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The research conducted in our laboratory will address the following NE1048 Objectives. <br/>Objective 1: Characterization of host mechanisms associated with mastitis susceptibility and resistance. (ii) Host-Pathogen Interactions at the Cellular Level. <br/>Objective 2: Characterization and manipulation of virulence factors of mastitis pathogens for enhancing host defenses. (i)Characterization of Pathogen Virulence Factors (ii) Antimicrobial Resistance (iii) Use of Molecular Epidemiology & Diagnostic Tools. <br/>Objective 3: Assessment and application of new technologies that advance mastitis control, milk quality and dairy food safety. (i) Reduction of Pathogenicity of Mastitis Organisms (ii) Technologies to Promote Host Defense Systems (iii) Mastitis Control and Dairy Food Safety. Staphylococcus aureus is a major cause of bovine mastitis; a disease that results in decreased milk yield and costs the dairy industry billions of dollars each year. For S. aureus to be successful at colonizing and infecting the host mammary gland it must successfully detect and respond to specific cues. Our research project will examine how S. aureus detects and responds to host and environmental changes at the cellular level. This project will address which stimuli promote virulence factor expression and will provide clues as to the host cellular factors that promote susceptibility or resistance to S. aureus mammary gland infections. Two-component regulatory (TCR) systems are signal transduction pathways that provide organisms with information about its surroundings. They typically consist of a membrane spanning sensor kinase and a DNA binding response regulator. Upon stimulation with either a small molecule or environmental change, the histidine kinase hydrolyzes ATP and phosphorylates a histidine residue which, transfers this phosphate group to the response regulator. This simple signal cascade results in changes in gene expression and ultimately resulting in a response. Some changes that we will monitor are expression of virulence factors, the ability to form a biofilm, and the susceptibility to killing by whole blood. <P>The first objective of our work is to determine the stimuli for the TCS and the cellular response. We hypothesize that some of these TCR systems are necessary for pathogenesis. The data produced will be available to researchers and will aid in the design of new technologies to promote host defense systems. Neutrophil granulocytes are white blood cells that provide mammals with a "first line" of defense against S. aureus infections. After bacterial phagocytosis of S. aureus, the neutrophil cell releases a number of hydrolytic enzymes and bombards the bacterium with the host generated oxidants superoxide, hypochlorous acid, nitric oxide, and hydrogen peroxide. S. aureus is able to withstand this stress and survive within the neutrophil. A second objective of our work is to determine which cellular components are required to withstand oxidative stress. These data will be used to determine the molecular mechanism for oxidative stress resistance.
Non-Technical Summary:<br/>
Two-component regulatory (TCR) systems are signal transduction pathways that provide organisms with information about its surroundings. We have created a library of Staphylococcus aureus mutant strains that contain individual deletions in the genes that encode two-component regulatory systems. We will use phenotypic analysis to determine if any of the strains in our mutant library have alterations when responding to cellular stressors or differ in virulence factor production. Strains that display phenotypes different from the wild-type will be further analyzed using classical and molecular genetics.
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Approach:<br/>
We have created a library of mutants that contain deletions in the genes that encode two-component regulatory systems. We will use phenotypic analysis to determine if any of the strains in our mutant library have alterations when responding to cellular stressors or differ in virulence factor production. Strains that display phenotypes different from the wild-type will be further analyzed using classical and molecular genetics.
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Progress:<br/>
2011/10 TO 2012/09<br/>
OUTPUTS: Outputs Activities. Our group has spent the last year conducting experiments to examine how the mammalian pathogen Staphylococcus aureus responds to changes in its environment and nutrient availability. During the past year, Professor Boyd mentored one postdoctoral researcher, five graduate students, and four undergraduate students in laboratory research. Prof. Boyd aided these students in conducting experiments and analyzing results. Prof. Boyd co-designed the curricula and taught one-half of the lectures for a graduate class in microbial physiology. Prof. Boyd designed the curricula and taught a class for undergraduates on microbial physiology.
