Extragenic suppressor mutations in ΔripA disrupt stability and function of LpxA

BMC Microbiology

Thomas H Kawula³  Edward J Collins³  Garry D Dotson¹  Corbin D Jones²  Artur Romanchuk⁴  Sharon A Taft-Benz³  Eric D LoVullo³  Ronald J Jenkins¹  Jason C Brunton³  Shaun P Steele³  Cheryl N Miller³ 

[1] Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor 48109, MI, USA;Carolina Center for Genome Sciences, University of North Caroline, Chapel Hill 27599, NC, USA;Department of Microbiology and Immunology, School of Medicine, University of North Carolina, Chapel Hill 27599, NC, USA;Department of Biology, University of North Carolina, Chapel Hill 27599, NC, USA

关键词: UDP-N-acetylglucosamine;    Lipid A biosynthesis;    LpxA;    Francisella tularensis;    RipA;    Membrane protein;    Enzyme mechanism;    Enzyme mutation;    Cell wall;    Extragenic suppressor mutations;   

Others  :  1137664
DOI  :  10.1186/s12866-014-0336-x

 received in 2014-09-11, accepted in 2014-12-18,  发布年份 2014

【 摘 要 】

Background

Francisella tularensis is a Gram-negative bacterium that infects hundreds of species including humans, and has evolved to grow efficiently within a plethora of cell types. RipA is a conserved membrane protein of F. tularensis, which is required for growth inside host cells. As a means to determine RipA function we isolated and mapped independent extragenic suppressor mutants in ∆ripA that restored growth in host cells. Each suppressor mutation mapped to one of two essential genes, lpxA or glmU, which are involved in lipid A synthesis. We repaired the suppressor mutation in lpxA (S102, LpxA T36N) and the mutation in glmU (S103, GlmU E57D), and demonstrated that each mutation was responsible for the suppressor phenotype in their respective strains. We hypothesize that the mutation in S102 altered the stability of LpxA, which can provide a clue to RipA function. LpxA is an UDP-N-acetylglucosamine acyltransferase that catalyzes the transfer of an acyl chain from acyl carrier protein (ACP) to UDP-N-acetylglucosamine (UDP-GlcNAc) to begin lipid A synthesis.

Results

LpxA was more abundant in the presence of RipA. Induced expression of lpxA in the ΔripA strain stopped bacterial division. The LpxA T36N S102 protein was less stable and therefore less abundant than wild type LpxA protein.

Conclusion

These data suggest RipA functions to modulate lipid A synthesis in F. tularensis as a way to adapt to the host cell environment by interacting with LpxA.

