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Controlling Staphylococcus Aureus Virulence by Way of the Pentose Phosphate Shunt

Investigators
Somerville, Greg
Institutions
University of Nebraska - Lincoln
Start date
2011
End date
2016
Objective

Objective #1 of this proposal is to determine the structure of the RpiR metabolite binding domain.

Objective #2 is to determine the metabolite(s) to which the RpiR proteins bind.

Objective #3 is to determine if the RpiR proteins bind to the capsule and PIA promoter regions and to determine if the metabolite(s) identified in objective 2 alters the DNA binding properties of the RpiR proteins.

The completion of these objectives will define the metabolite(s) that modulate RpiR regulatory activity. With this information, we anticipate developing strategies that alter the intracellular concentrations of these metabolites. By altering the intracellular concentration of these metabolites, we are altering the activity of these virulence regulators. We have successfully used this strategy to increase TCA cycle activity, transiently decrease PIA synthesis, and significantly reduce in vivo virulence in the endocarditis model in terms of achievable bacterial densities in biofilm-associated cardiac vegetations, kidneys and spleen. In summary, completion of these aims will allow us to exploit the close linkage of pentose phosphate pathway activity and virulence factor synthesis to alter infectious outcomes.

More information

NON-TECHNICAL SUMMARY:
Staphylococcus aureus pose major health risks and cause significant economic hardships in the dairy and food industries. The annual economic impact of bovine mastitis in the United States is approximately $2 billion (~$200 per milk cow per year) due to reduced production, animal replacement costs, discarded milk, treatment costs, and veterinary fees. The 2007 USDA census of agriculture reported that Nebraska had 493 farms with 54,410 dairy cows, thus the economic impact of bovine mastitis to Nebraska per year is approximately $10.9 million. In addition to causing bovine mastitis, Staphylococcus aureus is a major cause of food-borne diseases in the US. Food-borne diseases are a critical public health problem, affecting an estimated 76 million people each year in the US with more than 300,000 hospitalized and 5,000 deaths as a result of their illness. My laboratory has identified three regulators in Staphylococcus aureus belonging to the RpiR family, which respond to changes in pentose phosphate metabolites in Escherichia coli and Pseudomonas putida. Inactivation of these regulators increases capsule biosynthesis, RNAIII transcription or stability, and decreases protein A expression. These data demonstrate a close association between the pentose phosphate pathway and virulence factor synthesis in Staphylococcus aureus. Previously, we developed a strategy to exploit a metabolic linkage to alter the normal temporal pattern of virulence factor synthesis and the infectious outcome in a rabbit endocarditis model (18). The work contained in this proposal will provide the necessary information to take advantage of the linkage between the pentose phosphate pathway and virulence factor synthesis. We anticipate this information will lead to a novel means to control bovine mastitis.

APPROACH:
This proposal will use standard protein over-expression and purification methods. NMR will be used to determine a solution structure for the RpiR proteins and for determining which metabolite(s) bind to the different RpiR proteins. Once metabolites have been identified to which RpiR bind, the puried proteins will be used in mobility shift assays using the capsule and ica promoter regions as probes. We anticipate that RpiR (i.e., SAV2315) will bind to and repress capA, but not icaA. The reason for this is that the intracellular concentration of ribose, or more likely ribose-5-phosphate, is greatest during the exponential phase when the pentose phosphate pathway is most active and capsule synthesis is repressed. Additionally, we anticipate that binding of the RpiR proteins to the ligands identified in objective #2 will decrease the affinity of the regulators for the operator site. All data relating to this project will be communicated in scientific journals and presented at meetings.

PROGRESS: 2012/10 TO 2013/09
Target Audience: Researchers and scientists in academia and industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This research has allowed two post-doctoral fellows, one graduate student, and two undergraduate students to be trained in molecular microbiology and to understand the importance of animal health in state and national priorities. In addition, our results have been communicated to veterinarians and scientists by publication in peer-reviewed journals. How have the results been disseminated to communities of interest? The results have been published in peer-reviewed scientific journals and presented at meetings and seminars. What do you plan to do during the next reporting period to accomplish the goals? We will assess the ability of purified RpiR proteins to bind promoters using electrophoretic mobility shift assays. We will continue to use NMR to gain insight into the structures of the RpiR proteins.

