biosynthesis, β-lactams disrupt the cell wall, killing susceptible organisms. Bacteria have developed resistance mechanisms to β-lactams, of which β-lactamases are the most prevalent and problematic. β-Lactamases hydrolyze the namesake, four-membered, cyclic amide β-lactam ring essential to the activity of β-lactam antibiotics, rendering them ineffective. Unfortunately, β-lactamases are constantly evolving in clinics and the environment and substrate expansion means fewer antibiotics are available to treat a given infection. Pseudomonas aeruginosa is a commonly multi-drug resistant (MDR) pathogen and Pseudomonas-derived cephalosporinase (PDC) is a chromosomal β-lactamase and major resistance determinant in the species. Herein, we take a comprehensive and multidisciplinary approach to understanding and overcoming PDC-mediated antibiotic resistance. We begin by proposing a standardized numbering scheme for class C β-lactamases, seeking eliminate the confusion that commonly arises when different research groups refer to the same amino acids using different designations. Next, we microbiologically and biochemically characterize and elucidate the mechanism of two PDC variants associated with oxyimino-cephalosporin resistance: a tyrosine to histidine substitution at amino acid 221 (associated with ceftolozane-tazobactam and ceftazidime-avibactam resistance) and the deletion of threonine 289 and proline 290 (associated with cefepime resistance). These variants modify the Ω-loop and R2-loop (regions bounding opposite sides of the active site and playing a crucial role in substrate specificity), respectively. Both variants lead to substrate expansion through a kcat or k3 driven mechanism ass (open full item for complete abstract)

Committee: Robert Bonomo (Advisor); Focco van den Akker (Committee Member); Krisztina Papp-Wallace (Committee Member); W. Henry Boom (Committee Chair) Subjects: Biochemistry; Bioinformatics; Microbiology; Molecular Biology

Doctor of Philosophy, Miami University, 2024, Microbiology

Acinetobacter baumannii is a Gram-negative opportunistic pathogen commonly associated with severe nosocomial infections such as pneumonia, urinary tract infections, wound infections, and meningitis in immunocompromised individuals. This bacterium poses a serious global health threat due to its intrinsic and acquired resistance to currently available antibiotics, resulting in a high mortality rate. Given that the treatment options are limited, a comprehensive understanding of host immune responses is essential to develop alternative therapeutics against this human pathogen. Because we aim to better understand host immune responses, with a long-term goal of developing novel immunotherapeutics, our study has focused on determining the functions of innate B cells and the signaling lymphocyte activation molecule family member 9 (SLAMF9) receptor during A. baumannii infection. The work presented in this dissertation shows that B cell-deficient mice are highly susceptible to A. baumannii respiratory infection. Our work also demonstrates that passive transfer of natural antibodies (NAbs) can rescue mice from severe infection, and that NAbs exert this protective function through complement-mediated opsonophagocytosis of A. baumannii. RNA sequencing and flow cytometric analyses of mouse lung tissues reveal that B cells are crucial for regulating pulmonary recruitment of multiple innate immune cells, including eosinophils and natural killer cells, following A. baumannii infection. They are also important for preventing extrapulmonary dissemination of A. baumannii at early stages of infection. In this study, we also examined the role of SLAMF9, a poorly characterized leukocyte receptor, using an intranasally infected mouse pneumonia model. The SLAMF9-deficient (Slamf9-/-) mice exhibit enhanced clearance of A. baumannii from the lung, liver, and spleen at 24 hours post-infection. Transcriptomic analysis of whole lung tissue shows significantly higher expression of several interf (open full item for complete abstract)

Committee: Timothy Wilson, PhD (Advisor); Luis Actis, PhD (Advisor); Paul James, PhD (Committee Member); Joseph Carlin, PhD (Committee Member); Mitchell Balish, PhD (Committee Member) Subjects: Microbiology