<br/>Events. Prof. Boyd attended the East Coast Fe-S enzyme meeting in Blacksburg VA the Gordon Conference on Iron-Sulfur Enzymes, the 60th anniversary of Selman Waksman Nobel Prize meeting, and the International Conference on Gram-Positive Pathogens. Professor Boyd presented his work at all four meetings. One student attended the International Meeting on Gram Positive Pathogens and presented his work. Prof. Boyd and 3 graduate students attended the 2011 Rutgers University Microbiology Symposium and all four individuals presented their work. During the past year this project has allowed me to enter active collaborations with Victor Torres, New York University; Alex Horswill, University of Iowa; William Nauseef, University of Iowa; Greg Sommerville, University of Nebraska; Antonio Perik, Philipps-Universitat Marburg, Germany; William Belden, Rutgers University; Alastair McEwan, the University of Queensland; Eric Skaar, Vanderbilt University; and Ann Stock, Rutgers University/UMDNJ. In collaboration with William Belden, I devised a way to clone any gene into any plasmid, using a yeast genetic system. We will be seek a patent for this technology. Dissemination. Participated in Ag field day and educated the public about the impact of microbiology on their daily lives. Organized voter education seminar series to inform the public about the three microbes that we have nominated to become the state microbe of NJ. I also aided in designing a website that allows interested parties to educate themselves about the importance of microbes, that we isolated at Rutgers University, on their daily lives. The website can be found here: http://dbm.rutgers.edu/stateMicrobe.php
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PARTICIPANTS: Jeff Boyd. Principal investigator Harsimranjit K. Chahal. Postdoctoral researcher Ameya Mashruwala. Ph.D. student Zuelay Rosario-Cruz. Ph.D. student Shiven Bhatt. M.S. student Shiming Tang, M.S. student Adriana van de Guchte, Undergraduate researcher Bhavana Narala, Undergraduate researcher Valerie T. Raziano, Undergraduate researcher Colaborators. Victor Torres, New York University; Alex Horswill, University of Iowa; William Nauseef, University of Iowa; Greg Sommerville, University of Nebraska; Antonio Perik, Philipps-Universitat Marburg, Germany. William Belden, Rutgers University; Alastair McEwan, the University of Queensland Ann Stock, Rutgers University/UMDNJ. Eric Skaar, Vanderbilt University. Training or professional development. The following individuals were trained in my lab during the past year. Ameya Mashruwala. Ph.D. student Zuelay Rosario-Cruz. Ph.D. student Shiven Bhatt. M.S. student Shiming Tang, M.S. student Adriana van de Guchte, Undergraduate researcher Bhavana Narala, Undergraduate researcher Valerie T. Raziano, Undergraduate researcher Jeffery Matthews, Undergraduate researcher Eric Huselid, Rotating Ph.D. student Ibrahim Alsawaf, Rotating Ph.D. student
<br/>TARGET AUDIENCES: Target audiences. Staphylococcus aureus is a major cause of bovine mastitis; a disease that results in decreased milk yield and costs the dairy industry billions of dollars each year. S. aureus infections are also the leading cause of human infectious disease related death in the USA. The research conducted by my group impacts the health and pocketbooks of all Americans.
<br/>PROJECT MODIFICATIONS: Not relevant to this project.
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IMPACT: For successful colonization of host tissues, S. aureus must acquire and effectively and efficiently metabolize iron (Fe). Although much is known about how S. aureus acquires Fe from the host, little is known about what happens to the iron once internalized. Much of the Fe that S. aureus internalizes is used to build small inorganic cofactors called iron-sulfur ([Fe-S]) clusters. The innate immune system of mammals produces chemical oxidents such as hydrogen peroxide, hypochlorous acid (bleach), superoxide, and nitric oxide to kill invading bacteria, in part, by damaging [Fe-S] clusters, which are required for numerous metabolic processes. Once the clusters are damaged the iron is released and can freely react with hydrogen peroxide to produce hydroxyl radicals that result in cell death. We created a series of mutant S. aureus strains that are defective in [Fe-S] cluster metabolism and used these stains to test the hypothesis that an S. aureus strain that is defective intracellular Fe metabolism has altered virulence properties. We have focus our efforts on two genes nfu (SAUSA300 0839) and sufT (SAUSA300 0875). The mutants had lower activity of the Fe-S cluster dependent enzymes aconitase and isopropylmalate isomerase. These mutant strains had destabilized and increased intracellular unchelated Fe pools and were sensitive to the chemicals hydrogen peroxide, superoxide and nitric oxide. Biochemical analysis found that the nfu protein bound a Fe-S cluster, but the type of Fe-S cluster is currently unknown. We used classic and molecular genetics to examine the Fe-S cluster interactome. We found that potentially two pathways exist to build Fe-S clusters. The SufT protein interacted with the Fe-S cluster trafficking proteins consistant with the hypothesis that it is involved in the late stages of Fe-S cluster insertion into apo-proteins. We have also found that a S. aureus strain defective in producing the small molecule bacillithiol is defective in intracellular metal metabolism. The bacillithiol mutant was sensitive to copper, cobalt, and cadmium. It also had a growth defect with out exogenous added iron.