Variations in amount of TSST-1 produced by clinical methicillin resistant Staphylococcus aureus (MRSA) isolates and allelic variation in accessory gene regulator (agr) locus
© Nagao et al; licensee BioMed Central Ltd. 2009
Received: 01 November 2008
Accepted: 10 March 2009
Published: 10 March 2009
Staphylococcus aureus (S. aureus) is an important pathogen associated with both nosocomial and community-acquired infections and its pathogenicity is attributed to its potential to produce virulence factors. Since the amount of toxin produced is related to virulence, evaluating toxin production should be useful for controlling S. aureus infection. We previously found that some strains produce relatively large amounts of TSST-1; however, no reports have described the amount of TSST-1 produced by clinical isolates.
Amounts of TSST-1 produced by clinical methicillin resistant S. aureus (MRSA) isolates were measured by Western blotting. We determined their accessory gene regulator (agr) class by PCR and investigated whether TSST-1 production correlates with variations in the class and structure of the agr.
We found that 75% of surveyed MRSA isolates (n = 152) possessed the tst gene and that 96.7% belonged to agr class 2. The concentrations of TSST-1 secreted into culture supernatants by 34 strains measured by Western blotting differed 170-fold. Sequencing the entire agr locus (n = 9) revealed that some had allelic variations regardless of the amount of TSST-1 produced whereas sequencing the sar, sigma factor B and the tst promoter region revealed no significant changes.
The amounts of TSST-1 produced by clinical MRSA isolates varied. The present results suggest that TSST-1 production is not directly associated with the agr structure, but is instead controlled by unknown transcriptional/translational regulatory systems, or synthesized by multiple regulatory mechanisms that are interlinked in a complex manner.
Staphylococcus aureus (S. aureus) is responsible for many nosocomial and community-acquired infections. Its pathogenicity is attributed to its ability to produce many membrane-associated components and extracellular substances, several of which have been implicated as virulence factors [1, 2]. One of the most unique manifestations among the various staphylococcal infections is staphylococcal toxic shock syndrome (TSS). The associated toxin TSS toxin-1 (TSST-1) is encoded by the tst gene, and might also be involved in the genesis of some autoimmune diseases [1, 3, 4]. The accessory gene regulator (agr) operon among several potentially associated factors is thought to positively regulate TSST-1 production [2, 3]. The agr locus comprises 5 genes (AgrA, AgrB, AgrC, AgrD, and hld) that function in both transcription and translation to regulate numerous toxins, enzymes and cell surface proteins. A polymorphism in a variable region of the agr locus comprises nucleotide sequences encoding AgrD, the C-terminal two-thirds of AgrB, and a portion of the N-terminal half of AgrC, which has led to the assignation of S. aureus isolates into four classes [2, 5]. In addition to the agr polymorphism, mutations of wild-type S. aureus strains resulting in agr deletions alter exoprotein biosynthesis . However, the relationship between the agr polymorphism and TSST-1 production is unknown.
We previously analyzed images from two-dimensional electrophoresis (2-DE) and found that two clinical methicillin-resistant S. aureus (MRSA) isolates produce relatively large amounts of superantigenic exotoxins . Since the amount of toxins produced is probably directly related to the virulence of S. aureus, evaluating the concentration of toxins produced by each strain might be useful for controlling infection.
The aim of this study was to determine whether TSST-1 production varies among clinical MRSA strains and whether it is related to variations in agr class and structure.
Detection of the tst gene and agrclasses
We detected the tst gene in 115 (75.7%) of 152 strains after PCR amplification. Among them, 53 of 66 strains from the nation-wide collection (80.3%) and 62 isolated from 86 blood samples (72.0%) harbored the gene. We identified 147 of 152 isolates (96.7%) as agr class 2, and 3 isolates as agr class 1 (1.9%). We did not identify any isolates of agr classes 3 or 4. The classes of 2 strains were unidentifiable. Among 112 tst-positive strains, 111 belonged to agr class 2. These results indicated the clonal dissemination of a specific group of tst-positive and agr class 2 MRSA in Japanese hospitals.
Evaluation of TSST-1 production
We measured the amount of TSST-1 produced in 34 randomly selected strains. The densities of the bands detected by Western blotting correlated in a semi-log manner with the amount of rTSST-1 produced. The amounts of TSST-1 secreted into culture supernatants evaluated by comparison with the standard curve ranged from 0.8 to 14.0 μg/ml. Thus, the amount of TSST-1 produced varied 170-fold among clinical MRSA isolates that were cultured under the same conditions.
Sequencing of the agroperon
Production of TSST-1 evaluated by Western blotting.
