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Evaluating The Role Of Gras And Fadxdeba In Promoting Staphylococcus Aureus Adaptation To Host-Derived Fatty Acids Encountered At Sites Of Colonization And Infection, Robert C. Kuiack

Electronic Thesis and Dissertation Repository

Staphylococcus aureus is an opportunistic pathogen that asymptomatically colonizes 30% of humans, where it is well adapted to survive on the skin in the presence of innate defense mechanisms such as antimicrobial free fatty acids (FFA). While antimicrobial FFA function to inhibit the growth of S. aureus, they also provide a valuable source of lipids for membrane synthesis and energy production. We hypothesized that S. aureus possesses a novel antimicrobial FFA resistance pathway that is activated under conditions found on human skin, and that under these conditions, S. aureus can metabolize exogenous fatty acids to fuel growth and virulence …

Tetr Family Regulator Farr Variation Controls Antimicrobial Fatty Acid Efflux In Staphylococcus Aureus, Camryn M. Bonn

Electronic Thesis and Dissertation Repository

To colonize human skin and survive within abscesses, Staphylococcus aureus has evolved mechanisms to evade host innate defenses. USA300 has become the predominate community-acquired methicillin-resistant S. aureus (CA-MRSA) clone, which can be in part attributed to detoxification of unsaturated free fatty acids (uFFA) found in sebum and the nares. Our lab has previously identified the TetR family regulator FarR responsible for induction of the resistance-nodulation-division (RND) superfamily efflux pump FarE to promote efflux of toxic uFFA. However, RND family efflux pumps remain poorly characterized in Gram-positive species and the mechanism by which FarR regulates FarE has yet to be determined. …

Screening For Purine Transporters In S. Aureus, Tothong Sonpaveerawong

Electronic Thesis and Dissertation Repository

Staphylococcus aureus is an important human pathogen and crucial part of its pathogenesis depends on its ability to acquire purines to cause disease. In this study, I screened a library of individual mutants under purine import-dependent conditions by inhibiting the purine biosynthesis using the pharmacological agents methotrexate and 6-mercaptopurine and supplementing inosine monophosphate and/or guanine as an exogenous purine source. I identified an ATP-Binding Cassette transporter mutant that failed to grow under the selective purine transport conditions. Further growth characterization of the mutant revealed that the growth defect was not due to an inability to transport purines but rather to …

The Staphylococcus Aureus Lrgab Operon Encodes A Holin-Like Protein Involved In Pyruvate Transport, Jennifer L. Endres

Theses & Dissertations

The Staphylococcus aureus cidABC and lrgAB operons encode a well-conserved family of proteins involved in programmed cell death (PCD) during biofilm development. Based on the structural similarities that CidA and LrgA share with bacteriophage holins, we have hypothesized that these proteins function by forming pores within the cytoplasmic membrane. To test this, we utilized a “lysis cassette” system that demonstrated the abilities of the cidA and lrgA genes to support bacteriophage endolysin-induced cell lysis. In addition, the CidA and LrgA proteins were shown to localize to the surface of membrane vesicles and cause leakage of small molecules, providing direct evidence …

Elucidating The Role Of The Msaabcr Operon In Persister Cells Formation And Stress Response In Staphylococcus Aureus, Shanti Pandey

Dissertations

Staphylococcus aureus is a major human pathogen that causes lethal systemic conditions such as infective endocarditis, osteomyelitis, sepsis and pneumonia. Chronic in nature, these diseases are often refractory to the antibiotic treatment. Such recalcitrance is mainly caused due to the presence of persister cells, which are a bacterial subpopulation that exhibits extreme, yet transient antibiotic tolerance accompanied by a transient halt in the growth. However, upon cessation of antibiotic treatment, resumption in growth of persister cells causes recurrence of infections and treatment failure, displaying tremendous clinical significance. In this study, we show the involvement of the msaABCR operon in persister …

