Karla L De Anda-Mora, Faviola Tavares-Carreón, Carlos Alvarez, Samantha Barahona, Miguel A Becerril-García, Rogelio J Treviño-Rangel, Rodolfo García-Contreras, Angel Andrade
{"title":"Serratia marcescens 临床菌株 HU1848 蛋白质分解活性的提高与 eepR 的高表达有关。","authors":"Karla L De Anda-Mora, Faviola Tavares-Carreón, Carlos Alvarez, Samantha Barahona, Miguel A Becerril-García, Rogelio J Treviño-Rangel, Rodolfo García-Contreras, Angel Andrade","doi":"10.33073/pjm-2024-002","DOIUrl":null,"url":null,"abstract":"<p><p><i>Serratia marcescens</i> is a global opportunistic pathogen. <i>In vitro</i> cytotoxicity of this bacterium is mainly related to metalloprotease serralysin (PrtS) activity. Proteolytic capability varies among the different isolates. Here, we characterized protease production and transcriptional regulators at 37°C of two <i>S. marcescens</i> isolates from bronchial expectorations, HU1848 and SmUNAM836. As a reference strain the insect pathogen <i>S. marcescens</i> Db10 was included. Zymography of supernatant cultures revealed a single (SmUNAM836) or double proteolytic zones (HU1848 and Db10). Mass spectrometry confirmed the identity of PrtS and the serralysin-like protease SlpB from supernatant samples. Elevated proteolytic activity and <i>prtS</i> expression were evidenced in the HU1848 strain through azocasein degradation and qRT-PCR, respectively. Evaluation of transcriptional regulators revealed higher <i>eepR</i> expression in HU1848, whereas <i>cpxR</i> and <i>hexS</i> transcriptional levels were similar between studied strains. Higher <i>eepR</i> expression in HU1848 was further confirmed through an <i>in vivo</i> transcriptional assay. Moreover, two putative CpxR binding motifs were identified within the <i>eepR</i> regulatory region. EMSA validated the interaction of CpxR with both motifs. The evaluation of <i>eepR</i> transcription in a <i>cpxR</i> deletion strain indicated that CpxR negatively regulates <i>eepR</i>. Sequence conservation suggests that regulation of <i>eepR</i> by CpxR is common along <i>S. marcescens</i> species. Overall, our data incorporates CpxR to the complex regulatory mechanisms governing <i>eepR</i> expression and associates the increased proteolytic activity of the HU1848 strain with higher <i>eepR</i> transcription. Based on the global impact of EepR in secondary metabolites production, our work contributes to understanding virulence factors variances across <i>S. marcescens</i> isolates.</p>","PeriodicalId":94173,"journal":{"name":"Polish journal of microbiology","volume":"73 1","pages":"11-20"},"PeriodicalIF":0.0000,"publicationDate":"2024-03-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10911700/pdf/","citationCount":"0","resultStr":"{\"title\":\"Increased Proteolytic Activity of <i>Serratia marcescens</i> Clinical Isolate HU1848 Is Associated with Higher <i>eepR</i> Expression.\",\"authors\":\"Karla L De Anda-Mora, Faviola Tavares-Carreón, Carlos Alvarez, Samantha Barahona, Miguel A Becerril-García, Rogelio J Treviño-Rangel, Rodolfo García-Contreras, Angel Andrade\",\"doi\":\"10.33073/pjm-2024-002\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"<p><p><i>Serratia marcescens</i> is a global opportunistic pathogen. <i>In vitro</i> cytotoxicity of this bacterium is mainly related to metalloprotease serralysin (PrtS) activity. Proteolytic capability varies among the different isolates. Here, we characterized protease production and transcriptional regulators at 37°C of two <i>S. marcescens</i> isolates from bronchial expectorations, HU1848 and SmUNAM836. As a reference strain the insect pathogen <i>S. marcescens</i> Db10 was included. Zymography of supernatant cultures revealed a single (SmUNAM836) or double proteolytic zones (HU1848 and Db10). Mass spectrometry confirmed the identity of PrtS and the serralysin-like protease SlpB from supernatant samples. Elevated proteolytic activity and <i>prtS</i> expression were evidenced in the HU1848 strain through azocasein degradation and qRT-PCR, respectively. Evaluation of transcriptional regulators revealed higher <i>eepR</i> expression in HU1848, whereas <i>cpxR</i> and <i>hexS</i> transcriptional levels were similar between studied strains. Higher <i>eepR</i> expression in HU1848 was further confirmed through an <i>in vivo</i> transcriptional assay. Moreover, two putative CpxR binding motifs were identified within the <i>eepR</i> regulatory region. EMSA validated the interaction of CpxR with both motifs. The evaluation of <i>eepR</i> transcription in a <i>cpxR</i> deletion strain indicated that CpxR negatively regulates <i>eepR</i>. Sequence conservation suggests that regulation of <i>eepR</i> by CpxR is common along <i>S. marcescens</i> species. Overall, our data incorporates CpxR to the complex regulatory mechanisms governing <i>eepR</i> expression and associates the increased proteolytic activity of the HU1848 strain with higher <i>eepR</i> transcription. Based on the global impact of EepR in secondary metabolites production, our work contributes to understanding virulence factors variances across <i>S. marcescens</i> isolates.</p>\",\"PeriodicalId\":94173,\"journal\":{\"name\":\"Polish journal of microbiology\",\"volume\":\"73 1\",\"pages\":\"11-20\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2024-03-04\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10911700/pdf/\",\"citationCount\":\"0\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Polish journal of microbiology\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.33073/pjm-2024-002\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"2024/3/1 0:00:00\",\"PubModel\":\"eCollection\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Polish journal of microbiology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.33073/pjm-2024-002","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2024/3/1 0:00:00","PubModel":"eCollection","JCR":"","JCRName":"","Score":null,"Total":0}
Increased Proteolytic Activity of Serratia marcescens Clinical Isolate HU1848 Is Associated with Higher eepR Expression.
Serratia marcescens is a global opportunistic pathogen. In vitro cytotoxicity of this bacterium is mainly related to metalloprotease serralysin (PrtS) activity. Proteolytic capability varies among the different isolates. Here, we characterized protease production and transcriptional regulators at 37°C of two S. marcescens isolates from bronchial expectorations, HU1848 and SmUNAM836. As a reference strain the insect pathogen S. marcescens Db10 was included. Zymography of supernatant cultures revealed a single (SmUNAM836) or double proteolytic zones (HU1848 and Db10). Mass spectrometry confirmed the identity of PrtS and the serralysin-like protease SlpB from supernatant samples. Elevated proteolytic activity and prtS expression were evidenced in the HU1848 strain through azocasein degradation and qRT-PCR, respectively. Evaluation of transcriptional regulators revealed higher eepR expression in HU1848, whereas cpxR and hexS transcriptional levels were similar between studied strains. Higher eepR expression in HU1848 was further confirmed through an in vivo transcriptional assay. Moreover, two putative CpxR binding motifs were identified within the eepR regulatory region. EMSA validated the interaction of CpxR with both motifs. The evaluation of eepR transcription in a cpxR deletion strain indicated that CpxR negatively regulates eepR. Sequence conservation suggests that regulation of eepR by CpxR is common along S. marcescens species. Overall, our data incorporates CpxR to the complex regulatory mechanisms governing eepR expression and associates the increased proteolytic activity of the HU1848 strain with higher eepR transcription. Based on the global impact of EepR in secondary metabolites production, our work contributes to understanding virulence factors variances across S. marcescens isolates.