Honors Theses, Ohio Dominican University, 2023, Honors Theses

Antibiotic resistance is one of the world's fastest growing and most prevalent problems today. The influx of antibiotics within our environment from inadequate antibiotic stewardship has led to surge of drug resistant microorganisms. New drug development is imperative to combat infections caused by drug resistant pathogens. Bacterial translation, the process of protein synthesis by the ribosome, is a common target for new antibiotic development. Elongation factor P (EF-P) is a universally conserved protein that alleviates ribosomal pausing by aiding in peptide bonding at polyproline motifs. We characterize the role of EF-P in clinically related phenotypes within A. baylyi. EF-P is required for biofilm formation, surface associated motility, and resistance to beta-lactams and carbapenems within A. baylyi. The data we present holds hope for future drug development targeting EF-P in pathogens closely related to A. baylyi.

Committee: Anne Witzky (Advisor); Emily Post (Committee Member); Michael Doughtery (Committee Member) Subjects: Biology; Microbiology

Doctor of Philosophy, Miami University, 2021, Microbiology

Acinetobacter baumannii is Gram-negative bacterial human pathogen that can be found in myriad environmental niches and, more notoriously, thrives in the hospital setting. This opportunistic pathogen is the causative agent in a range of disease outcomes including wound infections, urinary tract infections, meningitis, and pneumonia. The ability of A. baumannii to persist on medical equipment in the hospital environment, colonize the human host, and express a number of antibiotic resistance mechanisms makes it a formidable nosocomial threat and justifies the investigation of the molecular mechanisms underlying its virulence and overall physiology. A. baumannii uses various sensing and regulatory systems to coordinate a response to cues from its local environment. Of particular importance in the context of this study are the cues of light and temperature. The goal of the work presented in this manuscript was to elucidate the molecular mechanisms underlying light sensing and regulation in A. baumannii at two different temperatures, 24°C and 37°C. The dependence of the bacterial oxidative stress response on the blue light using flavin protein BlsA at 24°C was tested using catalase activity, peroxide resistance, and superoxide dismutase (SOD) assays. BlsA's regulation of the catalase enzyme KatE was dependent on the BlsA residue lysine 144, and its regulation of SOD activity was dependent on the last five BlsA residues. Blue light-dependent, BlsAindependent regulation at 24°C was examined by RNA sequencing analysis and investigation of the role of the putative photolyase PhrB in light-dependent functions. Global blue light-dependent transcriptional regulation was apparent even in the absence of BlsA at 24°C. Furthermore, PhrB did not play a detectable role in canonical photolyase activity yet did impact bacterial surface-associated motility, a light-regulated phenotype in A. baumannii. For bacteria cultured at 37°C, a temperature at which BlsA is not (open full item for complete abstract)

Committee: Luis Actis PhD (Advisor); Kelly Abshire PhD (Committee Member); Mitchell Balish PhD (Committee Member); Rick Page PhD (Committee Member); Timothy Wilson PhD (Committee Member) Subjects: Microbiology

Master of Science, Miami University, 2019, Microbiology

The BfmRS two-component regulatory system of Acinetobacter baumannii plays roles in biofilm formation, pili production, capsule production, and other functions essential for colonization and persistence. Based on previously-collected RNA sequencing data comparing wild-type A. baumannii ATCC 19606T to an isogenic BfmR-deficient strain, we hypothesized that BfmR negatively regulates transcription of the genes for acinetobactin biosynthesis, secretion, and uptake for A. baumannii ATCC 19606T. Considering the recent identification of a BfmR-binding inverted repeat sequence, we also hypothesized that BfmR directly regulates the transcription of genes containing the BfmR-binding site upstream of the predicted start codon. Experimental analyses performed in this work included immunoblotting assays, qRT-PCR, electrophoretic mobility shift assays, molecular cloning techniques, and biofilm formation assays. The data acquired in this thesis demonstrate that BfmR is capable of repressing the expression of acinetobactin biosynthesis, secretion, and uptake genes. We also present information regarding the ability of BfmR to exhibit direct and indirect transcriptional regulation of metabolite transport and pili production genes.

