| Streptococcus suis is a Gram-positive bacterium.As an important zoonosis pathogen,it can cause meningitis,septicemia and other infections in pigs and humans.It not only brings huge economic losses to pig industry,but also seriously endangers public health.According to the difference of capsular polysaccharide antigens,S.suis can be divided into 29 serotypes.Among them,serotype 2 is the serotype with the highest clinical isolation rate in the world.Transition metals are essential nutrients for organisms,and the host can inhibit the growth of pathogens by limiting metal ions.But excessive metals can also cause toxicity to cells.Studies have found that the application of metal toxicity is another host strategy against pathogens.Bacteria,on the other hand,have evolved complex mechanisms to efflux excessive metals from bacteria or isolate them in order to maintain metal homeostasis.Therefore,metal efflux systems play important roles in the pathogenic process of pathogens.By homologous sequence alignment,we found that the protein encoded by B9H01 RS01605 of SC19 has high homology with ferrous[Fe(Ⅱ)]pump found in several Gram-positive bacteria and we named it pmtA.By qRT-PCR we found that pmtA expression is significantly induced by Fe(Ⅱ),cobalt[Co(Ⅱ)],and nickel[Ni(Ⅱ)].Then we constructed pmtA gene deletion and complementation strains.By measuring the growth curves,we found that the growth of ApmtA deletion strain was significantly inhibited under Fe(Ⅱ)and Co(Ⅱ)conditions,and slightly inhibited under ferric iron[Fe(Ⅲ)]or zinc[Zn(Ⅱ)]conditions.However,the spot dilution tests showed that PmtA inhibited the bactericidal effect induced by Fe(Ⅱ)and Fe(Ⅲ)and the bacteriostatic effect induced by Co(Ⅱ)and Zn(Ⅱ).The result of ICP-OES showed that pmtA gene deletion resulted in increased intracellular iron and cobalt levels.Moreover,△pmtA was more sensitive to the antibiotic streptomycin activated by Fe(Ⅱ).When Fe(Ⅱ)or Co(Ⅱ)is excessive,manganese[Mn(Ⅱ)]can alleviate the growth defects of ΔpmtA.In addition,PmtA plays a role in H2O2 oxidative stress tolerance in S.suis,but is not involved in the virulence of S.suis.These results suggest that PmtA of S.suis functions of Fe(II)and Co(II)efflux pump and is related to oxidative stress tolerance.In order to study the changes of transcription-level of S.suis under Fe(Ⅱ)and Co(II)conditions,we carried out RNA sequencing.The results showed that the expressions of many genes were different under the conditions of excessive iron and cobalt.Most genes responding to cobalt toxicity have the same expression trend as those responding to ferrous toxicity.qRT-PCR analysis of the selected genes confirmed the accuracy of RNA sequencing results.Bioinformatics analysis of differentially expressed genes showed that iron and cobalt had similar effects on the celluar process of S.suis.Ferrous treatment resulted in the down regulation of genes related to oxidative stress tolerance and the up regulation of amino acid ABC transporter operon genes.In addition,ferrous and cobalt treatments lead to the down-regulation of several genes in the arginine deaminase system.These results suggest that S.suis alters the expression of multiple genes in response to ferrous and cobalt toxicity.In this study,pmtA gene deletion and complementary strains were constructed to identify and study the function of PmtA,the metal efflux system of S.suis.The transcriptome-level changes of S.suis under Ferrous and cobalt conditions were analyzed by RNA sequencing.These results revealed that S.suis could efflux excessive metal out of the cell and change the expression of related genes to resist the toxicity of ferrous and cobalt. |
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