| Streptococcus suis serotype 2 (SS2) is a leading swine pathogen that occasionally infects humans who engage in close contact with swine and pork-derived products. Before our report of two epidemic outbreaks of human SS2 in China, it was thought that SS2 could only cause sporadic human cases. In view of this epidemiological history of human SS2 infection, both outbreaks and sporadic cases are now believed to take place in China. Comparative genomics analyses from three different groups have suggested that virulent Chinese strains of SS2 feature a specific, 89-kb-long DNA fragment (dubbed 89K by our group). Further genetic studies showed preliminary evidence that 89K may function as a pathogenicity island. Besides well-known virulence-associated factors, such as capsular polysaccharide and suilysin, 17 additional virulence-associated components have been identified, including a two-component signal transduction system (TCSTS), SalK-SalR; an orphan regulator, CovR; lipoteichoic acid; and others.Like many other pathogenic bacteria, SS2 is transmitted via the respiratory route and remains localized in the palatine tonsils but subsequently escapes from the immune system and disseminates via the blood circulation system, finally invading various host organs. In order to colonize and infect different regions within the host, SS2 requires a regulatory network that senses changing surroundings and responds to environmental signals. Such responses typically involve genome-wide changes in transcriptional activation or repression of specific genes through interactions among multiple regulators. In recent years, thorough research and analyses of global regulatory networks in Streptococcus pyogenes have been made; however, the corresponding knowledge regarding SS2 is relatively limited. Rgg-like regulators, a family of transcriptional regulator, are widely distributed in Gram-positive bacteria, including S. pyogenes, Streptococcus gordonii, Lactococcus lactis, and others. Rgg-like proteins have a conserved helix-turn-helix motif and several invariant residues in the amino terminus that are necessary for specific binding to the promoter regions of Rgg-regulated genes. Rgg protein plays various roles in many bacteria. In fact, Rgg could affect the transcription of multiple genes throughout the genome by altering the expression of known and putative transcriptional regulators, thereby achieving bacterial adaptive responses to various environmental conditions.In the present study, we characterized a functional member of the Rgg protein family of transcriptional regulators from a Chinese SS2 isolate, 05ZYH33. And the following experiments are conducted:1. Characterization, Cloning and prokaryotic expression of the rgg gene in 05ZYH33: The rgg gene on the chromosome of 05ZYH33 was predicted to encode a 287-amino-acid protein product. Bioinformatics analysis showed that this Rgg protein possesses a DNA-binding helix-turn-helix motif and three invariant residues which are critical for fully active Rgg-like regulators. To identify and demonstrated the properties of Rgg protein in SS2, we prokaryotic expressed the rgg gene using the pET-32a system. SDS-PAGE results showed that the purified protein forms homodimers in vitro, consistent with data for other members of the Rgg family.2. Knockout of rgg and functional complementation: The Spc resistance (Spc~R) gene cassette was inserted into a pUC18 vector to create the recombinant plasmid pUC18-Spc. Two DNA fragments flanking the rgg gene were cloned into pUC18-Spc to generate the knockout plasmid, pUC::rgg. The pUC::rgg plasmid was electroporated into 05ZYH33 competent cells. The fidelity of the double-crossover recombination was confirmed in the mutants by PCR using flanking primers lying outside the homologous regions, followed by direct DNA sequencing. Reverse transcription-PCR (RT-PCR) detection was carried out to confirm the successful inactivation of the rgg gene in the deletion mutant, designated?rgg. To create a construct for functional complementation, the DNA fragment covering the rgg coding region plus its 286-bp upstream promoter sequence was amplified from the 05ZYH33 chromosomal DNA The resulting PCR product was cloned into E. coli-S. suis shuttle vector pSET1. This recombinant plasmid was electrotransformed into the?rgg mutant to screen the complemented strain C?rgg with double selection pressure of Spc and Cm and RT-PCR detection.3. The effects of rgg deletion on the biological properties and virulence of 05ZYH33: The basic biological properties of the wild type strain 05ZYH33 and the?rgg mutant were compared under the same conditions. Firstly, Morphological examination by Gram staining and transmission electron microscopy showed that the?rgg mutant grew as much longer chains of individual cells that lost the typical shape characteristic of SS2, showing an altered cell diameter and irregular margins. Secondly, we found that the growth of the?rgg strain was 40% slower in the first 8 to 9 h than the wild type (WT) strain by measuring OD600 values. However, when we determined the growth rates of?rgg and WT cultures by counting the CFU, no obvious differences in the CFU counts were observed during the initial 8 to 9 h of growth, but the?rgg strain continued to reproduce until the culture reached an OD600 similar to that of the WT strain. Thirdly, deletion of the rgg gene in SS2 increased its adhesion to Hep-2 cells and hemolytic activity in vitro. However, we observed that the?rgg strain had significantly lower lethality than the WT strain in an experimental piglet infection model. Pathological examination of the two groups of sacrificed piglets revealed that many organs suffered less damage in the mutant-infected group. Therefore, we can conclude that inactivation of Rgg in SS2 attenuates its pathogenicity in the piglet infection model. In addition, these microscopic phenotypes of SS2 were restored after complementation.4. Inactivation of rgg alters the global gene transcription profile: To obtain a comprehensive view of the extent to which Rgg impacts gene expression in SS2, global gene transcription profiles were determined by DNA microarray analysis and quantitative PCR for the?rgg mutant and WT strains grown to post-exponential phase. Globally, 15.87% of the genes represented on the microarray (n = 345) were differentially transcribed in the?rgg strain compared to the WT strain. Among these, 195 genes were upregulated, and 150 genes were downregulated. Within each functional category, the tendency for genes to be up- or downregulated was easily discerned: (i) Many transcripts encoding proteins associated with information storage and processing were the main part of upregulated genes in the?rgg strain, including protein biosynthesis and modification, DNA replication/repair, defense mechanisms, etc.; (ii) Downregulated transcripts principally focused on genes related to glycometabolism; (iii) The?rgg mutant showed changes in transcription for several genes that play critical roles in the maintenance of proper cell division and shape, which provided a possible explanation for the morphological changes seen in the?rgg mutant; (iv) Deletion of rgg influences transcripts of 29 genes encoding known or putative transcriptional regulators, suggested that Rgg is part of the regulatory network in SS2 and may influence the activity of other regulatory systems to exert rigorous global regulation.On the basis of the present experimental and microarray analysis results, we suggest that the Rgg regulator in SS2 plays a critical role in global regulatory networks to activate transcription of genes involved in the metabolism of nonglucose carbohydrates. At the same time, it appears to decrease unnecessary expression of bacterial surface proteins responsible for adherence or internalization, thus facilitating the survival of SS2 under nutritional stress conditions within the host. |
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