Accessory Gene Regulator Locus of Staphylococcus intermedius

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INFECTION AND IMMUNITY, May 2006, p Vol. 74, No /06/$ doi: /iai Copyright 2006, American Society for Microbiology. All Rights Reserved. Accessory
INFECTION AND IMMUNITY, May 2006, p Vol. 74, No /06/$ doi: /iai Copyright 2006, American Society for Microbiology. All Rights Reserved. Accessory Gene Regulator Locus of Staphylococcus intermedius Julia M. L. Sung, 1 Peter D. Chantler, 2 and David H. Lloyd 1 * Departments of Veterinary Clinical Sciences 1 and Veterinary Basic Sciences, 2 Royal Veterinary College, University of London, London, United Kingdom Received 15 December 2005/Returned for modification 26 January 2006/Accepted 15 February 2006 The accessory gene regulator (agr) locus, a candidate system for the regulation of the production of virulence factors in Staphylococcus intermedius, has been characterized. Using PCR-based genome walking, we have obtained the first complete sequence (3,436 bp) of the accessory gene regulator (agr) gene in this organism. Sequence analysis of the agr gene has identified five open reading frames (ORFs), agrb, agrd, agrc, agra, and hld. The translated ORF contained amino acid motifs characteristic of the response regulator and histidine protein kinase signal transducer of the classic two-component regulatory system. Sequencing of the agrd PCR products amplified from DNA from 20 different isolates has facilitated detection of genetic variation in the putative autoinducing peptide (AIP) within the agr gene of S. intermedius, revealing the presence of at least three agr specificity groups within this species. Classification of the agr gene from S. intermedius was supported by phylogenetic analysis. Real-time PCR also revealed that the effector molecule of the agr system, RNAIII, was regulated in an autocrine manner in S. intermedius and demonstrated positive correlation with the temporal gene expression patterns of luk and entc. Transcription of RNAIII was also dependent on self secreted cues. Cyclic self and nonself peptides were synthesized on the basis of the novel AIPs produced by S. intermedius, which lack the cysteine necessary to form the thiolactone ring in analogous peptides from Staphylococcus aureus and Staphylococcus epidermidis. Experiments with these synthetic cyclic peptides indicated that self peptides led to up-regulation of RNAIII findings in support of the assumption that activation of the agr gene is initiated by growth- and species-specific factors generated during bacterial growth. Staphylococcus intermedius, first described in 1976 (10), is the most common cause of skin infection in dogs, with canine pyoderma being caused almost exclusively by S. intermedius (31). S. intermedius is also frequently isolated from dogs with otitis media and externa (3, 4, 28) and is commonly associated with seborrhoea, a cutaneous scaling disorder in dogs. Little is known, however, about the pathogenesis of S. intermedius. The results of studies investigating the production of virulence factors are unclear, and no distinct differences in the nature of the toxins produced by isolates from healthy dogs and dogs with pyoderma have been found (1). Thus far, there are differing opinions as to the underlying causes of canine pyoderma. Ihrke suggested factors that predispose dogs to staphylococcal pyoderma infections were due to a wide variety of underlying diseases, such as allergy, metabolic diseases, disruption of normal skin flora, immunodeficiency, and endoparasitic infestation (12), whereas according to Greene and Lammler, hostbacterium interactions and the immune status of the host appeared to be the factors most crucial in determining the outcome of infection (9). Many of the factors believed to be involved in S. intermedius virulence have also not been well characterized, and proof of their association with disease pathogenesis is often lacking. It is thus difficult for veterinary clinicians and microbiologists to determine