Committee: Luis Actis (Advisor); Mitchell Balish (Committee Member); Timothy Wilson (Committee Member); Michael Crowder (Committee Member) Subjects: Microbiology

Doctor of Philosophy, Miami University, 2019, Microbiology

Acinetobacter baumannii is a prevalent human pathogen commonly associated with severe nosocomial infections such as ventilator-associated pneumonia and wound infections in immunocompromised individuals. This pathogen is a global concern due to its ability to acquire resistance to antimicrobials and persist within the hospital environment for weeks under different pressures such as nutrient limitation and desiccation. The capacity of A. baumannii to survive in different niches, including in and on human hosts or abiotic environments within clinical settings suggests that this pathogen senses and responds to its surroundings to modulate its physiology to survive. Recently, it has been reported that A. baumannii senses and responds to blue light through the photoreceptor protein BlsA, with both factors playing significant roles in the regulation of surface motility, biofilm formation, metabolism and antibiotic resistance responses. The work presented here shows that the interaction of the BlsA N-terminal region with flavin chromophores determines light sensory functions and protein stability, while the C-terminal region could play critical photocycling and downstream regulatory functions. This work also demonstrates that the ability of A. baumannii to differentially display surface motility and biofilm formation on plastic at 24°C depends on the active expression of the PrpABCD type I pilus assembly system. Unexpectedly, analysis of a PrpA deficient mutant resulted in the detection of light regulated motility responses by bacteria cultured at 37°C, a condition that impairs BlsA production and its sensory functions. These unexpected observations suggest that A. baumannii senses and responds to illumination when incubated at 24°C and 37°C using different light sensing and regulatory systems. The application of a shotgun proteomics approach not only confirmed the predicted light-mediated BlsA-dependent differential expression of proteins at 24°C, but also showed that ligh (open full item for complete abstract)

Committee: Luis Actis Dr. (Advisor) Subjects: Microbiology

MS, University of Cincinnati, 2017, Medicine: Molecular Genetics, Biochemistry, and Microbiology

A novel biocide, AB569 containing acidified nitrite (A-NO2-) and Ethylenediaminetetraacetic Acid (EDTA) combined, has demonstrated bactericidal activity in previous studies with Pseudomonas aeruginosa (Pa) (1) Multi-Drug Resistant Organisms (MDRO) present a challenge to the all-inclusive killing abilities of AB569. Thus, Acinetobacter baumannii (Ab) and Acinetobacter spp. present a model with widely known resistance to antibiotics. Killing studies with AB569 reveal bactericidal action with extended exposure time, 24 to 48 hr, and effective results at key concentrations during shorter inhibitory exposure time, 1 mM EDTA and 32 mM A-NO2-. AB569 as a treatment for Ab is a viable option to prevent bacterial growth, with tests revealing average Fractional Inhibitory Concentration (FIC) concentrations of 0.25 mM EDTA plus 4 mM A-NO2 - across several reference and clinical strains. In addition, toxicity testing on Adult Human Dermal Fibroblasts (HDFa) revealed an upper toxicity limit of 3 mM EDTA plus 64 mM A-NO2 -. AB569 FIC concentrations are within the HDFa cell toxicity range for effective Ab and Acinetobacter spp. treatment. Following treatment with AB569, quantitative PCR revealed up-regulated siderophore production on EDTA treated sample in Siderophore Biosynthesis Non-Ribosomal Peptide Synthetase Module (SBNRPSM) and Siderophore Biosynthesis Protein, Monooxygenase (SBPM) genes compared to untreated control during inhibitory conditions, which was not observed with AB569 treatment. Treating Ab infections with AB569 at inhibitory concentrations reveals the potential clinical benefit of preventing Ab from gaining a growth advantage early in a mixed infection followed by bactericidal activity with extended treatments

Committee: Daniel Hassett Ph.D. (Committee Chair); Edmund Choi Ph.D. (Committee Member); Alison Weiss Ph.D. (Committee Member) Subjects: Molecular Biology

