Kritika Singh, Shubham Vashishtha, Ankan Chakraborty, Ashish Kumar, Sheetal Thakur and Bishwajit Kundu*,
{"title":"The Salmonella typhi Cell Division Activator Protein StCAP Impacts Pathogenesis by Influencing Critical Molecular Events","authors":"Kritika Singh, Shubham Vashishtha, Ankan Chakraborty, Ashish Kumar, Sheetal Thakur and Bishwajit Kundu*, ","doi":"10.1021/acsinfecdis.4c00001","DOIUrl":null,"url":null,"abstract":"<p >Conserved molecular signatures in multidrug-resistant <i>Salmonella typhi</i> can serve as novel therapeutic targets for mitigation of infection. In this regard, we present the <i>S. typhi</i> cell division activator protein (StCAP) as a conserved target across <i>S. typhi</i> variants. From <i>in silico</i> and fluorimetric assessments, we found that StCAP is a DNA-binding protein. Replacement of the identified DNA-interacting residue Arg<sup>34</sup> of StCAP with Ala<sup>34</sup> showed a dramatic (15-fold) increase in <i>K</i><sub>d</sub> value compared to the wild type (<i>K</i><sub>d</sub> 546 nm) as well as a decrease in thermal stability (10 °C shift). Out of the two screened molecules against the DNA-binding pocket of StCAP, eltrombopag, and nilotinib, the former displayed better binding. Eltrombopag inhibited the stand-alone <i>S. typhi</i> culture with an IC<sub>50</sub> of 38 μM. The effect was much more pronounced on THP-1-derived macrophages (T1Mac) infected with <i>S. typhi</i> where colony formation was severely hindered with IC<sub>50</sub> reduced further to 10 μM. Apoptotic protease activating factor1 (Apaf1), a key molecule for intrinsic apoptosis, was identified as an StCAP-interacting partner by pull-down assay against T1Mac. Further, StCAP-transfected T1Mac showed a significant increase in LC3 II (autophagy marker) expression and downregulation of caspase 3 protein. From these experiments, we conclude that StCAP provides a crucial survival advantage to <i>S. typhi</i> during infection, thereby making it a potent alternative therapeutic target.</p>","PeriodicalId":17,"journal":{"name":"ACS Infectious Diseases","volume":null,"pages":null},"PeriodicalIF":4.0000,"publicationDate":"2024-05-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"ACS Infectious Diseases","FirstCategoryId":"3","ListUrlMain":"https://pubs.acs.org/doi/10.1021/acsinfecdis.4c00001","RegionNum":2,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"CHEMISTRY, MEDICINAL","Score":null,"Total":0}
引用次数: 0
Abstract
Conserved molecular signatures in multidrug-resistant Salmonella typhi can serve as novel therapeutic targets for mitigation of infection. In this regard, we present the S. typhi cell division activator protein (StCAP) as a conserved target across S. typhi variants. From in silico and fluorimetric assessments, we found that StCAP is a DNA-binding protein. Replacement of the identified DNA-interacting residue Arg34 of StCAP with Ala34 showed a dramatic (15-fold) increase in Kd value compared to the wild type (Kd 546 nm) as well as a decrease in thermal stability (10 °C shift). Out of the two screened molecules against the DNA-binding pocket of StCAP, eltrombopag, and nilotinib, the former displayed better binding. Eltrombopag inhibited the stand-alone S. typhi culture with an IC50 of 38 μM. The effect was much more pronounced on THP-1-derived macrophages (T1Mac) infected with S. typhi where colony formation was severely hindered with IC50 reduced further to 10 μM. Apoptotic protease activating factor1 (Apaf1), a key molecule for intrinsic apoptosis, was identified as an StCAP-interacting partner by pull-down assay against T1Mac. Further, StCAP-transfected T1Mac showed a significant increase in LC3 II (autophagy marker) expression and downregulation of caspase 3 protein. From these experiments, we conclude that StCAP provides a crucial survival advantage to S. typhi during infection, thereby making it a potent alternative therapeutic target.
期刊介绍:
ACS Infectious Diseases will be the first journal to highlight chemistry and its role in this multidisciplinary and collaborative research area. The journal will cover a diverse array of topics including, but not limited to:
* Discovery and development of new antimicrobial agents — identified through target- or phenotypic-based approaches as well as compounds that induce synergy with antimicrobials.
* Characterization and validation of drug target or pathways — use of single target and genome-wide knockdown and knockouts, biochemical studies, structural biology, new technologies to facilitate characterization and prioritization of potential drug targets.
* Mechanism of drug resistance — fundamental research that advances our understanding of resistance; strategies to prevent resistance.
* Mechanisms of action — use of genetic, metabolomic, and activity- and affinity-based protein profiling to elucidate the mechanism of action of clinical and experimental antimicrobial agents.
* Host-pathogen interactions — tools for studying host-pathogen interactions, cellular biochemistry of hosts and pathogens, and molecular interactions of pathogens with host microbiota.
* Small molecule vaccine adjuvants for infectious disease.
* Viral and bacterial biochemistry and molecular biology.