The transthyretin-like protein (TLP) from Salmonella enterica subspecies I is a periplasmic protein with high level structural similarity to a protein found in mammals and fish. In humans, the protein homologue, transthyretin, binds and carries retinol and thyroxine, and a series of other, unrelated aromatic compounds. Here we show that the amino acid sequence of the TLP from different species, subspecies and serovars of the Salmonella genus is highly conserved and demonstrate that the TLP gene is constitutively expressed in S. Typhimurium and that copper and other divalent metal ions severely inhibit enzyme activity of the TLP, a cyclic amidohydrolase that hydrolyses 5-hydroxyisourate (5-HIU). In order to determine the in vivo role of the S. Typhimurium TLP, we constructed a strain of mouse-virulent S. Typhimurium SL1344 bearing a mutation in the TLP gene (SL1344 ΔyedX). We assessed the virulence of this strain via oral inoculation of mice and chickens. Whilst SL1344 ΔyedX induced a systemic infection in both organisms, the bacterial load detected in the faeces of infected chickens was significantly reduced when compared to the load of S. Typhimurium SL1344. These data demonstrate that the TLP gene is required for survival of S. Typhimurium in a high uric acid environment such as chicken faeces, and that metabolic traits of Salmonellae in natural and contrived hosts may be fundamentally different. Our data also highlight the importance of using appropriate animal models for the study of bacterial pathogenesis especially where host-specific virulence factors or traits are the subject of the study.
References
[1]
Barrow PA, Huggins MB, Lovell MA, Simpson JM (1987) Observations on the pathogenesis of experimental Salmonella typhimurium infection in chickens. Res Vet Sci 42: 194–199.
[2]
Mead PS, Slutsker L, Dietz V, McCaig LF, Bresee JS, et al. (1999) Food-related illness and death in the United States. Emerg Infect Dis 5: 607–625.
[3]
Ochman H, Wilson AC (1987) Evolution in bacteria: evidence for a universal substitution rate in cellular genomes. J Mol Evol 26: 74–86.
[4]
Hennebry SC (2009) Evolutionary changes to transthyretin: structure and function of a transthyretin-like ancestral protein. FEBS J 276: 5367–5379.
[5]
Hennebry SC, Wright HM, Likic VA, Richardson SJ (2006) Structural and functional evolution of transthyretin and transthyretin-like proteins. Proteins 64: 1024–1045.
[6]
Hennebry SC, Law RH, Richardson SJ, Buckle AM, Whisstock JC (2006) The crystal structure of the transthyretin-like protein from Salmonella dublin, a prokaryote 5-hydroxyisourate hydrolase. J Mol Biol 359: 1389–1399.
[7]
Zanotti G, Cendron L, Ramazzina I, Folli C, Percudani R, et al. (2006) Structure of zebra fish HIUase: insights into evolution of an enzyme to a hormone transporter. J Mol Biol 363: 1–9.
[8]
Ramazzina I, Folli C, Secchi A, Berni R, Percudani R (2006) Completing the uric acid degradation pathway through phylogenetic comparison of whole genomes. Nat Chem Biol 2: 144–148.
[9]
Lee Y, Lee DH, Kho CW, Lee AY, Jang M, et al. (2005) Transthyretin-related proteins function to facilitate the hydrolysis of 5-hydroxyisourate, the end product of the uricase reaction. FEBS Lett 579: 4769–4774.
[10]
Kahn K, Tipton PA (1998) Spectroscopic characterization of intermediates in the urate oxidase reaction. Biochemistry 37: 11651–11659.
[11]
Santos CX, Anjos EI, Augusto O (1999) Uric acid oxidation by peroxynitrite: multiple reactions, free radical formation, and amplification of lipid oxidation. Arch Biochem Biophys 372: 285–294.
[12]
Schultz AC, Nygaard P, Saxild HH (2001) Functional analysis of 14 genes that constitute the purine catabolic pathway in Bacillus subtilis and evidence for a novel regulon controlled by the PucR transcription activator. J Bacteriol 183: 3293–3302.
[13]
Wilkinson SP, Grove A (2004) HucR, a novel uric acid-responsive member of the MarR family of transcriptional regulators from Deinococcus radiodurans. J Biol Chem 279: 51442–51450.
