| Staphylococcus aureus is an important zoonotic pathogen that causes many diseases and has been a serious challenge for animal and human health. As an opportunistic pathogenic bacterium, the microbial characteristics of S. aureus are very complicated, and the pathogenicity of S. aureus is closely related to the the expression of virulence. The ability of S. aureus to cause such a wide range of infections has been ascribed to its huge armoury of different virulence factors, many of which are under the control of the quorum-sensing accessory gene regulator(Agr) system. The agr operon consists of two divergent promoters—P2 and P3, where the expression from P2 produces the components of a quorum-sensing system(Agr B, D, C, A), and the expression from P3 produces the agr effector molecule RNAIII. Agr B is required for processing of the propeptide AgrD to produce the quorum signal molecule, autoinducing peptides(AIPs); AgrC and Agr A comprise a classical two component signal transduction system, where ligand bound AgrC phosphorylates and activates the DNA-binding response regulator Agr A, which regulates expressions of phenol soluble modulins(PSMs), P2 and P3. RNAIII is responsible for posttranscriptional regulation of multiple virulence factors. There are many different phenotypes of S. aureus, and the pathogenesis between the various phenotypic S. aureus is also different. According to seven housekeeping genes, S. aureus isolates were molecularly characterized as 2957 different sequence types(STs). Epidemiological studies have demonstrated that the infections of S. aureus are diverse in different countries and regions, and different clones may have variable virulence. In 2007, Schefold et al first reported a multiple organ failure case of a 51-year-old male caused by S. aureus ST121. Concei??o et al revealed that the high prevalence of ST121 in CA–MSSA lineages is responsible for skin and soft tissue infections in Europe. However, there are no data about ST121 infections reported in China. In this study, a clinical ST121 type S. aureus strain(XQ) was isolated and characterized. The biological characteristics and the possible virulence variation mechanisms of XQ are also investigated. The major methods and result s are as following:1. Case report, bacterial isolation and identificationXQ strain was isolated from a healthy 17-y-old male adolescent presenting with wheal in the skin of his head, face, and upper extremities in our emergency department. Physical examination on admission revealed the patient was malnourished and in a superficial coma with a Glasgow Coma Scale score of 8. His temperature was 38 °C, heart rate was 150 beats per min, and respiratory rate was 50 breaths per min. Cardiac, chest and abdominal examination were normal except for a sprinkle moist rale in the area of low right lung. Chest X-ray investigation revealed patchy bilateral pulmonary infiltrates, and an enlarged podoid. Ultrasound investigation of the heart suggested hydropericardium. The p atient remained in a comatose state with deteriorating respiration treated with meropenem and died approximately 65 h after admission. The isolates from the patient were Gram-positive cocci, and identified strain as a PVL-positive, methicillin-sensitive S. aureus(MSSA) by specific genes detection(femB, mec A, pvl)—named XQ strain, and the case was reported(Rev Med Microbiol,2015).2. Molecular typing of XQ strainThe molecular typing methods, such as pulsed field gel electrophoresis(PFGE), multilocus sequence typing(MLST), X region encoding staphylococal protein A(spa) typing, accessory gene regulator system typing(Agr typing) were applied to XQ strain. PFGE technology is a method for separating large molecules of DNA, after enzyme digestion of bacterial chromosome, the DNA fragments get good separation in a particular electrophoresis system. MLST is a highly discriminatory method for characterizing bacterial isolates on the basis of the sequences of ~450-bp internal fragments of seven housekeeping genes(arc、aro E、glp F、gmk、pta、tpi、yqiL). For each gene fragment, the different sequences are assigned as distinct alleles, and each isolate is defined by the alleles at each of the seven housekeeping loci. Staphylococcal protein A(SPA) is the component of S. aureus cell wall, its polymorphic X region consists of a variable number of 24-bp repeats and the two ends of the repetitive sequence for spa are conservative, the existence of well-conserved regions flanking the X region coding