Doctor of Philosophy, The Ohio State University, 1975, Graduate School

Committee: Not Provided (Other) Subjects: Biology

Doctor of Philosophy, Miami University, 2016, Microbiology

Acinetobacter baumannii is an opportunistic nosocomial pathogen that has a global impact on human health. The capacity of A. baumannii to cause a wide variety of severe infections, combined with the emergence of multi-drug resistant isolates, present significant challenges toward the development of new therapies to treat A. baumannii infections. Biofilm formation on abiotic surfaces, which is a key factor in the persistence of this pathogen, is controlled by the BfmRS two-component regulatory system (TCS). However, the role of this TCS in other Acinetobacter isolates is not understood. The work presented herein demonstrates that the BfmR response regulator provides conserved global regulatory functions, which includes the regulation of adherence and pathogenicity factors, in both A. baumannii and Acinetobacter nosocomialis. In contrast to the interaction of A. baumannii with abiotic surfaces, the mechanism by which this pathogen regulates attachment and virulence factors in biotic environments is unknown. Our work provides insight into this pathway by demonstrating that A. baumannii responds to mucin, a major component secreted by lung epithelial cells particularly during respiratory infections, which triggers a cascade of responses that allow this pathogen to acquire critical nutrients and enhance the expression of virulence factors in the host environment. Furthermore, along with data collected by others, our findings shed light on the important role of mucin in the effectiveness of antibiotics toward A. baumannii in biotic environments where this glycoprotein is present. Taken together, these findings provide a deeper understanding of how A. baumannii uses a complex regulation of adherence and virulence factors in response to specific environmental signals to promote its persistence and pathogenicity within abiotic and biotic environments.

Committee: Luis Actis PhD (Advisor); Mitchell Balish PhD (Committee Member); Rachael Morgan-Kiss PhD (Committee Member); Joseph Carlin PhD (Committee Member); Nicholas Money PhD (Committee Member) Subjects: Genetics; Molecular Biology

Doctor of Philosophy, The Ohio State University, 2015, Integrated Biomedical Science Graduate Program

Acinetobacter nosocomialis is a member of the Acinetobacter calcoaceticus-baumannii complex, which are Gram-negative opportunistic pathogens of increasing relevance worldwide. Medically relevant Acinetobacter species form biofilms, are resistant to desiccation, and easily acquire antibiotic resistance genes, all of which contribute to its ability to cause disease. Despite many reports on the epidemiology and antibiotic resistance phenotypes of Acinetobacter, there are limited reports characterizing the virulence mechanisms of these important nosocomial pathogens. Type IV pili (Tfp) are transenvelope protein complexes that can act as surface appendages mediating many bacterial processes. Analysis of the genomes of fully sequenced medically relevant Acinetobacter strains reveals the presence of genes that encode proteins predicted to be involved with the biogenesis of Tfp. Furthermore, many medically relevant Acinetobacter species have been shown to exhibit twitching motility and natural transformation, two classical Tfp-associated phenotypes. Therefore we utilized mutagenesis strategies to selectively delete genes encoding proteins predicted to be involved in Tfp biogenesis and probed for Tfp functionality. In our analysis we determined that A. nosocomialis strain M2 did produce functional Tfp, which were required for natural transformation and twitching motility. During the course of our studies we also identified that the major pilin subunit, PilA, of the Tfp fiber was post-translationally modified. Subsequently we determined that PilA was glycosylated by the pilin-specific oligosaccharyltransferase, TfpO, at the carboxy-terminal serine. Lastly, we demonstrated that many Acinetobacter species encode two functional oligosaccharyltransferases, one devoted exclusively to pilin glycosylation and the other to general protein glycosylation. This study is the first to describe the production of functional Tfp production by a medically relevant Acinetobacter species and a (open full item for complete abstract)