3.5 ± 0.22
1.4 ± 0.19
14 ± 1.01
1.3 ± 0.05
5 ± 0.12
1.0 ± 0.25
1.3 ± 0.31
1.0 ± 0.01
1.1 ± 0.20
0.8 ± 0.02
1.2 ± 0.02
1.6 ± 0.23
2.0 ± 0.03
2.0 ± 0.18
4.0 ± 0.22
1.6 ± 0.22
2.8 ± 0.19
7.6 ± 0.07
14 ± 1.21
9.8 ± 0.28
12 ± 0.20
3.1 ± 0.16
7.0 ± 0.25
5.0 ± 1.12
7.6 ± 0.30
2.8 ± 0.23
5.2 ± 0.11
4.0 ± 0.13
8.0 ± 0.21
1.0 ± 0.19
1.3 ± 0.34
2.2 ± 0.20
4.0 ± 0.22
6.4 ± 0.08
Summary of nucleotide changes and predicted outcomes of mutations in the agr locus.
Amount of TSST-1 produced (μg/ml)
Changes in agrC region nucleotide sequence
Deletion of leu-lys-ile
The proportion of tst-positive isolates among clinical MRSA isolates varies from < 20% to 90% according to country and clinical background [11, 12]. The present study found that over 75% of clinical MRSA isolates carried the tst gene. This ratio is compatible with that of recent reports from Japan and it is obviously higher than those of other countries [11, 12]. The ratio of tst-positive isolates is increasing annually and thus it is important to understand how TSST-1 production is regulated.
The mere presence of a toxin gene does not mean that the protein will be expressed and if it is, toxin levels could widely from strain to strain. In fact, the quantity of Panton-Valentine Leukocidin (PVL) produced in vitro varies up to 10-fold among MRSA strains .
In the present study, we identified a 170-fold difference in the amount of TSST-1 produced among MRSA isolates by Western blotting. Expression of the tst gene is activated by agr so we sequenced the agr locus of various TSST-1 producers to determine whether it is associated with variations in TSST-1 production. Allelic variations in the agrC region were identified irrespective of the amount of TSST-1 produced. One producer of a relatively large amount of TSST-1 had an insertion of nucleotides in the agrC that resulted in a frameshift, which in turn generated many stop codons. Other strains had allelic variations that resulted in replacement of an amino acid irrespective of the amount of TSST-1 and a frameshift in the agrC of a high producer was predicted to generate truncated AgrC. Therefore, the agr locus is probably not functional with respect to TSST-1 production in those strains. Recent findings have shown that about 25% of 105 human isolates are deficient in the production of delta-toxin, indicating that agr mediated regulation is disrupted [14, 15]. These facts imply that mechanisms other than the agr locus are involved in TSST-1 production in our isolates. We also tried to evaluate tst gene expression by Northern blotting, but the results were not reproducible, perhaps because of high levels of expression or difficulty in removing nuclease contamination. In addition, the sequences of both the promoter region of the tst gene and the entire sar locus were conserved among these strains, indicating that these regions are not associated with variations in the amount of TSST-1 production.
The previous and present results indicate that unknown transcriptional/translational regulatory systems control TSST-1 production or that multiple regulatory mechanisms are linked in a complex manner to synthesize and produce toxin. Moreover, secretion mechanisms and proteolytic degradation would also be involved in the amount of TSST-1 produced.
A recent study has shown that variation in the amount of extracellular PVL does not correlate with the severity of infection . In addition, Pragman and Schlievert noted that the transcriptional analysis of virulence regulators in animal models in vivo or in human infection do not correlate with transcriptional analysis accomplished in vitro . From these viewpoints, further investigation is required to determine whether different amounts of TSST-1 are produced in vivo and if so, whether they are related to clinical symptoms of diseases.
The present results suggest that TSST-1 production is not directly associated with the agr structure, but is instead controlled by unknown transcriptional/translational regulatory systems, or synthesized by multiple regulatory mechanisms that are interlinked in a complex manner.
Of 152 clinical MRSA isolates that we analyzed, 66 were randomly selected from the nationwide MRSA collection representing various regions of Japan in 2003, and the remainder was isolated from the bloodstream of patients in different wards at a university hospital between 1996 and 2003.