the clinical significance of many S. intermedius isolates. * Corresponding author. Mailing address: Department of Veterinary Clinical Sciences, Hawkshead Lane, North Mymms, Hatfield, Hertfordshire AL9 7TA, United Kingdom. Phone: 44(0) Fax: 44(0) Present address: Department of Cellular and Molecular Medicine, St. George s Hospital Medical School, Cranmer Terrace, London SW17 0RE, United Kingdom. S. intermedius produces a diverse arsenal of virulence factors involving a large number of cell-bound and extracellular proteins (6, 11, 29, 32), which may be likely contributory factors that predispose dogs to staphylococcal pyoderma. The virulent components and products are thought to share overlapping roles, acting either in concert or alone, and may convert host tissue components into nutrients required for bacterial growth. On the basis of studies performed with corresponding virulence factors in Staphylococcus aureus, considerable knowledge is available about their contribution to the development of infection in humans (21). Less is known about the virulence factors of S. intermedius and their relative importance in canine pyoderma. In the human pathogen S. aureus, the production of virulence factors is known to be controlled, in part, by the accessory gene regulator system. One of a number of two-component regulatory systems identified thus far, the agr system has received much attention owing to it being assigned, historically, a central role in the model of S. aureus pathogenesis. It is recognized as a quorum-sensing gene cluster that up-regulates production of secreted virulence factors and down-regulates production of cell-associated virulence factors in a cell densitydependent manner (15, 19, 25). When the staphylococci are in lag phase, it is thought that staphylococci initiate infection by synthesizing surface proteins. Once colonization is established, the bacteria multiply and enter exponential phase, activating a density-sensing mechanism that stimulates toxic exoprotein production, thereby enabling them to spread to new sites to prevent overcrowding (30). Synthesis of essential factors only as and when required is important from an economic perspective, as it avoids overwhelming the organism metabolically with unnecessary energy expenditure. 2947 2948 SUNG ET AL. INFECT. IMMUN. FIG. 1. Schematic representation of the agr locus in S. intermedius. The extremities of agr, which consists of two divergent transcripts, RNAII and RNAIII, were amplified and sequenced using genome walking. For downstream genome walking, the D1 primer was initially designed, allowing amplification of the A fragment. The A fragment was partially sequenced, and from the sequences obtained, the D2 primer was designed and synthesized. Further downstream sequencing of the A fragment was performed using the D2 primer. Sequential primer design (D3) and sequencing were repeated until the whole downstream agr sequence in S. intermedius was elucidated. For upstream genome walking, PCR amplification with the U1 primer produced the B fragment. From the B fragment, the S. intermedius-specific U2 primer was designed, enabling sequencing of the C fragment. The agrb, agrd, agrc, and agra ORFs are transcribed in one direction; while the RNAIII is transcribed in the opposite direction. The individual ORFs are represented by block arrows. The size of each ORF can be seen by the base pair ruler in the middle of the figure. S. intermedius produces many toxins comparable to those of S. aureus, so we hypothesized that it is likely that the production of toxins in the former is under the regulation of a similar global regulator. To determine whether this is so, here we have ascertained the presence of the agr locus and analyzed its sequence. Our starting point was the agrd gene present in each strain of a collection of 20 S. intermedius strains isolated from cases of canine pyoderma. The elucidation of the agr locus facilitated assessment of its regulation during the growth of S. intermedius. In S. aureus, mutation and