Increasing The Frequency Of Periodic Spatial Disturbance Decreases Surface Attachment Protein Expression In Staphylococcus Aureus, Ivana M. Barraza

All HCAS Student Capstones, Theses, and Dissertations

Staphylococcus aureus is an opportunistic pathogen that can cause a variety of acute and chronic illnesses. The severity of these illnesses such as sepsis, necrotizing pneumonia, and toxic shock syndrome is measured through the virulence that S. aureus inflicts on its host. Methicillin-resistant S. aureus (MRSA) is commonly associated with secondary infections and is challenging to treat given the limited selection of antibiotics that are effective against it. Accordingly, novel approaches to reduce S. aureus pathogenicity are required. S. aureus regulates pathogenesis through a cell-to-cell communication system referred to as quorum sensing. Effective communication determines the production of two broad …

The Role Of The Msaabcr Operon In Implant-Associated Chronic Osteomyelitis In Staphylococcus Aureus Usa300 Lac, Gyan S. Sahukhal, Michelle Tucci, Hamed Benghuzzi, Gerri Wilson, Mohamed O. Elasri

Faculty Publications

The msaABCR operon regulates several staphylococcal phenotypes such as biofilm formation, capsule production, protease production, pigmentation, antibiotic resistance, and persister cells formation. The msaABCR operon is required for maintaining the cell wall integrity via affecting peptidoglycan cross-linking. The msaABCR operon also plays a role in oxidative stress defense mechanism, which is required to facilitate persistent and recurrent staphylococcal infections. Staphylococcus aureus is the most frequent cause of chronic implant-associated osteomyelitis (OM). The CA-MRSA USA300 strains are predominant in the United States and cause severe infections, including bone and joint infections.

Staphylococcus Aureus Small Colony Variants Use Heme And Staphyloferrin B For Iron Acquisition, Izabela Z. Batko

Electronic Thesis and Dissertation Repository

Iron is an essential nutrient for the bacterium Staphylococcus aureus. Wild-type S. aureus utilizes various iron acquisition systems to support growth in iron deplete conditions. S. aureus small colony variants (SCVs) are associated with chronic infections, yet the mechanisms by which these variants acquire iron are unknown. Mutation of hemB, involved in heme biosynthesis, generated a stable SCV that was auxotrophic for hemin and formed small colonies on solid media. To support growth under iron deplete conditions, my data revealed that S. aureus hemB synthesizes the siderophore staphyloferrin B, but not staphyloferrin A, although both siderophores could be …

Investigation Of The Growth And Survival Of Staphylococcus Aureus In Cftr-Deficient Macrophages, Bita Azad

Electronic Thesis and Dissertation Repository

Staphylococcus aureus and its small colony variants (SCVs) are commonly isolated from the lungs of cystic fibrosis (CF) patients. Although studies have suggested that cystic fibrosis transmembrane regulator (CFTR)-deficient macrophages are diminished in their ability to kill intracellular pathogens, whether this is true for CFTR-deficient macrophages infected with S. aureus or its SCVs is unknown. I employed gentamicin protection and eFluorÔ-670-based proliferation assays to assess the intracellular replication of S. aureus in CFTR inhibitor treated THP-1 and primary human macrophages, and in primary macrophages derived from CF patient blood. My work shows that the susceptibility of CFTR-deficient macrophages to the …

Acid Resistance Mechanisms In Staphylococcus Aureus, Chunyi Zhou

Theses & Dissertations

Staphylococcus aureus is a leading cause of opportunistic infections in community and health care settings. To thrive in a great variety of environments, S. aureus has developed the capability of tolerating temporary pH changes, as well as resisting constant acid stress. To evaluate the impact of strong and weak acid stress on S. aureus, growth patterns of JE2 were monitored when cultured in chemically defined media (CDM) at various pH in the presence of hydrochloric acid (HCl), acetic acid, and lactic acid. Our results showed that S. aureus responds to strong and weak acids in different manners. S. aureus …