Committee: Daniel Wozniak Ph.D. (Advisor); Amal Amer M.D., Ph.D. (Committee Member); Larry Schlesinger M.D. (Committee Chair); Kevin Mason Ph.D. (Committee Member) Subjects: Biomedical Research; Microbiology

MS, University of Cincinnati, 2012, Medicine: Molecular Genetics, Biochemistry, and Microbiology

In the past two decades Acinetobacter baumannii (Ab) has grown from relative obscurity to one of the most important global nosocomial infections. Ab is classified as a low grade pathogen, infecting only the immunocompromised. Overtime, Ab has evolved the capability to incorporate extraneous DNA into its own genome which has led to a dramatic increase in resistance to conventional antibiotics. To compound the problem, Ab can form biofilms which provide an even greater level of resistance against antibiotics and environmental pressures including biocides and desiccation. Adherent properties of Ab biofilms enable the organism to grow on a variety of surfaces and medical devices composed of glass, plastic or steel and instruments including stethoscopes and ventilator tubing. A previous study was conducted using High-Throughput Screening (HTS) to identify novel compounds that are capable of killing and/or inhibiting biofilm formation of Pseudomonas aeruginosa (Pa) or Staphylococcus epidermidis (Se) and Acinetobacter baumannii (Ab) biofilms. The novel compounds were divided into 5 groups based on the HTS results against one or more of the microorganisms. The first group of compounds (designated 3, 7, 8 and 11) were effective against Pa, Se and Ab biofilms. Group 2 compounds (1, 2, 9, 10, 12, and 13) were effective against only Pa and Ab biofilms. Group 3 compounds (14, 15 and 16) were effective only against Pa biofilms. Group 4 compounds (Se1 to Se16) were effective only against Se biofilm. Finally, group 5 compounds (17 to 34) were effective only against Ab biofilm. The biofilms were challenged in two phases. The first phase determined the compounds' ability to inhibit the initial formation of a biofilm. The second phase determined the compounds' ability to kill a mature biofilm. Confocal Laser Scanning Microscopy (CLSM) was used to study the effects of the compounds on initial biofilm formation and killing of mature biofilm. Of the compounds examined for inhibiting in (open full item for complete abstract)

Committee: Daniel Hassett PhD (Committee Chair); Thomas Lamkin PhD (Committee Member); Edmund Choi PhD (Committee Member) Subjects: Microbiology

Bachelor of Arts, Miami University, 2011, College of Arts and Sciences - Microbiology

Acinetobacter baumannii is a gram-negative bacterium that causes severe infections in immunocompromised patients, such as newborns, burn patients, and the elderly. Because the bacteria are strongly resistant to antibiotics, there is a dire need to develop new therapeutics to treat A. baumannii infections. Potential targets are proteins involved in bacterial iron metabolism, since iron is an essential micro-nutrient. Accordingly, random insertion mutagenesis analysis showed that the NfuA protein is needed when cells were cultured in the presence of 2,2'-dipyridyl, a synthetic iron chelator that generates iron-limiting conditions, and hydrogen peroxide and cumene hydroperoxide, which were used to mimic oxidative conditions. The role of NfuA was further confirmed by the observation that the genetic complementation of an A. baumannii ATCC 19606T mutant with the parental allele was enough to restore the iron metabolism and oxidative stress phenotypes expressed by the wild-type strain. Electron paramagnetic resonance (EPR) analysis of overexpressed and purified NfuA demonstrated that this protein harbors an iron-sulfur cluster, which is a prosthetic group required in central metabolic processes. Interestingly, the inactivation of NfuA did not affect bacterial growth under non-oxidative and non-chelated conditions and did not impair the ability of the mutant to express the acinetobactin siderophore-mediated iron acquisition system. On the other hand, the ability of the A. baumannii ATCC 19606T nfuA mutant to replicate inside human epithelial cells was significantly impaired when compared with the parental strain. Taken together, these observations suggest that NfuA plays a defined and important role in iron metabolism, resistance to oxidation, and intracellular replication without affecting bacterial iron acquisition processes. By understanding the function of NfuA and its importance to the A. baumannii virulence properties, we will come closer to understanding basic metab (open full item for complete abstract)

