SUMMER I- 2010 ACS Project SEED Department of Chemistry and Biochemistry Southern Illinois University

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Finding the KH Domain structure in CvfA gene of Streptococcus pyogenes

Rebecca C. Weber[1,2], Stephanie Geiser[1],Gabriela C. Pérez·Alvarado[1], Kyu Hong Cho[3], Brian Lee[1] [1]Department of Chemistry and Biochemistry, Southern Illinois University, Carbondale, IL 62901 [2]Herrin High School, Herrin, IL 62948 [3]Department of Microbiology, Southern Illinois University Medical School, Carbondale, IL 62901

August 6, 2010

Final Written Research Report for the Summer I-2010 ACS Project SEED

Abstract

Streptococcus pyogenes is a pathogenic bacterium that causes several types of infection in the human body. Within S. pyogenes lies the Conserved virulence factor A (CvfA) gene that responds to the environment surrounding it. This study looks at the second of three domains located in the CvfA gene: the K homology (KH) domain. The KH domain is the specific part of CvfA that binds and interacts with RNA. The process of achieving this involves isolating the KH domain and amplify the products. Protein expressions were checked after lysis and then purified by sonication and afterward the KH domain was cleaved. The experimental results showed that the KH domain could be cloned. When it comes to expression, only cytoplasmic expression works in contrast with periplasmic expression. In a later study, we hope to find how RNA and the KH Domain interact together.

Introduction

Streptococcus pyogenes is a spherical bacterium that groups with others in a string-like arrangement. It is the main cause for most streptococcal infections and human diseases. This bacterium can cause strep throat (pharyngitis), a contagious skin infection (impetigo), as well as many other respiratory and skin infections and diseases. This pathogenic bacterium is responsible for 1,000 to 1,800 deaths every year in the United States alone.[4] Conserved virulence factor A (CvfA) is the gene located in S. pyogenes that is responsible for regulating host virulence in response to growth phase and environmental conditions including nutritional status. Little is known about CvfA since it has only been studied previously with Staphylococcus aureus and a related protein called YmdA in Bacillus subtilis.[1] CvfA has three domains: the transmembrane (TM) domain at the beginning, a K homology (KH) domain

in the middle, and a His-Asp-containing phosphohydrolase (HD) domain at the end. The domain studied in this project is the KH Domain. The CvfA ortholog, YmdA, from Bacillus subtilis has been shown to identify mRNA transcripts for degradation during the regulation of S-box gene expression in response to the binding of S-adenosylmethionine (SAM) to an anti-anti-terminator structure. The KH Domain is presumed to participate in the recognition of the anti-anti-terminator RNA structure. The goal of this study is to find the structure of CvfA starting with the KH Domain and also characterize its interactions with RNA.

Experimental Methods

The DNA constructs of the KH Domain of CvfA were isolated by PCR amplification with Phusion polymerase (Finnzymes) from genomic DNA of Streptococcus pyogenes strains M3 and HSC5. The KH Domain constructs included combinations with the start site at Ala 205 and Gly 218; and end sites at Gly 292 and Ala 297. PCR products were cloned into pET 24a (Novagen), pMAL-c5X, pMAL-p5X, and pMAL-c5G (NEB) and amplified in XL1 Blue cells (Stratagene). Proteins were expressed at 37ºC, 20ºC or 15ºC in M9 minimal media cultures of BL21 (DE3) cells (Novagen) after induction with 1mM IPTG (USB).

Expression levels were checked by

SDS-PAGE gel after lysis with B-PER reagent (Pierce). Maltose binding protein (MBP) fusions were purified after lysis by sonication (Branford) through affinity chromatography with amylose resin (NEB) using an AKTA FPLC (GE Healthcare). Elution fractions were checked for protein concentration by a Bradford assay (Biorad). The KH Domain was cleaved from the MBP fusion by factor Xa protease (NEB).

Results and Discussion

Figure 1 S. pyogenes causes many deaths each year and several thousand more infections. Figures in green are the least harmful, followed by yellow, orange, and finally red- which is the most lethal. The figure below shows only of few of the infections and diseases that S. pyogenes can cause. Arrows show how one problem can lead to another starting with one type of bacterium.

Figure 2 Agarose gel showing the DNA after a PCR of the KH Domain in CvfA. The four strong bands show good DNA amplification from the strains M3 and HSC5 as noted. The start site for these constructs is Gly 218 and the end sites are at Gly 292 and Ala 297.