Biofilm Producing Clinical Staphylococcus Aureus Isolates Augmented Prevalence Of Antibiotic Resistant Cases In Tertiary Care Hospitals Of Nepal, Sarita Manandhar, Anjana Singh, Ajit Varma, Shanti Pandey, Neeraj Shrivastava

Student Publications

Staphylococcus aureus, a notorious human pathogen, is a major cause of the community as well as healthcare associated infections. It can cause a diversity of recalcitrant infections mainly due to the acquisition of resistance to multiple drugs, its diverse range of virulence factors, and the ability to produce biofilm in indwelling medical devices. Such biofilm associated chronic infections often lead to increase in morbidity and mortality posing a high socio-economic burden, especially in developing countries. Since biofilm formation and antibiotic resistance function dependent on each other, detection of biofilm expression in clinical isolates would be advantageous in treatment decision. …

The Characterization Of The Transcription Factor Msab And Its Role In Staphylococcal Virulence, Justin Batte

Dissertations

Staphylococcus aureus is a common human pathogen that is responsible for a wide range of infections, ranging from relative minor skin infections to life-threatening disease such as bacteremia, septicemia, and endocarditis. S. aureus possesses many different virulent factors that aid in its ability to cause this wide array of infections. One major virulence factor includes the production of capsular polysaccharide (CP). The production of CP plays a major role in the virulence response during infection specifically by providing S. aureus an antiphagocytic mechanism that allows the pathogen to evade phagocytosis during an infection. S. aureus has developed complex genetic regulatory …

Gas Plasma Pre-Treatment Increases Antibiotic Sensitivity And Persister Eradication In Methicillin-Resistant Staphylococcus Aureus, Li Guo, Ruobing Xu, Yiming Zhao, Dingxin Liu, Zhijie Liu, Xiaohua Wang, Hailan Chen, Michael G. Kong

Bioelectrics Publications

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious nosocomial infections, and recurrent MRSA infections primarily result from the survival of persister cells after antibiotic treatment. Gas plasma, a novel source of ROS (reactive oxygen species) and RNS (reactive nitrogen species) generation, not only inactivates pathogenic microbes but also restore the sensitivity of MRSA to antibiotics. This study further found that sublethal treatment of MRSA with both plasma and plasma-activated saline increased the antibiotic sensitivity and promoted the eradication of persister cells by tetracycline, gentamycin, clindamycin, chloramphenicol, ciprofloxacin, rifampicin, and vancomycin. The short-lived ROS and RNS generated by plasma …

Identification And Characterization Of Farr And Fare As A Regulator And Effector Of Fatty Acid Resistance In Staphylococcus Aureus, Heba Alnaseri

Electronic Thesis and Dissertation Repository

Although Staphylococcus aureus is exposed to antimicrobial fatty acids on the skin, in nasal secretions and in abscesses, specific mechanisms for regulating gene expression and intrinsic resistance in response to these fatty acids have not been reported. Through in vitro selection for increased resistance of S. aureus to linoleic acid, I identified fatty acid resistant clone FAR7, where a single nucleotide polymorphism caused a His₁₂₁Tyr substitution in an uncharacterized member of the TetR family of transcriptional regulators, which is divergently transcribed from a gene encoding a member of the resistance-nodulation-division superfamily of multi-drug efflux pumps. I named these …

Development Of A Cholera Toxin Cta2/B Based Staphylococcus Aureus Vaccine To Prevent Bovine Mastitis, Neha Misra

Boise State University Theses and Dissertations

Staphylococcus aureus is an important pathogen causing chronic and invasive disease worldwide. This bacterium is a leading cause of community and hospital acquired infections in humans, and is also known to infect wild and domestic animals. Bovine mastitis, or inflammation of the udder, is one of the most economically relevant diseases of the dairy industry, with a high incidence worldwide. S. aureus is a major etiological agent causing this disease. S. aureus mastitis is highly contagious and difficult or impossible to treat. Management practices at dairy farms, that include good sanitation and antibiotic use, have been partially successful in reducing …