sequence in spa allows the use of primers for PCR amplification and direct sequence typing. Accessory gene regulator system(Agr) is an important global regulatory system of S. aureus, according to the diversity of agrD and agr C amino acid sequence and the length, S. aureus can be divided into four Agr types. The molecular typing results show that XQ strain is an ST121, spa t159, agr IV, MSSA.3. Biological characteristics of XQ strainBlood plate to cultivate XQ strain is used to observe its hemolysis activity, and BHI culture medium is used to determine its growth curve, BALB/c mice are injected in subcutaneous and tail vein to observe the toxicity of XQ strain. Results showed that the basic biological characteristics of XQ strain(including hemolytic, growth curve, etc.) and the control bacteria ATCC25923, RN4220 and N315 are different, the hemolytic activity of XQ strain is enhenced, and its growth rate is moderate. 1 x 107 CFU of XQ strain and ATCC25923 were infected into mice in subcutaneous respectively, after 2 weeks, the ulcer of XQ strain group is a wide and deep, while the wound of ATCC25923 group tends to recover; 1 x 105 CFU of XQ strain and ATCC25923 were infected into mice via tail vein respectively, the mice of XQ strain group died within two days, while the mice of ATCC25923 group still survived 7 days post infection, which showed that XQ strain has strong pathogenicity.4. Continuous passage in vitro and virulence variation of XQ strainIn order to determine whether the virulence of the XQ strain is stable, XQ strain was subcultured continuously to 200 generations in vitro with LB culture medium. PFGE showed that the genomes of XQ strains were unaltered, but the animal experiments indicated the virulence of the 180 th generation of XQ strain(XQ180) was significantly decreased. SDS-PAGE analysis demonstrated that the protein expression patterns in the culture supernatants were different between XQ and XQ180. We hypothesized that the high expression of virulence factors may be closely related to the strong pathogenesis of XQ strain. The culture supernatants of 20-generation interval strains were analyzed with SDS-PAGE, and the results showed that after the 20 th generations, the secretory proteins in the culture supernatant of XQ strain were decreased significantly. Further analysis found that the culture supernatant of secretory protein profiles in the fifth generation was changed. There were two genetically stable colonies in TSB plate of the fifth generation bacteria: one was golden colored and normal sized, the other was yellow with bigger colonies. Animal experiments showed that the pathogenicity of the golden strain was similar with the wild-type XQ strain, while the yellow showed weakened virulence. This yellow and low virulent strain was named as XQM strain.5. The mechanisms for the virulence variation of XQ strainThe protein expression profiles of XQ and XQM strains were significantly different, which may be an explaination of the significantly decreased virulence in XQM strain. The high expression proteins in the supernatant of XQ strian were subjected to mass spectrometry analysis, results showed that these proteins were three acyl glyceride enzyme(triacylglycerol lipase, 76.6 kDa), PVL(Panton-Valentine leukocidin, 37 k Da), alpha hemolysin(alpha-hemolysin, 37 kDa), enterotoxin B(superantigen enterotoxin SEB, 31 k Da), gamma hemolysin(gamma-hemolysin, 36 kDa), staphylococcus of cysteine protease(staphylococcal serine proteinase A, Sspa, 44 kDa), etc., which are virulence proteins of S. aureus regulated by the Agr system. Whether the decrease expressions of these virulence factors in XQM strain related to the Agr system mutation remains unclear. To test this hypothesis, the agrA, agrB, agrC, and agrD genes were amplified and sequenced. The results indicated that an extra 188 bp repeated sequence was inserted into the 443 th of nucleotide agr C gene in strain of XQM, resulting in a truncated mutant of AgrC protein. Morever, the gene expression levels controlled by Agr were markedly reduced in XQM strain, and the agr C mutation caused by an insertion of repetitive sequence was rarely reported. In our study, we tried to use agrC gene of wild-type XQ strain to replace that in XQM, unfortunately, the transformation was always unsuccessful. The further mechanisms, such as that guide the specific insertion of a sequence into agr C gene still need to be investigated. |
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