deletion analyses performed suggested that RNAIII itself, rather than any translation product, is the effector of exoprotein gene regulation (25). We have thereby quantified RNAIII, the effector molecule of agr, together with luk and entc, encoding leukotoxin and enterotoxin C, respectively, using real-time quantitative PCR. We have also synthesized two novel cyclic autoinducing peptides (AIPs), shown to be found in S. intermedius (17), but not in S. aureus or Staphylococcus epidermidis, and used these in functional assays monitoring RNAIII production. MATERIALS AND METHODS Bacterial strains and growth conditions. Twenty clinical isolates of S. intermedius were collected from the skin of dogs with pyoderma at the Royal Veterinary College. Long-term storage of the isolates was maintained in 20% glycerol in brain heart infusion broth at 70 C, and cultures for routine use were maintained on sheep blood agar plates. Isolates were identified as S. intermedius according to their colony morphology, biochemical reactions (production of coagulase and DNase), and on the basis of the analytical profile index (API) (biomerieux, France). PCR and sequencing. The collection of 20 S. intermedius isolates was screened by direct colony PCR. It was found that the initial hot start of the PCR was sufficient to break down bacterial cell walls from a pinprick colony of S. intermedius, allowing these cells to be used as the direct source of DNA. We used the following primers: 5 -TTCCAATCAATCGGTACT-3 and 5 -TTCCTCCTTCT AACAACTC-3 ; their design was based on regions of the partial agr sequence derived from a pigeon isolate of S. intermedius (GenBank accession number AF346723). PCR was carried out in the GeneAmp PCR system 9700 thermocycler (Perkin-Elmer Biosystems, Foster City, California). Amplification was performed in a final volume of 50 l containing datp, dctp, dgtp, and dttp (each at a concentration of 200 M), 10 M of the corresponding primer, 5 l of 10 Expand high-fidelity buffer (Roche, Nutley, NJ), and 2.6 U of Expand high-fidelity enzyme mix (Roche). The cycling parameters for PCR were as follows: (i) an initial denaturation step of 2 min at 94 C; (ii) 35 cycles, with 1 cycle consisting of 15 s at 94 C, 30 s at 45 C, and 60 s at 72 C; and (iii) a final extension step of 7 min at 72 C. The amplified fragments were cloned into the pgem-t Easy vector (Promega, Madison, Wis.). Recombinant DNA molecules were transformed into electrocompetent Escherichia coli JM109 cells and sequenced (MWG-Biotech, Ebersberg, Germany). Genome walking. Genome walking was initiated from the amplified and sequenced agrd DNA fragment using the Universal Genome-Walker kit (BD Biosciences Clontech, Palo Alto, CA). Total genomic DNA of S. intermedius strain 3 was isolated with Genomic-Tip 100/G (QIAGEN, Crawley, United Kingdom) and digested with DraI, EcoRV, PvuII, and StuI to obtain blunt ends. An aliquot of each DNA sample was run on a 0.7% agarose gel to ascertain quality. After purification, the fragments were ligated to a Genome-Walker adapter provided by the manufacturer (31). The Genome-Walker protocol consists of a series of PCR amplifications, starting initially with the adapter primers (AP1 or AP2) and two nested gene-specific primers designed on the basis of the agrd sequence of S. intermedius (see above), D1 (5 -CCAATCAATCGGT ACTTTTGCTAGAATCCCTATTAGTACTGGA-3 ) and U1 (5 -AGATGAG CCAGAAATCCCTGCCGAGTTGTTAGAAGAGGAC-3 ). Sequence extension occurred through further walks using novel gene-specific primers, and the primers were designed on the basis of initial sequence data obtained from previous steps. Care was taken to ensure that the sequences overlapped to some degree. Downstream chromosomal walking was continued with the following primers: D2 (5 -CGTGAAGATGATTTAGAAGGTTTGAAGCAA-3 ) and D3 (5 -GCTACCAATGACCCTTATGCTATCTTAGAAACTTCT-3 ). The upstream region of agrd was analyzed in a similar way using primer U2 (5 -CAA CAACTTACGCACTGGTTAACACAAGGAC-3 ). Each amplification was performed in a final volume of 50 l