Molecular Subtyping Of Staphylococcus Aureus Isolates From The U.P. Community For The Presence Of Toxin-Encoding Genes, Carol I. Kessel

All NMU Master's Theses

Staphylococcus aureus is the most common cause of human bacterial infections; however, humans can also be asymptomatically colonized with S. aureus. Asymptomatic carriers can potentially spread S. aureus infection to others. These infections can range from mild to severe. The pathology of a S. aureus infection is often dependent on which toxins are expressed and the virulence factors with which they are associated. One goal of this study was to isolate S. aureus from healthy, consenting adult volunteers who submitted nasal swabs for culture and qRT-PCR analysis to determine which strains are present in the community. This knowledge could …

Amino Acid Catabolism In Staphylococcus Aureus, Cortney Halsey

Theses & Dissertations

Staphylococcus aureus must rapidly adapt to a variety of carbon and nitrogen sources during invasion of a host. Within a staphylococcal abscess, preferred carbon sources such as glucose are limiting, suggesting S. aureus survives through the catabolism of secondary carbon sources. S. aureus encodes pathways to catabolize multiple amino acids including those that generate pyruvate, 2-oxoglutarate, and oxaloacetate. To assess amino acid catabolism, S. aureus JE2 and mutants were grown in complete defined medium containing 18 amino acids but lacking glucose (CDM). A mutation in glutamate dehydrogenase (gudB), which generates 2-oxoglutarate from glutamate, abrogated growth in CDM suggesting …

Spatiotemporal Control Of Staphylococcus Aureus Biofilm Development, Derek E. Moormeier

Theses & Dissertations

Chronic biofilm-related infections caused by the human pathogen Staphylococcus aureus often lead to significant increases in morbidity and mortality in both hospital- and community-associated settings. Typically, S. aureus biofilm development occurs in three stages: 1) attachment, 2) tower maturation, and 3) dispersal. Here, utilizing BioFlux1000 time-lapse microscopy we have expanded upon these fundamental stages of biofilm development and also unveiled and characterized two additional stages (multiplication and exodus).

The attachment and multiplication stages were shown to be protease sensitive but independent of most cell surface-associated proteins. Following multiplication, an exodus of the biofilm population that followed the transition of the …

Characterization Of Fatty Acid Resistant Staphylococcus Aureus Mutants Containing Snps In Cvfa, Melissa N. Loyzer

Electronic Thesis and Dissertation Repository

Upon colonization, Staphylococcus aureus must withstand the actions of many host defense mechanisms, including the unsaturated free fatty acids (uFFAs) secreted in sebum. Linoleic acid, a representative uFFA, is toxic to S. aureus at concentrations ≥50 µM; however, we selected for mutants capable of growing at elevated concentrations of linoleic acid. Five mutants had single nucleotide polymorphisms (SNPs) in the cvfA gene encoding an RNase that is a component of the RNA degradosome, and expression of the SNP-containing cvfA genes from a plasmid engendered S. aureus with high level resistance to linoleic acid. The SNPs were located within a region …

Regulation Of Vancomycin Resistance And Stress Response By The Msaabcr Operon In Staphylococcus Aureus, Dhritiman Samanta

Dissertations

Staphylococcus aureus is the predominant cause of public health problems around the world. Vancomycin has been an important antibiotic against Methicillin Resistant Staphylococcus aureus (MRSA) infections. However, Vancomycin Intermediate S. aureus (VISA) strains have been reported. These strains are characterized by thick cell walls, reduced autolytic rate, reduced PBP4 activity, and increased amount of D-Ala-D-Ala termini in the cell wall. In this study, we show that the msaABCR operon regulates vancomycin resistance in two clinical VISA strains. Deletion of the msaABCR operon in strains Mu50 and HIP6297 resulted in a significant decrease in the minimum inhibitory concentration (MIC) for vancomycin. …

