Salmonella typhimurium's transthyretin-like protein is a host-specific factor important in fecal survival in chickens.

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

TLP hydrolytic activity in the presence of divalent metal ions.

<p>The production and hydrolysis of 5-HIU was measured at 312 nm and is shown over a 10 minute period. Enzyme reactions were performed as described in the text. Briefly, 0.04 U uricase was equilibrated in 50 mM potassium phosphate buffer, pH 7.8 in the presence or absence of 100 µM divalent metal ions (Cu²⁺; results for other divalent metal cations not shown). Enzyme reactions were initiated with the addition of 100 µM freshly diluted uric acid (at time 0). Reactions were performed at 22°C in a Biorad spectrophotometer. Open squares (□): production of 5-HIU in the absence of metal ions. Closed squares (▪): production of 5-HIU in the presence of Cu²⁺. After approximately 3 minutes, the amount of 5-HIU peaked and underwent slow, spontaneous decomposition. Open circles (○): addition of 5.2 nM recombinant <i>S.</i> Typhimurium TLP at time 2.5 minutes in the absence of metal ions resulted in rapid hydrolysis of 5-HIU. Closed circles (•): in the presence of Cu²⁺ no hydrolysis occurred following addition of TLP. (Data for other metal ions not shown).</p

Phylogenetic relationship of cytoplasmic and periplasmic TLPs.

<p>Neighbor-joining tree of representative periplasmic and cytosolic TLP sequences from various classes of proteobacteria. One hundred bootstrap replicates were performed to assess the robustness of the tree and the results are indicated at each node in the tree. The TLP sequence from <i>Marinomonas</i> sp. was used as the outgroup and to root the tree. Periplasmic TLP sequences are indicated with an asterisk. The taxonomic classes of the bacteria represented in the tree are indicated on the far right hand side of the Figure (Gammaproteobacteria, Betaproteobacteria, Epsilonproteobacteria, Alphaproteobacteria). The taxonomic orders within these subsets are indicated to the immediate right hand side of the tree (Enterobacteriales, Pseudomonadales, Rhizobiales, Oceanospiralles, Rhodobacterales, Vibrionales, Rhodobacterales, Burkholderiales). In the instances where TLP sequences grouped in a region of the tree alongside TLP sequences from unrelated bacteria, both the order and class of the bacterium are indicated (e.g. Oceanospiralles, Gammaproteobacteria). The tree demonstrates that periplasmic TLP sequences evolved along a separate evolutionary pathway to cytoplasmic TLP sequences.</p

Purine metabolism in bacteria.

<p><b>A</b>. Schematic illustrating the enzymes involved in purine metabolism, specifically in conversion of uric acid to allantoin. The COG (cluster of orthologous groups) numbers indicate the protein family which functions at different steps in the pathway. Asterisks denote homologs which have not been identified in the genomes of members of the <i>Salmonella</i> genus. <b>B</b>. Comparison of the genetic context of i) genes encoding cytoplasmic TLPs with ii) genes encoding periplasmic TLPs. Genes encoding cytoplasmic TLPs are generally located within purine metabolism operons. Genes encoding periplasmic TLPs are not located within operons and do not appear to have a consensus genomic context.</p

Characterisation of the promoter of the <i>S.</i> Typhimurium TLP gene.

<p><b>A</b>. The TLP gene promoter overlaps the 5′ untranslated region of the CopR gene. The bold arrow indicates the relative position of the TLP gene promoter and the dashed arrow indicates the relative position of the CopR gene promoter on the complementary strand of DNA. <b>B</b>. The results of primer extension analysis using [γ³²P]-radiolabelled primer StyphPrimEx1 (data not shown) Samples were electrophoresed in an 8% urea/polyacrylamide gel and visualised by exposure of Kodak XAR-5 MR film. In lane 1, the GA ladder is shown; lane 2: reverse transcription product using RNA from strain SL1344/pSCH (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046675#pone-0046675-t001" target="_blank">Table 1</a>); lane 3: reverse transcription product using RNA from SL1344; lane 4: no RNA control. Migration of the single product (indicated “P” in lane 2) was compared to the GA ladder and indicated that the start site of transcription was 49 nucleotides upstream of the ATG codon. This was determined by comparing the genomic DNA sequence upstream of the StyphPrimEx1 sequence to the GA ladder. <b>C</b>. The DNA sequence upstream and downstream of the ATG (initiation of translation) codon for the <i>yedX</i> (TLP) gene is shown. The +1 site (start site of transcription) determined by primer extension analysis is indicated. The −10 and −35 regions of the TLP gene promoter are indicated with a single underline and a double underline, respectively. The shaded grey box represents the putative palindromic copper-box sequence that may influence the transcription of both the TLP and CopR genes. The start site of translation of the CopR gene is indicated (CopR←) and is on the complementary strand to the TLP gene promoter. Thus, the 5′ untranslated region of the CopR gene is also the sequence complementary to the TLP gene promoter.</p