【 授权许可】

   
2014 Miller et al.; licensee BioMed Central.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

【 参考文献 】

• [1]Fuller JR, Craven RR, Hall JD, Kijek TM, Taft-Benz S, Kawula TH: RipA, a Cytoplasmic Membrane Protein Conserved among Francisella Species, Is Required for Intracellular Survival. Infect Immun 2008, 76:4934-4943.
• [2]Mortensen BL, Fuller JR, Taft-Benz S, Collins EJ, Kawula TH: Francisella tularensis RipA Protein Topology and Identification of Functional Domains. J Bacteriol 2012, 194:1474-1484.
• [3]Fuller JR, Kijek TM, Taft-Benz S, Kawula TH: Environmental and intracellular regulation of Francisella tularensis ripA. BMC Microbiol 2009, 9:216. BioMed Central Full Text
• [4]Huang MTH, Mortensen BL, Taxman DJ, Craven RR, Taft-Benz S, Kijek TM, Fuller JR, Davis BK, Allen IC, Brickey WJ, Gris D, Wen H, Kawula TH, Ting JPY: Deletion of ripA Alleviates Suppression of the Inflammasome and MAPK by Francisella tularensis. J Immunol 2010, 185:5476-5485.
• [5]Peng K, Broz P, Jones J, Joubert LM, Monack D: Elevated AIM2-mediated pyroptosis triggered by hypercytotoxic Francisella mutant strains is attributed to increased intracellular bacteriolysis. Cell Microbiol 2011, 13:1586-1600.
• [6]Anderson MS, Bulawa CE, Raetz CR: The biosynthesis of gram-negative endotoxin. Formation of lipid A precursors from UDP-GlcNAc in extracts of Escherichia coli. J Biol Chem 1985, 260:15536-15541.
• [7]Mengin-Lecreulx D, van Heijenoort J: Identification of the glmU gene encoding N-acetylglucosamine-1-phosphate uridyltransferase in Escherichia coli. J Bacteriol 1993, 175:6150-6157.
• [8]Crowell DN, Anderson MS, Raetz CR: Molecular cloning of the genes for lipid A disaccharide synthase and UDP-N-acetylglucosamine acyltransferase in Escherichia coli. J Bacteriol 1986, 168:152-159.
• [9]Wyckoff TJO, Lin S, Cotter RJ, Dotson GD, Raetz CRH: Hydrocarbon Rulers in UDP-N-acetylglucosamine Acyltransferases. J Biol Chem 1998, 273:32369-32372.
• [10]Raetz CRH, Roderick SL: A Left-Handed Parallel β Helix in the Structure of UDP-N-Acetylglucosamine Acyltransferase. Science 1995, 270:997-1000.
• [11]Wyckoff TJO, Raetz CRH: The Active Site of Escherichia coliUDP-N-acetylglucosamine Acyltransferase: chemical modification and site-directed mutagenesis. J Biol Chem 1999, 274:27047-27055.
• [12]Phillips NJ: Schilling B. McLendon MK, Novel modification of lipid A of Francisella tularensis. Infection and immunity; 2004.
• [13]Jantzen BPBTO E: Cellular fatty acid composition of Francisella tularensis. J Clin Microbiol 1979, 10:928.
• [14]Anderson R, Bhatti AR: Fatty acid distribution in the phospholipids ofFrancisella tularensis. Lipids 1986, 21:669-671.
• [15]Wang X, Ribeiro AA, Guan Z, McGrath SC, Cotter RJ, Raetz CRH: Structure and Biosynthesis of Free Lipid A Molecules That Replace Lipopolysaccharide in Francisella tularensissubsp. novicida†. Biochemistry 2006, 45:14427-14440.
• [16]Schilling B, McLendon MK, Phillips NJ, Apicella MA, Gibson BW: Characterization of Lipid A Acylation Patterns in Francisella tularensis, Francisellanovicida, and FrancisellaphilomiragiaUsing Multiple-Stage Mass Spectrometry and Matrix-Assisted Laser Desorption/Ionization on an Intermediate Vacuum Source Linear Ion Trap. Anal Chem 2007, 79:1034-1042.
• [17]Barker JH, Kaufman JW, Zhang D-S, Weiss JP: Metabolic labeling to characterize the overall composition of Francisella lipid A and LPS grown in broth and in human phagocytes. Innate Immun 2014, 20:88-103.
• [18]Lee CH, Tsai CM: Quantification of bacterial lipopolysaccharides by the purpald assay: measuring formaldehyde generated from 2-keto-3-deoxyoctonate and heptose at the inner core …. Analytical Biochemistry 1999, 267(1):161–168. doi:10.1006/abio.1998.2961.
• [19]LoVullo ED, Miller CN, Pavelka MS, Kawula TH: TetR-Based Gene Regulation Systems for Francisella tularensis. Appl Environ Microbiol 2012, 78:6883-6889.
• [20]Das A, Mukhopadhyay C: LpxA: A natural nanotube. Biopolymers 2010, 93:845-853.
• [21]Khurana R, Fink AL: Do Parallel β-Helix Proteins Have a Unique Fourier Transform Infrared Spectrum? Biophys J 2000, 78:994-1000.
• [22]Jenkins RJ, Dotson GD: A continuous fluorescent enzyme assay for early steps of lipid A biosynthesis. Anal Biochem 2012, 425:21-27.
• [23]Zhang Y-M, Rock CO: Membrane lipid homeostasis in bacteria. Nat Rev Micro 2008, 6:222-233.
• [24]Emiola A, Falcarin P, Tocher J, George J: A model for the proteolytic regulation of LpxC in the lipopolysaccharide pathway of Escherichia coli. Comput Biol Chem 2013, 47:1-7.
• [25]Chamberlain RE: Evaluation of Live Tularemia Vaccine Prepared in a Chemically Defined Medium. Appl Microbiol 1965, 13:232-235.
• [26]Li R, Li Y, Kristiansen K, Wang J: SOAP: short oligonucleotide alignment program. Bioinformatics 2008, 24:713-714.
• [27]Kent WJ: BLAT—The BLAST-Like Alignment Tool. Genome Res 2002, 12:656-664.
• [28]LoVullo ED, Molins-Schneekloth CR, Schweizer HP, Martin S, Pavelka J: Single-copy chromosomal integration systems for Francisella tularensis. Microbiology 2009, 155:1152-1163.
• [29]LoVullo ED, Sherrill LA, Perez LL, Martin S, Pavelka J: Genetic tools for highly pathogenic Francisella tularensis subsp tularensis. Microbiology 2006, 152:3425-3435.
• [30]Zhao J, Raetz CRH: A two-component Kdo hydrolase in the inner membrane of Francisella novicida. Mol Microbiol 2010, 78:820-836.
• [31]LoVullo ED, Sherrill LA, Pavelka MS Jr: Improved shuttle vectors for Francisella tularensisgenetics. FEMS Microbiol Lett 2009, 291:95-102.