Committee: Luis Actis PhD (Advisor); Daniel Zimbler (Committee Member); William Penwell (Committee Member) Subjects: Microbiology

Doctor of Philosophy, Miami University, 2010, Microbiology

Acinetobacter baumannii is a gram-negative opportunistic pathogen that has emerged as a problematic organism, causing severe infections in human hosts. This is compounded by the fact that clinical isolates of this organism are often resistant to multiple types of antimicrobial therapies. To better understand the molecular mechanisms involved in pathogenicity with the long-term goal of finding targets for novel therapies of the future, the work presented in this manuscript focuses on the factors involved in the adherence to abiotic and biotic surfaces, invasion of epithelial cells, and killing of invertebrate animal models. In addition, this work presents novel data about the regulation of those factors. Adherence to biotic surfaces is often the first step of pathogenesis. It is apparent that outer membrane protein A is required for adherence of A. baumannii ATCC 19606T cells to human respiratory epithelia. In addition, cells lacking the productionof this protein are deficient in their ability to kill eukaryotic cells such as A549 human alveolar cells and also Candida albicans tup1 filaments. Furthermore, it was proven that the death of these cells is a result of an induction of apoptosis and secreted proteins in addition to OmpA are implicated in that process. After the bacterial cell invades the A549 eukaryotic cell, it requires the function of an iron acquisition system, specifically acinetobactin biosynthesis and transport. Without these functions, bacterial cells can adhere to the surface of A549 cells, but cannot persist and replicate in the cytoplasm of the eukaryotic cell. In addition, the apoptotic induction in A549 cells is lowered when infections are performed with mutants lacking iron acquisition functions. Interestingly, A. baumannii cells lacking siderophore biosynthesis can be crossfed during infections of A549 cells via co-infection with parental strain expressing full function of siderophore biosynthesis. These results are also supported by animal mo (open full item for complete abstract)

Committee: Luis Actis PhD (Advisor); Gary Janssen PhD (Committee Member); Rachael Morgan-Kiss PhD (Committee Member); Annette Bollmann PhD (Committee Member); David Pennock PhD (Committee Member) Subjects: Microbiology

Doctor of Philosophy, Miami University, 2010, Microbiology

Acinetobacter baumannii is an opportunistic nosocomial pathogen of substantial concern due to its increasing antibiotic resistance and ability to survive unfavorable conditions in the hospital environment, likely in the form of a biofilm, which potentiates its ability to spread. However, the information about its virulence factors and the regulatory networks that could control their expression is insufficient. This lack of data is compounded by large variations in genotypic and phenotypic traits among clinical isolates as illustrated by the differences in biofilm structures they form on plastic or glass. These biofilm variations did not correlate with properties of abiotic surfaces or other cell properties, such as surface hydrophobicity, pellicle formation, surface-associated motility, or somatic appendages. It is apparent that environmental signals, such as the concentration of extracellular free iron, could affect the aforementioned interactions, as measured by changes in cell motility on semisolid media and biofilm formation on plastic. These responses are independent of pilT and pilU orthologs, which impact motility and biofilm functions in other pathogens. However, these two genes play a role in the interaction of A. baumannii ATCC 17978 cells with human alveolar epithelial cells which results in the apoptotic death of these epithelial cells. The A. baumannii responses to multiple environmental cues may be attributable to the sensing and response function of regulators such as that coded by the bfmL locus present in the ATCC 19606T strain. Interestingly, this LysR-type transcriptional regulator (LTTR) is needed for the post-transcriptional expression of the CsuA/BABCDE chaperone-usher pilus assembly system needed for cell attachment and biofilm formation on plastics. Equally interesting is that inactivation of bfmL results in the production of pili different from those produced when the aforementioned assembly system is expressed. Taken together, these resu (open full item for complete abstract)