containing 5 l 10 genomic PCR buffer, 1.1 mm magnesium acetate, 10 mm 50 deoxynucleoside triphosphate (dntp) VOL. 74, 2006 STAPHYLOCOCCUS INTERMEDIUS agr LOCUS 2949 Bacterial source (GenBank no.) TABLE 1. Alignment and comparison of the primary sequences of agrd from staphylococci Primary sequence S. aureus Group I (x52543)...mntlfnlffd FITGILKNIG NIAAYSTCDF IMDEVEVPKE LTQLHE Group II (AP003135)...MNTLVNMFFD FIIKLAKAIG IVGGVNACSS LFDEPKVPAE LTNLYDK Group III (AP004829)...MKKLLNKVIE LLVDFFNSIG YRAAYINCDF LLDEAEVPKE LTQLHE Group IV (AF288215)...MNTLYKSFFD FITGVLKNIG NVASYSTCYF IMDEVEIPKE LTQL S. lugdunensis (AF173933)...MNLLSGLFTK GISAIFEFIG NFSAQDICNA YFDEPEVPQE LIDLQRKQLIESV S. epidermidis (Z49220)...MNLLGGLLLK LFSNFMAVIG NAAKYNPCAS YLDEPQVPEE LTKLDE S. intermedius pigeon (AF346723)...MR ILEVLFN LITNLFQSIG TFARIPTSTG FFDEPEIPAE LLEEEK S. intermedius canine strains S3...MR ILEVLFN LITNLFQSIG TFARIPISTG FFDEPEIPAE LLEEDK S11...MR ILEVLFN LITNLFQSIG TFAKIPTSTG FFDEPEIPAE LLEE S24...MR ILEVLFN LITNLFQSIG TFARIPTSTG FFDELEIPAE LLEE S27...MR ILEVLFN LITNLFQSIG TFAKIPTSTG FFDEPEIPEE LLEE S31...MR ILEVLFN LITNLFQSIG TFAKIPTSTG FFDEPEIPEE LLEE S33...MR ILEVLFN LITNLFQSIG TFAKIPTSTG FFDEPEIPEE LLEE S36...MR ILEVLFN LITNLFQSIG TFAKIPTSTG FFDEPEIPEE LLEE S38...MR ILEVLFN LITNLFQSIG TFARIPISTG FFDEPEIPAE LLEE S40...MR ILEVLFN LITNLFQSIG TFARIPISTG FFDEPEIPAE LLEE S55...MR ILEVLFN LITNLFQSIG TFAKIPTSTG FFDEPEIPAE LLEE S64...MR ILEVLFN LITNLFQSIG TFARIPISTG FFDEPEIPAE LLEE S65...MR ILEVLFN LITNLFQSIG TFARIPISTG FFDEPEIPAE LFEE S69...MR ILEVLFN LITNLFQSIG TFARIPISTG FFDEPEIPAE LLEE S73...MR ILEVLFN LITNLFQSIG TFAKIPTSTG FFDEPEIPEE LLEE S77...MR ILEVLFN LITNLFQSIG TFAKIPTSTG FFDEPEIPEE LLEE S81...MR ILEVLFN LITNLFQSIG TFARIPISTG FFDEPEIPAE LLEE S83...MR ILEVLFN LITNLFQSIG TFAKIPTSTG FFDEPEIPEE LLEE S85...MR ILEVLFN LITNLFQSIG TFAKIPTSTG FFDEPEIPAE LLEE S88...MR ILEVLFN LITNLFQSIG TFAKIPTSTG FFDEPEIPEE LLEE S95...MR ILEVLFN LITNLFQSIG TFAKIPTSTG FFDEPEIPEE LLEE a Primary sequences of agrd from S. aureus group I (GenBank accession number X52543), S. aureus group II (AP003135), S. aureus group III (AP004829), S. aureus group IV (AF288215), S. lugdunensis (AF173933), S. epidermidis (Z49220), S. intermedius of pigeon origin (AF346723), and isolates of S. intermedius of canine origin. Conserved amino acid residues in all organisms are in boldface type. Sequences of autoinducing peptides are underlined. Gaps introduced to maximize alignment are indicated by dashes. mix, 1 l50 Advantage genomic polymerase mix (BD Biosciences), and 10 M of the corresponding primers. A typical PCR cycle consisted of 7 cycles of 94 C for 2 s and 72 C for 3 min, followed by 32 cycles of 94 C for 2 s and 67 C for 3 min, and a final elongation step at 67 C for 4 min. To facilitate PCR amplification of sequences that proved difficult to amplify, we used the Advantage GC genomic polymerase (BD Biosciences). PCR products from genome walking were purified using the QIAquick gel extraction kit (QIAGEN) and directly sequenced with both primers (adapter and S. intermedius specific). For PCR amplicons that were longer than 0.6 kb, sequencing was performed via primer walking, involving repeated cycles of custom oligonucleotide synthesis and direct sequencing. Sequences obtained were confirmed by comparison of independent PCR experiments with different primers and genomic DNA. Alignments of amino acid sequences from the individual protein-coding genes were realized by using the ClustalW algorithm ( and default gap penalties (28). Regions of sequence that were difficult to align were removed from the data file and the sequences realigned. Ambiguous alignment regions, such as the N and C termini, were excluded from the alignments. Overlapping nucleotide sequences of DNA generated were manually aligned to form a contiguous agr operon and assessed for complete open reading frames (ORFs). DNA and protein sequence similarity searches were performed by the BLASTN and BLASTP (National Center for Biotechnology Information server). Conserved