Detection of the tst gene and agr-genotyping by PCR
Bacterial chromosomal DNA was extracted after overnight growth on Luria Bertani agar as described . We detected the tst gene by PCR amplification using the specific primers, TGT AGA TCT ACA AAC GAT AAT ATA AAG GAT (forward) and ATT AAG CTT AAT TAA TTT CTG CTT CTA TAG TT (reverse). Genes were amplified by denaturation for 5 min at 94°C followed by 30 cycles of 30 s at 94°C, 30 s at 52°C, 60 s at 72°C and a final extension at 72°C for 5 min in a 25-μl mixture, comprising 1 μl template DNA, 0.2 mM dNTP mix, 1.5 mM 10× Ex buffer (Takara, Tokyo, Japan), 1.25 U Ex Taq (Takara) and 0.5 μM each of the forward and reverse primers. The agr class was determined by PCR amplification of the hypervariable domain of the agr locus using specific oligonucleotide primers as described .
Preparation of recombinant partial TSST-1 and anti TSST-1 antibody
Primers used in this study.
AAA AAG CCA GCT ATA CAG TG
AGT GAG GAG AGT GGT GTA AAA
CTG AAT TAC TGC CAC GTT CT
TCC GTT GTT ATT TAT GCA CCT
CGA AT TCC ATA GGC TTT TC
AGA AAG GTG TGT AGC ATA TGG
GCC TTT TAT CTC ACG TCG TT
TCC TGC AAT ACT CTT ACC AT
TTC TTA CCA AATATG TCG CC
CGA GAA TCT TAA AGT ACG TGA
AAA AGT GGC CAT AGC TAA GT
CGA AGA CGA TCC AAA ACA AA
AGG TGC ATA AAT AAC ACG G
GAT TGA ATT TGA ACG TGG AG
TGT AGA TCT ACA GAT TTT ACC CCT GTT
ATT AAG CTT CGC TAG TAT GTT GGC TTT
Quantitation of TSST-1 by Western blotting
Strains were incubated in 3 ml LB broth overnight and then 100 μl cultures were transferred into 5 ml fresh LB broth and incubated at 37°C with rotary shaking at 150 rpm. Bacterial growth was monitored until the cell density reached the early stationary phase. Culture supernatant was obtained by centrifugation at 8000 × g for 15 min to precipitate bacterial cells. Total exoproteins precipitated from the culture supernatant with 10% trichloroacetic acid (TCA) were washed with cold acetone and dissolved in 100 μl of Laemmli sample buffer . Proteins were resolved by electrophoresis and then Western blotted according to standard procedures with the minor modification described by Whiting et al . Serially diluted rTSST-1 samples were western blotted to produce a standard curve. The individual experiments to determine TSST-1 expression for each strain were repeated three times. The density of each immunostained band was evaluated using Imagemaster 1D Elite ver.3.00 (Amersham Bioscience, Tokyo, Japan) and mean values were adopted.
Sequence analysis of a variant agrlocus
Table 1 lists the specific primers used to sequence the entire region of agr A, B, C, and D. The region was amplified by PCR under the same conditions as described for detection of the tst gene. The products were purified using a QIAquick PCR purification kit (Qiagen) and sequenced on a CEQ 2000 DNA analysis system (Beckman Coulter, Fullerton, CA, USA) using Beckman Dye terminator cycle sequencing kits (CEQ DTCS kit, Tokyo, Japan) according to the manufacturer's instructions.
Potential conflicts of interest. None of the authors have any conflicts.
- Crossley KB, Archer GL: The Staphylococci in human disease. 2000, Churchill Livingstone, United States of AmericaGoogle Scholar
- Novick RP: Pathogenicity factors of Staphylococcus aureus and their regulation. Gram-positive pathogens. Edited by: Fischetti V. 2000, Washington D.C.: ASM Press, 392-07.Google Scholar
- Wright JD, Holland KT: The effect of cell density and specific growth rate on accessory gene regulator and toxic shock syndrome toxin-1 gene expression in Staphylococcus aureus. FEMS Microbiol Lett. 2003, 218: 377-383. 10.1016/S0378-1097(02)01193-X.PubMedView ArticleGoogle Scholar
- McCormick JK, Yarwood JM, Schlievert PM: Toxic shock syndrome and bacterial superantigens: an update. Annu Rev Microbiol. 2001, 55: 77-104. 10.1146/annurev.micro.55.1.77.PubMedView ArticleGoogle Scholar
- Ji G, Beavis R, Novick RP: Bacterial interference caused by autoinducing peptide variants. Science. 1997, 276: 2027-2030. 10.1126/science.276.5321.2027.PubMedView ArticleGoogle Scholar
