CONTROLLING STAPHYLOCOCCUS AUREUS VIRULENCE BY WAY OF THE PENTOSE PHOSPHATE SHUNT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0225011
• Project No.: NEB-39-159
• Project Start Date: Mar 1, 2011
• Project End Date: Feb 28, 2016

Project Director
Somerville, G. A.

Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583

Performing Department
Veterinary and Biomedical Sciences

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.

Animal Health Component

100%

Research Effort Categories

Basic

80%

Applied

5%

Developmental

15%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4010	1100	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

staphylococcus aureus

central metabolism

pathogenesis

mastitis

Goals / Objectives
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.

Project Methods
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 03/01/11 to 02/28/16

Outputs
Target Audience:Veterinarians, physicians, and scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the past year, the research contained in this project has allowed one post-doctoral fellow, 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, these results have been communicated to veterinarians and scientists by publication in peer-reviewed journals. How have the results been disseminated to communities of interest?These results were published in one peer-reviewed journal article, one book chapter, and in a book for which I am the editor. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? In the United States, the bacterium Staphylococcus aureus causes millions of infections in humans and animals every year. These infections are difficult for veterinarians and physicians to treat due to increasing resistance to antibiotics. New approaches to treat these infections are urgently needed. To address this need, we chose to attack a basic requirement of life; namely, all living organisms require food to survive. With bacteria, their food is derived from the immediate environment of wherever they happen to be. For this reason, as their environment changes their food sources change. Because bacteria live in ever-changing environments, they have evolved to rapidly respond to these environmental/food changes. Any change in the availability or types of food, necessitate changes in the type of enzymes necessary to digest, or catabolize, that food. Some of these enzymes that are necessary to catabolize food are also important for the bacterium's ability to cause infections in humans and animals. This linkage between bacterial food and the ability to cause an infection led us to examine how we could control the bacterium's ability to cause disease by manipulating what the bacterium can catabolize. To this end, we have determined that it is possible to alter how the bacterium S. aureus responds to changes in the availability of food and, importantly, this alteration changes the bacterium's ability to cause an infection. Specifically, the availability of food is determined by several metabolite-responsive regulators that mediate the linkage to virulence determinant synthesis. S. aureus possesses at least three members of the RpiR family of transcriptional regulators. Of the three RpiR homologs, RpiRc is a potential regulator of the pentose phosphate pathway, which also regulates RNAIII levels. RNAIII is the regulatory RNA of the agr quorum-sensing system that controls virulence determinant synthesis. The effect of RpiRc on RNAIII likely involves other regulators, as the regulators that bind the RNAIII promoter have been intensely studied. To determine which regulators might bridge the gap between RpiRc and RNAIII, sarA, sigB, mgrA, and acnA mutations were introduced into an rpiRc mutant background, and the effects on RNAIII were determined. Additionally, phenotypic and genotypic differences were examined in the single and double mutant strains, and the virulence of select strains was examined using two different murine infection models. The data suggest that RpiRc affects RNAIII transcription and the synthesis of virulence determinants in concert with σ(B), SarA, and the bacterial metabolic status to negatively affect virulence. The impacts of this proposal are the demonstrated importance of RpiR-family proteins in controlling S. aureus infections and how infectious outcomes can be altered by targeting this family of proteins. These data should facilitate the rationale design of inhibitors that will alter the ability of S. aureus to cause infections. In addition, the book and book chapter published in 2016 (2016. Staphylococcus aureus Metabolism and Physiology. Ed. G. A. Somerville. In Staphylococcus: Genetics and Physiology. Caister Academic Press) will allow students and entry-level staphylococcal researchers to learn from their more experienced colleagues. Objective #1 of this proposal is to determine the structure of the RpiR metabolite binding domain. We were unable to obtain the NMR solution structure of the binding domain due to the unstable nature of this region; although, computer modelling of the binding region was accomplished by another group. Objective #2 is to determine the metabolite(s) to which the RpiR proteins bind. Electrophoretic mobility shift assays were conducted with purified RpiRc and RpiRb using the rpiA promoter region as the probe. Both purified proteins bound specifically to the probe; however, RpiRb bound at a lower concentration. This indicated that RpiRb was more likely the regulator of rpiA. Of the metabolites that we assessed for their ability to enhance DNA binding activity, none increased DNA binding activity above background levels. These data have not been published, although a manuscript describing the effects of rpiRc inactivation on transcription were published (Infect. Immun. 2016. 84:2031-2041). 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. Electrophoretic mobility shift assays were conducted with purified RpiRc and RpiRb using the cap5 and cap8 promoter regions. The proteins bound non-specifically to the promoters, suggesting that RpiR proteins alter PIA and capsule biosynthesis independent of direct binding of the promoters of PIA and capsule biosynthetic genes. It is likely the dramatic effects of rpiR deletion on S. aureus PIA and capsule biosynthesis are likely due to a redirection of carbon flow in amino sugar biosynthesis, which are essential for capsule and PIA synthesis.