Figure 3 Protein expression test of HSC5 652-876 (lanes 1-7) and HSC5 652-891 (lanes 9-15) cloned into pMAL-p5X using BL21 (DE3) cells. 1. HSC5 876 p5X 0hr 2. HSC5 876 p5X 37° C 4hr 3. HSC5 876 p5X 37°C 4hr supernatant 4. HSC5 876 p5X 37°C 4hr inclusion bodies 5. HSC5 876 p5X 15°C overnight 6. HSC5 876 p5X 15°C overnight supernatant 7. HSC5 876 p5X 15°C overnight inclusion bodies 8. Molecular weight marker mark 12 9. HSC5 891 p5X 0hr 10. HSC5 891 p5X 37°C C 4hr 11. HSC5 891 p5X 37°C 4hr supernatant 12. HSC5 891 p5X 37°C 4hr inclusion bodies 13. HSC5 891 p5X 15°C overnight 14. HSC5 891 p5X 15°C overnight supernatant 15. HSC5 891 p5X 15°C overnight inclusion bodies

Figure 4 Protein expression test for HSC5 652-891 cloned into pMAL-c5X using BL21 (DE3) cells. 1.Total cell lysate 0hr 2.Total cell lysate 2hr 37ºC 3.Total cell lysate 4hr 37ºC 4.Soluble protein 4hr 37ºC 5.Inclusion bodies 4hr 37ºC 6.Total cell lysate 4hr 15ºC 7.Total cell lysate overnight 37ºC 8.Total cell lysate overnight 15ºC 9.Soluble protein overnight 15ºC 10.Inclusion bodies overnight 15ºC 11.Molecular weight marker mark 12

Figure 5 FPLC fractions from amylose affinity chromatography of 0.5L BL21 (DE3) growth culture of HSC5 652-891 (Gly 218 to Ala 297) cloned into pMAL-c5X. Lane 1: wash, lane 2-12: fractions 1-11, lane 13: cell lysate, lane 14: flow through, lane 15: molecular weight marker mark 12. Elution with a step gradient of 10mM maltose.

Figure 6 FPLC fractions from amylose affinity chromatography of 0.5L BL21 (DE3) growth culture of HSC5 652-891 (Gly 218 to Ala 297) cloned into pMAL-p5X. Lane 1: wash, lane 2-12: fractions 1-11, lane 13: cell lysate, lane 14: flow through, lane 15: molecular weight marker mark 12. Elution with a step gradient of 10mM maltose.

Figure 7 The sequence alignment shows the KH Domain region of CvfA from Streptococcus pyogenes compared to several related proteins from other bacteria including YmdA from Bacillus subtilis and CvfA from Staphylococcus aureus.

Figure 8 YmdA in Bacillus subtilis targets mRNA transcripts for degradation through recognition of the anti-anti-terminator structure stabilized by S-adenosylmethionine (SAM) in the presence of methionine.5

Figure 9 Shown here is the 3D structure of a type I KH Domain from Poly-C binding protein 2 (PDB code: 2PY9)[3]. This specific system contains four KH Domains and shows where they bind to RNA. Type I KH Domains have a ßaaßaß structure while type II KH Domains have an aßßaaß structure.

Conclusions

From this study we have learned that we can successfully clone the KH Domain as an MBP fusion. When dealing with the KH Domain cytoplasmic expression works because the fusion protein is not being targeted by any proteases in the bacteria. Periplasmic expression will not work because we are losing the KH domain in the bacterial membrane before the protein can be extracted when it is hydrolized into pieces. In the future we hope to do isotope labeling of the KH Domain in order to use NMR spectroscopy to find its structure. We can use this structure to learn how it interacts with RNA and target this interaction as a way to reduce virulence.

Overview of experience in the lab and with your mentor

The ACS Project SEED program was very helpful in the research are of doing science. I learned many things that I had never dome before along with learning new information about what we would be studying. I did gain experience that will definitely help me out in the future when I am in college studying different sciences. All of the lab mentors and students were very helpful and informative as well. Both Dr. Lee and Dr. Perez-Alvarado were able to explain things for me to understand. If I had questions about the research or experimental procedures, they could be answered by my mentors or usually by other students in the lab. Overall this lab experience was excellent. I did develop an even greater interest in the area of science and even gathered information in other science areas. I learned several things that will help me out in my future, and I hope to return for a second summer of research next year.

Acknowledgements

We acknowledge Daniel Merkel, Bryce Hilburn, Mateo Houle, Justin Hennings, Tori Nosovitsky, and Dr. Gabriela Perez-Alvarado. Financial support for this project was provided by the Department of Chemistry start up funds, Brian Lee. Rebecca C. Weber acknowledges support from the ACS Project SEED program, the Department of Chemistry and Biochemistry, the College of Science, and the Vice Chancellor for Research Office.

References 1.Kang, S., Caparon, M., Cho, K. (2010) Virulence Gene Regulation by CvfA, a Putative RNase: the CvfA-Enolase Complex in Streptococcus pyogenes Links Nutritional Stress, Growth-Phase Control, And Virulence Gene Expression. Infection and Immunity 78, 2754-2767. 2.Messias, A., Sattler, M. (2004) Structural Basis of Single-Stranded RNA Recognition. Accounts of Chemical Research 37, 279-287. 3.Du, Z., Lee, J.K., Fenn, S., Tjhen, R., Stroud, R.M., James, T.L. (2007) Protein-RNA Interaction involving KH1 domain from Human Poly(C)-Binding Protein-2. Rna 13: 1043-1051 4.“Group A Streptococcal (GAS) Disease.” cdc.gov. CDC, n.p. Web. 3 Apr. 2008. 5.Shahbabian, K., Jamalli, A., Zig, L., Putzer, H. (2009) RNase Y, a novel endoribonuclease, initiates riboswitch turnover in Bacillus subtilis. The EMBO Journal 28, 3523-3533.