- Somerville GA, Beres SB, Fitzgerald JR, DeLeo FR, Cole RL, Hoff SJ, Musser JM: In vitro serial passage of Staphylococcus aureus : changes in physiology, virulence factor production, and agr nucleotide sequence. J Bacteriol. 2002, 184: 1430-1437.PubMed CentralPubMedView ArticleGoogle Scholar
- Nakano M, Kawano Y, Kawagishi M, Hasegawa T, Iinuma Y, Ohta M: Two-dimensional analysis of exoproteins of methicillin-resistant Staphylococcus aureus (MRSA) for possible epidemiological application. Micro Immunol. 2002, 46: 11-22.View ArticleGoogle Scholar
- Blevins JS, Gillaspy AF, Rechtin TM, Hurlburt BK, Smeltzer MS: The staphylococcal accessory regulator (sar) represses transcription of the Staphylococcus aureus collagen adhesin gene (cna) in an agr-independent manner. Mol Microbiol. 1999, 33: 317-326. 10.1046/j.1365-2958.1999.01475.x.PubMedView ArticleGoogle Scholar
- Chan PF, Foster J: Role of SarA in virulence determinant production and environmental signal transduction in Staphylococcus aureus. J Bacteriol. 1998, 180: 6232-6241.PubMed CentralPubMedGoogle Scholar
- Bayer MG, Heinrichs JH, Cheung AL: The molecular architecture of the sar locus in Staphylococcus aureus. J Bacteriol. 1996, 178: 4563-4570.PubMed CentralPubMedGoogle Scholar
- Becker K, Friedrich AW, Lubritz G, Weilert M, Peters G, Christo von Eiff : Prevalence of genes encoding pyrogenic toxin superantigens and exfoliative toxins among strains of Staphylococcus aureus isolated from blood and nasal specimens. J Clin Microbiol. 2003, 41: 1434-1439. 10.1128/JCM.41.4.1434-1439.2003.PubMed CentralPubMedView ArticleGoogle Scholar
- Imura S: Changes in drug susceptibility and toxin genes in Staphylococcus aureus isolated from blood cultures at a university hospital. J Infect Chemother. 2004, 10: 8-10. 10.1007/s10156-003-0276-0.View ArticleGoogle Scholar
- Hamilton SM, Bryant AE, Carrol KC, Lockary V, Ma Y, Mcindoo E, Miller LG, Perdreau-Remington F, Pullman J, Risi GF, Salmi DB, Stevens DL: In vitro production of Panton-Valentine Leukocidin among strains of methicillin-resistant Staphylococcus aureus causing diverse infections. Clin Infect Dis. 2007, 45: 1550-1558. 10.1086/523581.PubMedView ArticleGoogle Scholar
- Strommenger B, Cuny C, Werner G, Witte W: Obvious lack of association between dynamics of epidemic methicillin-resistant Staphylococcus aureus in central Europe and agr specificitygroups. Eur J Clin Microbio Infect Di. 2003, 23: 15-19.Google Scholar
- McCalla C, Smyth DS, Robinson DA, Steenbergen J, Luperchio AS, Moise PA, Fowler VG, Sakoulas G: Microbiological and Genotypic Analysis of Methicillin-Resistant Staphylococcus aureus Bacteremia. Antimicrob Agents Chemother. 2008, 52: 3441-3443. 10.1128/AAC.00357-08.PubMed CentralPubMedView ArticleGoogle Scholar
- Pragman AA, Schlievert PM: Virulence regulation in Staphylococcus aureus: the need for in vivo analysis of virulence factor regulation. FEMS Immunol Med Microbiol. 2004, 42: 147-154. 10.1016/j.femsim.2004.05.005.PubMedView ArticleGoogle Scholar
- Louie L, Matsumura SO, Choi E, Louie M, Simor AE: Evaluation of three rapid methods for detection of methicillin resistance in Staphylococcus aureus. J Clin Microbiol. 2000, 38: 2170-2173.PubMed CentralPubMedGoogle Scholar
- Gilot P, Lina G, Cochard T, Poutrel B: Analysis of the genetic variability of genes encoding the RNA III-activating components ag r and TRAP in a population of Staphylococcus aureus strains isolated from cows with mastitis. J Clin Microbiol. 2002, 40: 4060-4067. 10.1128/JCM.40.11.4060-4067.2002.PubMed CentralPubMedView ArticleGoogle Scholar
- Laemmli UK: Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature. 1970, 227: 680-685. 10.1038/227680a0.PubMedView ArticleGoogle Scholar
- Whiting Jl, Rostenm PM, Chow AW: Determination by Western blot (immunoblot) of seroconversions to Toxic Shock Syndrome (TSS) Toxin 1 and enterotoxin A, B, or C during infection with TSS- and Non- TSS-associated Staphylococcus aureus. Infect Immu. 1989, 57: 231-234.Google Scholar
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