Committee: Luis Actis PhD (Advisor) Subjects: Microbiology

Doctor of Philosophy, Miami University, 2004, Microbiology

Acinetobacter baumannii is an important human pathogen that causes severe respiratory diseases in compromised patients. This bacterium is capable of surviving on nutrient-limited abiotic surfaces, such as bed linens, hospital equipment, and medical devices. This survival property suggested the possibility that this pathogen could form biofilms to survive such unfavorable conditions. Biofilms are aggregates of bacterial cells that are metabolically and physiologically distinct from their planktonic counterparts. Cells composing these structures work together to acquire nutrients and survive harsh environmental conditions. This hypothesis was proven by observing biofilm formation by A. baumannii on abiotic surfaces such as plastics. This formation seems to occur independently of cell motility, as we have been unable to detect such a capability in this organism. Random insertion mutagenesis revealed the presence of a chaperone-usher secretion system, which was found to be involved in the assembly of pili on the bacterial cell surface. These pili are essential for attachment and subsequent biofilm formation on abiotic surfaces, and are assembled using a cellular machinery similar to that described previously in other gram-negative pathogenic organisms. The same mutagenesis approach allowed for the discovery of a putative two-component regulatory system, which is also involved in this attachment process. Interestingly, this regulatory circuit controls pili production by activating the transcription of the chaperone-usher system described above. Additionally, this regulatory system controls cellular morphology in a nutrient-dependent fashion. The ability of A. baumannii to form biofilms on biotic surfaces was also tested in this work. It was found that this organism could attach to two distinct biotic surfaces, Candida albicans and HeLa cells, and that the presence of pili on the bacterial cell surface was not as essential to attachment as it was for abiotic surface colon (open full item for complete abstract)

Committee: Luis Actis (Advisor) Subjects: Biology, Microbiology

Doctor of Philosophy, Case Western Reserve University, 2010, Pathology

Since the introduction of penicillin, β-lactam antibiotics have been the antimicrobial agent of choice for the treatment of many infections. Unfortunately, the efficacy of these life-saving antibiotics is significantly threatened by bacterial β-lactamase enzymes. To overcome β-lactamase-mediated resistance, β-lactamase inhibitors were introduced (clavulanate, sulbactam, and tazobactam). These inhibitors greatly enhance the efficacy of their partner β-lactams in the treatment of Gram-negative infections. However, selective pressure from excess antibiotic use accelerated the emergence of resistance to β-lactam/β-lactamase inhibitor combinations. Furthermore, the prevalence of clinically relevant β-lactamases that are intrinsically resistant to inhibition is rapidly increasing. There is an urgent need for effective inhibitors that can restore the activity of β-lactams. Here, we demonstrate that the Asn276Asp substitution confers resistance to clavulanate in the class A SHV β-lactamase. Unlike the Asn276Asp substitution in the related TEM enzyme, and inhibitor-resistant β-lactamases in general, the SHV variant maintains a high level of catalytic efficiency for penicillins. This “fine-tuning” of the inhibitor-resistant phenotype may represent a significant evolutionary advance, as the enzyme maintains a balance of desired catalytic properties. By probing Asn276 with selectively designed inhibitors, we explored how the configuration of the conserved β-lactam carboxylate impacts binding. Despite relative distance from the active site, this second-shell residue exerts important effects on enzyme-ligand interactions. Our work also addresses the class C β-lactamases from Acinetobacter spp. and Pseudomonas aeruginosa, pathogens of increasing clinical concern for which few effective therapeutic options remain. The currently available β-lactamase inhibitors are inactive against these enzymes, and thus development of “second-generation” agents is a priority. We first studied (open full item for complete abstract)

Committee: Robert A. Bonomo M.D. (Advisor); Shu G. Chen Ph.D. (Committee Chair); Focco van den Akker Ph.D. (Committee Member); Michael Harris Ph.D. (Committee Member); Marion Skalweit M.D., Ph.D. (Committee Member) Subjects: Biochemistry; Microbiology