Transduction As The Method Of Horizontal Gene Transfer Of The Staphylococcal Chromosomal Cassette Mec (Sccmec), Amber B. Sauder

Senior Honors Projects, 2010-2019

Methicillin-resistant Staphylococcus aureus (MRSA) gains resistance to β-lactam antibiotics through a mutated penicillin binding protein (PBP2a) encoded on the SCCmec element. In combination with the recombinase encoded by ccr, these two genes are used as markers of the mobile genetic element (SCCmec). Due to recent increases in community acquired MRSA infections, the mechanisms of antibiotic resistance gene transfer have gained attention. Transduction, a method of horizontal gene transfer mediated by bacteriophage, is believed to be responsible for the movement of the SCCmec element. Recent studies have shown the transduction of the SCCmec element in clinical isolates; however, this study is …

Characterization Of Transfer Of The Mobile Genomic Island Encoding Methicillin Resistance Among Staphylococci, Melissa D. Ray

Theses and Dissertations

The gene encoding methicillin resistance in Staphylococcus aureus (MRSA) is carried in the chromosome on a large genomic island called SCCmec and is always inserted at the att site within orfX. SCCmec has been designated a mobile genetic element but a mechanism by which it moves among different strains and species of staphylococci has never been demonstrated. This work shows that bacteriophage 80α is capable of transducing SCCmec into a recipient cell, after which it can integrate into the bacterial chromosome via homologous recombination. More importantly, this work characterizes a conjugative mechanism of SCCmec transfer. …

Elucidation Of A Novel Pathway In Staphylococcus Aureus: The Essential Site-Specific Processing Of Ribosomal Protein L27, Erin A. Wall

Theses and Dissertations

Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center where it interacts with both A-site and P-site tRNAs as well as with 23S rRNA. We observed that L27 in S. aureus and other Firmicutes is encoded with a short N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. The extension contains a conserved cleavage motif; nine N-terminal amino acids are …

The Role Of Superantigens During Staphylococcus Aureus Nasal Colonization And Infection, Stacey Xu

Electronic Thesis and Dissertation Repository

Superantigens (SAgs) are potent toxins produced by bacteria such as Staphylococcus aureus that function to overactivate T cells resulting in massive cytokine production and immune activation. Despite decades of research on the structure and function of these proteins, as well as their role in severe diseases such as toxic shock syndrome, the question as to why strains of S. aureus produce SAgs and the role that they play in the life cycle of these bacteria remains unanswered. The contribution of SAgs towards pathogenicity and bacterial survival in vivo were assessed using isogenic SAg deletion knockouts in conjunction with SAg-sensitive humanized …

An Investigation Of Bacterial Ribonucleases As An Antibiotic Target, Ashley Denise Frazier

Electronic Theses and Dissertations

Antibiotics have been commonly used in medical practice for over 40 years. However, the misuse and overuse of current antibiotics is thought to be the primary cause for the increase in antibiotic resistance.

Many current antibiotics target the bacterial ribosome. Antibiotics such as aminoglycosides and macrolides specifically target the 30S or 50S subunits to inhibit bacterial growth. During the assembly of the bacterial ribosome, ribosomal RNA of the 30S and 50S ribosomal subunits is processed by bacterial ribonucleases (RNases). RNases are also involved in the degradation and turnover of this RNA during times of stress, such as the presence of …

Coordinated Regulation By Agra, Sara, And Sarr To Control Agr Expression In Staphylococcus Aureus, Dindo Reyes, Diego O. Andrey, Antoinette Monod, William L. Kelley, Gongyi Zhang, Ambrose L. Cheung