Publications

• Type: Book Chapters Status: Published Year Published: 2016 Citation: G. A. Somerville. 2016. Staphylococcus aureus Metabolism and Physiology. Ed. G. A. Somerville. In Staphylococcus: Genetics and Physiology. Caister Academic Press. ISBN: 978-1-910190-49-4.
• Type: Books Status: Published Year Published: 2016 Citation: Staphylococcus: Genetics and Physiology. 2016. Ed. G. A. Somerville. Caister Academic Press. ISBN: 978-1-910190-49-4.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: R. Gaupp, J. Wirf, B. Wonnenberg, T. Biegel, J. Graham, M. Bischoff, C. Y. Lee, C. Beisswenger, C. Wolz, T. Tschernig, M. Herrmann, and G. A. Somerville. 2016. RpiRc is a pleiotropic effector of virulence determinant synthesis and attenuates pathogenicity in Staphylococcus aureus. Infect. Immun. 84:2031-2041. PMCID: PMC4936357.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:These results have been communicated to veterinarians and scientists by publication in peer-reviewed journals. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The research contained in this project has allowed 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. The graduate student presented his results at the Molecular Biology of Bacteria and Phages meeting in Madison, WI. How have the results been disseminated to communities of interest?These results were presented at an ASM meeting and at a seminar given at the University of Rochester Medical School. In addition, two manuscripts were published during this reporting period. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Staphylococcus aureus is a human and animal pathogen capable of causing a wide range of infections. These infections often involve a transition between diverse host environments (e.g., from the nose to the blood), leading to changes in nutrient availability. Previously, we identified three S. aureus metabolite-responsive regulators that are homologs of the ribose phosphate isomerase regulator (RpiR).During this reporting period, we have established that the virulence regulator RpiRc is a major contributor to virulence during infections. Specifically, we observed that inactivation of rpiRc results in higher bacterial loads of S. aureus within liver and kidneys of experimentally infected mice. This implies that increasing the activity of RpiRc could alter the infectious outcome in humans and animals.

Publications

• Type: Book Chapters Status: Published Year Published: 2015 Citation: A. R. Richardson, G. A. Somerville, and A. L. Sonenshein. Regulating the intersection of metabolism and pathogenesis in Gram-positive bacteria. 2015. Eds. T. Conway and P. S. Cohen. Microbiol. Spec. 3 doi:10.1128/microbiolspec.MBP-0004-2014. PMCID: PMC4540601.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: R. Gaupp, S. Lei, J. M. Reed, H. Peisker, S. Boyle-Vavra, A. S. Bayer, M. Bischoff, M. Herrmann, R. S. Daum, R. Powers, and G. A. Somerville. 2015. Staphylococcus aureus metabolic adaptations during the transition from a daptomycin susceptible phenotype to a daptomycin non-susceptible phenotype. Antimicrob. Agents Chemother. 59: 4226-4238. PMCID: PMC4468685.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Veterinarians and scientists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The research contained in this project has allowed one post-doctoral fellow, 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. How have the results been disseminated to communities of interest? These results have been communicated to veterinarians and scientists by publication in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Staphylococcus aureus is a versatile pathogen capable of colonizing or infecting most human and animal tissues. The success of S. aureus as a pathogen is due in part to its many virulence determinants and resistance to antibiotics. Regulation of S. aureus virulence determinants is complex involving transcriptional, post-transcriptional, translational, post-translational, and epigenetic regulation. Previously, we identified three transcriptional regulators in RpiR family of regulators that alter transcription of the S. aureus master regulator RNAIII. The RpiR family of regulators has a sugar isomerase binding domain, suggesting that their regulatory functions are modulated by binding to sugars. To determine what sugars the RpiR homologs bind, we are attempting to define the structure of the sugar isomerase binding domain. This structure will allow us to perfrom docking studies to predict what sugars may bind and modulate the activity of the RpiR homologs. This information will allow us to manipulate the bacterial metabolism to alter RpiR activity and transcription of RNAIII, and it will provide logical starting points for the design of vaccines again bovine mastitis. Results from this reporting period: 1. RpiRa has been isotopically labeled RpiRa with 15N, purified, and subjected to NMR structural analysis. A manuscript describing the methods and initial results is in preparation. 2. Mobility shift assays using purified RpiRc and RpiRb were conducted using the RNAIII promoter region as a probe. No shifts were detected. 3. Purified RpiRb does bind and shift the promoter region of SAV2337, a gene thought to be a molybdenum cofactor sulfurase. 4. We analyzed the antagonistic and/or synergistic effects of rpiRb and/or rpiRc mutations in S. aureus regulatory mutants using RT-PCR. These experiments have provided preliminary data regarding the structure of RpiRa. In addition, we have learned that the effects of RpiRc inactivation are dependent upon the alternative sigma factor sigB.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: J. L. Stark, J. C. Copeland, A. Eletsky, G. A. Somerville, T. Szyperski, and R. Powers. 2014. Identification of low molecular weight compounds inhibiting growth of Corynebacteria: lead compounds for novel antibiotics. ChemMedChem. doi: 10.1002/cmdc.201300386. PMCID: PMC3977743.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: N. Ledala, B. Zhang, J. Seravalli, R. Powers, and G. A. Somerville. 2014. The influence of iron and aeration on Staphylococcus aureus growth, metabolism, and transcription. J. Bact. 196:2178-2189. PMCID: PMC4054190.