Doctor of Philosophy, Miami University, 2013, Microbiology

Acinetobacter baumannii is a clinically important Gram-negative opportunistic nosocomial pathogen partly because of the advent of pan-drug resistant clinical isolates. This pathogen persists and grows under harsh conditions including iron limitation imposed by the environment and the human host. The ability of A. baumannii ATCC 19606T to grow under iron-limiting conditions requires the production of the siderophore, acinetobactin, which was also demonstrated to be essential for this strain to cause a successful infection. Initial studies have examined components involved in synthesis and uptake of acinetobactin; however, other components of this siderophore-mediated system have yet to be identified or characterized. The acinetobactin chromosomal gene cluster harbors all the traits needed for biosynthesis, export, and transport of this siderophore with the exception of an entA ortholog, which is needed for the production of acinetobactin precursor, 2,3-dihydroxybenzoic acid. Accordingly, the entA ortholog in ATCC 19606T was identified using genetic complementation and found located in a different genomic region next to genes coding for iron acquisition functions. Analysis of the nucleotide sequence of this ortholog with other A. baumannii sequenced genomes revealed that while most of the strains code for an active entA gene, the clinical isolate AYE has a natural entA mutation and does not produce acinetobactin. Despite not being able to produce acinetobactin, AYE is still able to grow under iron-limiting conditions, a phenotype that is in accordance with the fact that this strain produces an uncharacterized hydroxamate siderophore, which we called baumannoferrin. Comparison of the siderophore-mediated system between ATCC 19606T and AYE underline the ability of different A. baumannii isolates to acquire iron using different systems. The ATCC 19606T acinetobactin gene cluster also includes two genes coding for ABC-type efflux transport functions predicted to be (open full item for complete abstract)

Committee: Luis Actis PhD (Advisor); Mitchell Balish PhD (Committee Member); Joseph Carlin PhD (Committee Member); Annette Bollmann PhD (Committee Member); Jack Vaughn PhD (Committee Member) Subjects: Microbiology

Doctor of Philosophy, Miami University, 2013, Microbiology

Acinetobacter baumannii is an important opportunistic human pathogen that causes severe nosocomial infections. The bacterium must overcome iron starvation and oxidative stress conditions imposed by the host in order to propagate and cause disease. This work further investigates the transport and utilization of iron by A. baumannii, and their involvement in virulence. The transport of iron is an active process and requires energy; A. baumannii ATCC 19606T contains and expresses three gene loci encoding functions in the TonB energy-transducing complex to provide the energy needed for iron transport. Transformation of Escherichia coli KP1344 with plasmids harboring the TonB components of these A. baumannii TonB systems promoted cell growth under iron-chelated conditions, which shows that these systems provide the necessary energy needed for iron acquisition. Inactivation of tonB1 and tonB2 in A. baumannii resulted in growth restriction under iron-chelation, indicating these genes are involved in iron transport. The Galleria mellonella infection model showed that TonB1 and TonB2 are involved, but are not essential for bacterial virulence, indicating A. baumannii carries redundant functional TonBs. Furthermore, TonB2 plays an additional role in the interaction with A549 human alveolar cells. Inactivation of dppA1A2 and dppBC, components of an inner membrane ABC transporter, did not affect growth under iron-chelation, suggesting alternative transport functions. However, inactivation of cirA, which codes for an iron-regulated outer membrane receptor resulted in reduced growth under iron-chelated conditions, indicating CirA has a role in iron transport. Furthermore, A. baumannii expresses hemin utilization functions independent of production and transport of acinetobactin-siderophore. Following transport, iron must be integrated into the intracellular iron pool. NfuA, a [Fe-S] cluster carrier protein was found to be involved in the ability of cells to respond to (open full item for complete abstract)

Committee: Luis Actis PhD (Advisor); Kelly Abshire PhD (Committee Member); Rachael Morgan-Kiss PhD (Committee Member); Gary Janssen PhD (Committee Member); David Tierney PhD (Committee Member) Subjects: Microbiology