Dartmouth Scholarship

The agr locus of Staphylococcus aureus is composed of two divergent transcripts (RNAII and RNAIII) driven by the P2 and P3 promoters. The P2-P3 intergenic region comprises the SarA/SarR binding sites and the four AgrA boxes to which AgrA binds. We reported here the role of AgrA, SarA, and SarR on agr P2 and P3 transcription. Using real-time reverse transcription (RT)-PCR and promoter fusion studies with selected single, double, triple, and complemented mutants, we showed that AgrA is indispensable to agr P2 and P3 transcription, whereas SarA activates and SarR represses P2 transcription. In vitro runoff transcription assays revealed that …

Whole-Genome Sequencing Of Staphylococcus Aureus Strain Rn4220, A Key Laboratory Strain Used In Virulence Research, Identifies Mutations That Affect Not Only Virulence Factors But Also The Fitness Of The Strain, Dhanalakshmi Nair, Guido Memmi, David Hernandez, Jonathan Bard, Marie Beaume, Steven Gill, Patrice Francois, Ambrose L. Cheung

Dartmouth Scholarship

Staphylococcus aureus RN4220, a cloning intermediate, is sometimes used in virulence, resistance, and metabolic studies. Using whole-genome sequencing, we showed that RN4220 differs from NCTC8325 and contains a number of genetic polymorphisms that affect both virulence and general fitness, implying a need for caution in using this strain for such studies.

Proteolytic Regulation Of Toxin-Antitoxin Systems By Clppc In Staphylococcus Aureus, Niles P. Donegan, Earl T. Thompson, Zhibiao Fu, Ambrose L. Cheung

Dartmouth Scholarship

Bacterial toxin-antitoxin (TA) systems typically consist of a small, labile antitoxin that inactivates a specific longer-lived toxin. In Escherichia coli, such antitoxins are proteolytically regulated by the ATP-dependent proteases Lon and ClpP. Under normal conditions, antitoxin synthesis is sufficient to replace this loss from proteolysis, and the bacterium remains protected from the toxin. However, if TA production is interrupted, antitoxin levels decrease, and the cognate toxin is free to inhibit the specific cellular component, such as mRNA, DnaB, or gyrase. To date, antitoxin degradation has been studied only in E. coli, so it remains unclear whether similar mechanisms of regulation …

An Evaluation Of Methicillin Resistant Staphylococcus Aureus Survival On Five Environmental Surfaces Under Two Different Humidities, With And Without The Addition Of Bovine Serum Albumin, Courtney Ann Coughenour

UNLV Theses, Dissertations, Professional Papers, and Capstones

The spread of antibiotic resistant bacteria is a major public health concern, as they result in greater healthcare costs and increased morbidity and mortality rates. Methicillin resistant Staphylococcus aureus (MRSA) is one organism of particular concern, with the number of infections increasing in epidemic proportion. Bacterial surface contamination with MRSA is significant, as it may serve as a reservoir for transmission and have negative health implications. The purpose of this study was to evaluate survival of MRSA on five environmental surface materials; glass, wood, vinyl, plastic, and cloth. The effect of relative humidity (RH) and bovine serum albumin (BSA) were …

Regulation Of The Mazef Toxin-Antitoxin Module In Staphylococcus Aureus And Its Impact On Sigb Expression, Niles P. Donegan, Ambrose L. Cheung

Dartmouth Scholarship

In Staphylococcus aureus, the sigB operon codes for the alternative sigma factor σ^Band its regulators that enable the bacteria to rapidly respond to environmental stresses via redirection of transcriptional priorities. However, a full model of σ^Bregulation in S. aureus has not yet emerged. Earlier data has suggested that mazEF, a toxin-antitoxin (TA) module immediately upstream of the sigB operon, was transcribed with the sigB operon. Here we demonstrate that the promoter P_mazE upstream of mazEF is essential for full σ^B activity and that instead of utilizing autorepression typical of TA systems, sigB …