[14]
Reitzer L (2003) Nitrogen assimilation and global regulation in Escherichia coli. Annu Rev Microbiol 57: 155–176.
[15]
Xi H, Schneider BL, Reitzer L (2000) Purine catabolism in Escherichia coli and function of xanthine dehydrogenase in purine salvage. J Bacteriol 182: 5332–5341.
[16]
Kim GJ, Kim HS (1998) Identification of the structural similarity in the functionally related amidohydrolases acting on the cyclic amide ring. Biochem J 330(Pt 1): 295–302.
[17]
Lundberg E, Backstrom S, Sauer UH, Sauer-Eriksson AE (2006) The transthyretin-related protein: structural investigation of a novel protein family. J Struct Biol 155: 445–457.
[18]
Samocha-Bonet D, Lichtenberg D, Pinchuk I (2005) Kinetic studies of copper-induced oxidation of urate, ascorbate and their mixtures. J Inorg Biochem 99: 1963–1972.
[19]
Eiteman MA, Gordillo RM, Cabrera ML (1994) Analysis of oxonic acid, uric acid, creatine, allantoin, xanthine and hypoxanthine in poultry litter by reverse phase HPLC. Fresnius J Anal Chem 348: 680–638.
[20]
Richardson SJ (2007) Cell and molecular biology of transthyretin and thyroid hormones. Int Rev Cytol 258: 137–193.
[21]
Hennebry SC (2009) The Salmonella sp. TLP: A Periplasmic 5-Hydroxyisourate Hydrolase. In: Richardson SJ, Cody V, editors. Recent Advances in Transthyretin Evolution, Structure and Biological Functions. Heidelberg: Springer-Verlag.
[22]
Gutnick D, Calvo JM, Klopotowski T, Ames BN (1969) Compounds which serve as the sole source of carbon or nitrogen for Salmonella typhimurium LT-2. J Bacteriol 100: 215–219.
[23]
Simoyi MF, Falkenstein E, Van Dyke K, Blemings KP, Klandorf H (2003) Allantoin, the oxidation product of uric acid is present in chicken and turkey plasma. Comp Biochem Physiol B Biochem Mol Biol 135: 325–335.
[24]
Becker BF (1993) Towards the physiological function of uric acid. Free Radic Biol Med 14: 615–631.
[25]
Baumler AJ, Tsolis RM, Ficht TA, Adams LG (1998) Evolution of host adaptation in Salmonella enterica. Infect Immun 66: 4579–4587.
[26]
Barrow PA, Simpson JM, Lovell MA (1988) Intestinal colonisation in the chicken by food-poisoning Salmonella serotypes; microbial characteristics associated with faecal excretion. Avian Pathol 17: 571–588.
[27]
Braun EJ, Campbell CE (1989) Uric acid decomposition in the lower gastrointestinal tract. J Exp Zool Suppl 3: 70–74.
[28]
Ausubel FM (1988) Current Protocols in Molecular Biology; Ausubel FM, Brent R, Kingston RE, Moore DM, Smith JA, et al.., editors. New York: Greene Publishing Associates and Wiley Interscience.
[29]
Datsenko KA, Wanner BL (2000) One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97: 6640–6645.
[30]
Sambrook J, Fritsch E, Maniatis T (1989) Molecular Cloning: A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press.
[31]
Gowrishankar J, Pittard AJ (1998) Superimposition of tyrR protein-mediated regulation on osmoresponsive transcription of Escherichia coli proU in vivo. J Bacteriol 180: 6743–6748.
[32]
Miller TM (1974) Experiments in molecular genetics. Cold Spring Harbour, N.Y.: Cold Spring Harbour Laboratory.
[33]
Hudson GS, Davidson BE (1984) Nucleotide sequence and transcription of the phenylalanine and tyrosine operons of Escherichia coli K12. J Mol Biol 180: 1023–1051.
[34]
Govoni G, Canonne-Hergaux F, Pfeifer CG, Marcus SL, Mills SD, et al. (1999) Functional expression of Nramp1 in vitro in the murine macrophage line RAW264.7. Infect Immun 67: 2225–2232.
[35]
Wilcoxon F (1945) Individual comparison by ranking methods. Biometrics 1: 80–83.