Progress 10/01/12 to 09/30/13

Outputs
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.

Impacts
What was accomplished under these goals? During the first 2 years of this project, we have identified 5 regulators that coordinate staphylococcal metabolism and virulence factor synthesis. Four of these 5 regulators had previously not been described in staphylococci. Previously we initiated a bioinformatic study of PAS domain containing proteins in staphylococci with the goal of identifying a succinate-responsive regulator (all known succinate sensing regulators have 1 or more PAS/PDC domains; e.g., DcuS). While the results of this study demonstrate that staphylococci do not have a traditional succinate-responsive regulator, we have been able to use this bioinformatic study to evolution and function of PAS domain containing proteins. We have constructed expression vectors for all of the RpiR homologues. These have been used for over-expression of the proteins and preliminary structural studies.

Publications

• Type: Book Chapters Status: Published Year Published: 2013 Citation: G. A. Somerville and Robert Powers. 2014. Growth and preparation of Staphylococcus epidermidis for NMR metabolomic analysis. In Methods in Molecular Biology. Vol. 1106. Ed. P. Fey.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: G. A. Somerville and R. A. Proctor. 2013. Cultivation conditions and the diffusion of oxygen into culture media: The rationale for the flask-to-medium ratio in microbiology. BMC Microbiol. 13:9. PMCID: PMC3551634.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: N. Shah, R. Gaupp, H. Moriyama, K. M. Eskridge, E. N. Moriyama, and G. A. Somerville. 2013. Reductive evolution and the loss of PDC/PAS domains from the genus Staphylococcus. BMC Genomics. 14:524. PMCID: PMC3734008.

Progress 10/01/11 to 09/30/12

Outputs
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.

Impacts
Bacteria regulate virulence in response to environmental changes; thus, by knowing how bacteria sense their environment it is possible to manipulate bacterial virulence. Our data have demonstrated that the pentose phosphate pathway and virulence determinant synthesis are co-regulated by RpiRc. Previously, we published that bacterial environmental sensing can be manipulated by controlling their access to nutrients (e.g., glucose, amino acids, etc.) or co-factors (e.g., iron, magnesium, etc.) (Zhu, Y. et al., Infect. Immun. 2009. 77:4256-64.) Based on this information, we are looking for nutrient/co-factor transporters that would be suitable vaccine targets that can manipulate RpiRc regulation.

Publications

• Y. Zhu, R. Nandakumar, M. R. Sadykov, N. Madayiputhiya, T. T. Luong, R. Gaupp, C. Y. Lee, and G. A. Somerville. 2011. RpiR homologues may link Staphylococcus aureus RNAIII synthesis to the pentose phosphate pathway. J. Bact. 193:6187-6196. PMCID: PMC3209195
• R. Gaupp, N. Ledala, and G. A. Somerville. 2012. Staphylococcal response to oxidative stress. Front. Cell. Inf. Microbio. 2:33. doi: 10.3389/fcimb.2012.00033. PMCID: PMC3417528

Progress 10/01/10 to 09/30/11

Outputs
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.

Impacts
Bacteria regulate virulence in response to environmental changes; thus, by knowing how bacteria sense their environment it is possible to manipulate bacterial virulence. Previously, we established that one mechanism by which bacteria "sense" their environment is through stress (e.g., iron-limitation) induced changes in central metabolism. The changes in central metabolism alter the intracellular concentrations of metabolites that affect the activity of transcriptional regulators, such as CcpA, CodY, and the RpiR family. During the first year of this project, we constructed the plasmids necessary to over-express RpiRC and RpiRA. RpiRA has been purified and subjected to NMR structural analysis. Currently, additional NMR experiments are underway to optimize the structural information contained in the data.

Publications

• Y. Zhu, R. Nandakumar, M. R. Sadykov, N. Madayiputhiya, T. T. Luong, R. Gaupp, C. Y. Lee, and G. A. Somerville. 2011. RpiR homologues may link Staphylococcus aureus RNAIII synthesis to the pentose phosphate pathway. J. Bact. 193:6187-6196.
• B. Zhang, S. Halouska, C. E. Schiaffo, M. R. Sadykov, G. A. Somerville, R. Powers. 2011. NMR analysis of a stress response metabolic signaling network. J. Proteome. Res. 10:3743-3754.