domains in each ORFs were analyzed using reverse position-specific BLAST (rpsblast) (v1.65). Transmembrane topology prediction. The transmembrane topology of the various ORFs of the S. intermedius agr gene was analyzed using ConPredII ( ConPred2/), a program based on a consensus approach that combines the results of several prediction methods, such as KKD, TMpred, TopPredII, DAS, TMAP, MEMSAT 1.8, SOSUI, TMHMM 2.0, and HMMTOP 2.0 (2). Assessment of the regulation of leukotoxin, enterotoxin, and RNAIII transcription in S. intermedius. To examine growth phase-dependent expression, 2-ml cultures of S. intermedius (strain 3) were grown overnight with aeration in brain heart infusion broth at 37 C. Cells were then inoculated from the overnight culture to an initial optical density at 600 nm (OD 600 ) of 0.05 and grown with shaking to the desired phase of growth, e.g., early exponential (OD 600 of 0.5, 1 h), mid-exponential (OD 600 of 4.5; 4 h), or post-exponential phase (OD 600 of 8.0; 8 h). The various growth phases were determined in preliminary experiments (not shown). Growth was monitored turbidimetrically with a UV-160A spectrophotometer (Shimadzu) read at 600 nm. The results of growth phase effects were standardized for the number of bacteria, so that the increase in the total amount of protein is not simply a consequence of increasing number of bacterial cells. At indicated times, samples containing equal numbers of cells were immediately stabilized by the addition of 2 volumes of Bacterial Protect reagent (QIAGEN). The bacterial cells were subsequently harvested after 10 min by centrifugation at 13,000 g for 10 min at 4 C, prior to RNA extraction. Characterization of the activating factor for luk, entc, and RNAIII expression in S. intermedius. Cultures of S. intermedius strains 3 and 27 and S. aureus strain J3945 were inoculated from 1-day-old single colonies on sheep blood agar plates into 2 ml brain heart infusion broth. The cultures were grown overnight at 37 C with orbital shaking for 15 h. To synchronize cultures to an active mode of growth and to eliminate potential quorum signals that may have accumulated, 0.5 ml of overnight culture was centrifuged at 3,000 g for 5 min at 4 C. After the supernatant was discarded and the bacteria were resuspended in fresh prewarmed medium (50 ml), they were grown at 37 C with shaking. Aliquots (10 ml) were harvested at intervals of 1, 4, and 8 h corresponding to the early, mid-, and post-exponential phases of bacterial growth. Bacterial cells were removed by centrifugation at 13,000 g for 15 min at 4 C, and thereafter, the supernatants were filter sterilized through a m-pore-size filter (Millipore). All the supernatant filtrates obtained were lyophilized and stored at 80 C until use as a source of putative AIP. 2950 SUNG ET AL. INFECT. IMMUN. FIG. 2. Nucleotide sequence alignment of the RNAIII of S. aureus (S.a), S. lugdunensis (S.l), S. epidermidis (S.e), and S. intermedius (S.i). Identical nucleotides that are present in at least three of the sequences are indicated by shading. The predicted delta-lysin open reading frames (ATG) and the stop codons (TAA) are in boldface type. The putative ribosome-binding Shine-Dalgarno (SD) sites upstream of the delta-lysin are underlined. Arrowheads and double-underlined sequences indicate direct (DR) and indirect repeat (IR). Sequences are manipulated by introducing gaps (indicated by dashes) to fit transcription start points and the delta-lysin open reading frames. Putative transcriptional start sites ( 1) and the 10 and 35 promoter elements are underlined. VOL. 74, 2006 STAPHYLOCOCCUS INTERMEDIUS agr LOCUS 2951 FIG. 2 Continued. Synthesis of the AgrD cyclic peptides. The cyclic peptides RIPISTGFF and KIPTSTGFF were custom synthesized by Peptide Protein Research Company Ltd. (Eastleigh, United Kingdom). Both peptides were cyclized by a lactone ring between the serine side chain and the C terminus and purified in acetonitrile and water containin
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