Successful Application of Site-directed Mutagenesis Polymerase Chain Reaction to Mutate TMS 11 of the Staphylococcal Multidrug Efflux Protein QacA

Date Received: Mar 14, 2019

Date Published: Aug 12, 2020

DOI: https://doi.org/10.31817/vjas.2020.3.1.04

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Issue: Vol. 3 No. 1 (2020): Vietnam Journal of Agricultural Sciences

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ANIMAL SCIENCE – VETERINARY MEDICINE – AQUACULTURE

How to Cite:

Hang, N., Hanh, V., Phai, D., Huong, L., Phuong, N., Truong, L., … Brown, M. (2020). Successful Application of Site-directed Mutagenesis Polymerase Chain Reaction to Mutate TMS 11 of the Staphylococcal Multidrug Efflux Protein QacA. Vietnam Journal of Agricultural Sciences, 3(1), 512–519. https://doi.org/10.31817/vjas.2020.3.1.04

Successful Application of Site-directed Mutagenesis Polymerase Chain Reaction to Mutate TMS 11 of the Staphylococcal Multidrug Efflux Protein QacA

Nguyen Thi Hang (*) 1, 2 , Vu Duc Hanh 3 , Dam Van Phai 1 , Lai Thi Lan Huong 1 , Nguyen Thi Phuong 1 , Le Van Truong 1 , Mohsen Chitsaz 2   , Melissa H Brown 2

  • Corresponding author: hang.phar2012@gmail.com
  • 1 Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi 131000, Vietnam
  • 2 School of Biology, Flinders University, Sturt Road, Bedford Park, South Australia 5042, Australia
  • 3 1Faculty of Veterinary Medicine, Vietnam National University of Agriculture, Hanoi 131000, Vietnam

    • Keywords

    S.aureus, gene qacA, protein QacA, site-directed PCR

    Abstract


    Staphylococcus aureus is a major problem in both the clinical setting and within the community. S. aureus can quickly develop resistance to a wide range of antibiotics through a number of different mechanisms, of which, using transporters located in the cell membrane to pump antibiotics out of the cell is the most serious concern. In staphylococcal species, QacA, one such important transporter, is encoded by qacA. QacA is 55kD in size and has 14 transmembrane segments (TMS) (TMS 1-TMS 14). This research describes the mutation process of the amino acid residues in TMS 11 of QacA using site-direct PCR. In this research, 15 primers were successfully designed for site-directed mutagenesis PCR. The site-mutagenesis PCR was successfully conducted to create 15 qacA mutants. These mutants will be used in further functional research of QacA.

    References

    Abramson J., Smirnova I., Kasho V., Verner G., Kaback H. R. & Iwata S. (2003). Structure and mechanism of the lactose permease of Escherichia coli. Science. 301(5633): 610-615.

    Bassetti M., Righi E., Del Giacomo P., Sartor A., Ansaldi F., Trucchi C., Alicino C. , Trecarichi E. M. , Spanu T., Paganino C., Tumbarello M. & Carnelutti A. (2018). Predictors of mortality with Staphylococcus aureus bacteremia in elderly adults. Journal of the American Geriatrics Society. 66(7): 1284-1289.

    Brown M. H. & Skurray R. A. (2001). Staphylococcal multidrug efflux protein QacA. Journal of Molecular Microbiology and Biotechnology. 3(2): 163-170.

    Cervinkova D., Babak V., Marosevic D., Kubikova I. & Jaglic Z. (2013). The role of the qacA gene in mediating resistance to quaternary ammonium compounds. Microbial Drug Resistance. 19(3): 160-167.

    Costa S. S., Viveiros M., Amaral L. & Couto I. (2013). Multidrug efflux pumps in Staphylococcus aureus: an update. The Open Microbiology Journal. 7: 59.

    Frillingos S., Sahin‐Tóth M., Wu J. & Kaback H. R. (1998). Cys-scanning mutagenesis: a novel approach to structure–function relationships in polytopic membrane proteins. The FASEB Journal. 12(13): 1281-1299.

    Hanahan D. (1983). Studies on transformation of Escherichia coli with plasmids. Journal of Molecular Biology. 166(4): 557-580. DOI: 10.1016/S0022-2836(83)80284-8.

    Hassan K. A., Robinson K. L., Smith A. N., Gibson J. H., Skurray R. A. & Brown M. H. (2006). Glycine-rich transmembrane helix 10 in the staphylococcal tetracycline transporter TetA (K) lines a solvent-accessible channel. Biochemistry. 45(51): 15661-15669.

    Kahn M., Figurski D., Ito L. & Helinski D. (1979). Essential regions for replication of a stringent and a relaxed plasmid in Escherichia coli. Paper presented at the Cold Spring Harbor symposia on Quantitative Biology.

    Kumar S., Singh S., Kumar V., Datta S., Dhanjal D. S., Sharma P. & Singh J. (2020). Pathogenesis and Antibiotic Resistance of Staphylococcus aureus Model Organisms for Microbial Pathogenesis, Biofilm Formation and Antimicrobial Drug Discovery. Springer: 99-115.

    Lin S.-Y., Tu H.-P., Chen T.-C., Shen M.-C., Chiu Y.-W., Chen Y.-H. & Lu P.-L. (2018). Association of bacterial genotypes and epidemiological features with treatment failure in hemodialysis patients with methicillin-resistant Staphylococcus aureus bacteremia. PloS one. 13(6): e0198486.

    Nikaido H. (2009). Multidrug resistance in bacteria. Annual Review of Biochemistry. 78: 119.

    Pao S. S., Paulsen I. T. & Saier M. H. (1998). Major facilitator superfamily. Microbiology and Molecular Biology Reviews. 62(1): 1-34.

    Paulsen I. T., Brown M. H. & Skurray R. A. (1996). Proton-dependent multidrug efflux systems. Microbiology and Molecular Biology Reviews. 60(4): 575-608.

    Radestock S. & Forrest L. R. (2011). The alternating-access mechanism of MFS transporters arises from inverted-topology repeats. Journal of Molecular Biology. 407(5): 698-715.

    Reddy V. S., Shlykov M. A., Castillo R., Sun E. I. & Saier M. H. (2012). The major facilitator superfamily (MFS) revisited. FEBS Journal. 279(11): 2022-2035.

    Saier Jr. M. H., Beatty J. T., Goffeau A., Harley K. T., Heijne W., Huang S.-C., Jack D. L., Jähn P. S., Lew K., Liu J., Pao S. S., Paulsen I. T., Tseng T. T. & Virk P. S. (1999). The major facilitator superfamily. Journal of Molecular Microbiology and Biotechnology. 1(2): 257-279.

    Tennet J. M., Lyon B. R., Midgley M., Jones G., Purewal A. & Skurray R. A. (1989). Physical and biochemical characterization of the qacA gene encoding antiseptic and disinfectant resistance in Staphylococcus aureus. Microbiology. 135(1): 1-10.

    Vogel M., Schmitz R. P., Hagel S., Pletz M. W., Gagelmann N., Scherag A., Chlatttman P. & Brunkhorst F. M. (2016). Infectious disease consultation for Staphylococcus aureus bacteremia–A systematic review and meta-analysis. Journal of Infection. 72(1): 19-28.

    Zhu Q. & Casey J. R. (2007). Topology of transmembrane proteins by scanning cysteine accessibility mutagenesis methodology. Methods. 41(4). 439-450. DOI: 10.1016/j.ymeth.2006.08.004.