PROGRESS: 2011/10/01 TO 2012/09/30
OUTPUTS: Staphylococcus aureus is the leading cause of infectious mastitis in the United States, creating a significant economic impact in the dairy industry. Recently we demonstrated that S. aureus RpiR homologs (i.e., RpiRa, RpiRb, and RpiRc) regulate the pentose phosphate pathway and they also regulate virulence factor synthesis by bypassing the quorum sensing-dependent transcription of the master virulence regulator RNAIII (J. Bact. 193:6187-6196. PMCID: PMC3209195). To determine how the S. aureus RpiRc regulator bypasses the quorum sensing system and to integrate our findings with those of the staphylococcal community, we have constructed a series of rpiRc double mutants that also contain mutations in the most intensively studies S. aureus virulence regulators (i.e., agr, sarA, mgrA, sigB) and the tricarboxylic acid cycle (acnA/citB). These double mutants have allowed us to demonstrate that RpiRc has a greater effect on transcription of RNAIII than do SarA, SigmaB, MgrA, and aconitase. These results were presented at the Pathophysiology of Staphylococci in the Post-Genomic Era meeting in Bad Staffelstein, Germany in 2012. In addition, these results will be published in 2013. This research has allowed two post-doctoral fellows, one graduate student, and two undergraduate students to be trained in molecular microbiology and to understand the importance of animal health in state and national priorities. In addition, our results have been communicated to veterinarians and scientists by publication in peer-reviewed journals. PARTICIPANTS: Post-doctoral fellow Rosmarie Gaupp Post-doctoral fellow Nagender Ledala Graduate student Joe Reed TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

PROGRESS: 2011/10/01 TO 2012/09/30
OUTPUTS: Staphylococcus aureus is the leading cause of infectious mastitis in the United States, creating a significant economic impact in the dairy industry. Recently we demonstrated that S. aureus RpiR homologs (i.e., RpiRa, RpiRb, and RpiRc) regulate the pentose phosphate pathway and they also regulate virulence factor synthesis by bypassing the quorum sensing-dependent transcription of the master virulence regulator RNAIII (J. Bact. 193:6187-6196. PMCID: PMC3209195). To determine how the S. aureus RpiRc regulator bypasses the quorum sensing system and to integrate our findings with those of the staphylococcal community, we have constructed a series of rpiRc double mutants that also contain mutations in the most intensively studies S. aureus virulence regulators (i.e., agr, sarA, mgrA, sigB) and the tricarboxylic acid cycle (acnA/citB). These double mutants have allowed us to demonstrate that RpiRc has a greater effect on transcription of RNAIII than do SarA, SigmaB, MgrA, and aconitase. These results were presented at the Pathophysiology of Staphylococci in the Post-Genomic Era meeting in Bad Staffelstein, Germany in 2012. In addition, these results will be published in 2013. This research has allowed two post-doctoral fellows, one graduate student, and two undergraduate students to be trained in molecular microbiology and to understand the importance of animal health in state and national priorities. In addition, our results have been communicated to veterinarians and scientists by publication in peer-reviewed journals. PARTICIPANTS: Post-doctoral fellow Rosmarie Gaupp Post-doctoral fellow Nagender Ledala Graduate student Joe Reed TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

PROGRESS: 2010/10/01 TO 2011/09/30
OUTPUTS: The research contained in this project has allowed one post-doctoral fellow, one graduate student, and one undergraduate student to be trained in molecular microbiology and to understand the importance of animal health in state and national priorities. In addition, these results have been communicated to veterinarians and scientists by publication in peer-reviewed journals. PARTICIPANTS: Greg A. Somerville (PI) - supervised the research and editing/writing of manuscripts. Yefei Zhu (Graduate Student)- performed research and assisted in writing manuscripts Rosmarie Gaupp (Post-doctoral Fellow)- performed research and assisted in writing manuscripts McKenzie Steger (Undergraduate student) - assisted laboratory personnel with research projects TARGET AUDIENCES: Research from this project was communicated to scientists working on staphylococcal pathogenesis through the publication of manuscripts in peer-reviewed journals. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Funding Source
Nat'l. Inst. of Food and Agriculture
Project source
View this project
Project number
NEB-39-159
Accession number
225011
Categories
Bacterial Pathogens
Escherichia coli
Viruses and Prions
Staphylococcus
Commodities
Meat, Poultry, Game