Shiga-toxin producing E.coli – specific patterns of gene and regulatory non-coding RNAs expression and their involvement in virulence of bacteria
Project Status: 3 Approved without Funding
Duration in months: 36 months
Objective
Shiga–toxin producing Escherichia coli (STEC) Infection is among the leading causes of bloody diarrheal disease complicated by hemolytic uremic syndrome (HUS) in the country of Georgia. STEC-associated HUS was first recognized in Georgia in 2009 following the diagnosis of a cluster of eight cases of HUS that resulted in 7 deaths in Tbilisi. The study of these cases confirmed STEC O104 (stx2+) as a major causative agent of the outbreak (Chokoshvili et al.,2014). Two strains of O104 E.coli were isolated from this epidemiological outbreak in 2009 (2009EL-2050, 2009EL-2071) (Ahmed et al., 2012).
Later in 2015, eight STEC strains were isolated from stool samples from patients having different clinical complications. All these strains were genetically characterized using different molecular typing methods, such as multiplex PCR and Pulsed Field Gel Electrophoresis (PFGE). Five Stx2 producing strains were identified as enteroaggregative (EAEC) O104:H4 E. coli, and three strains as non-O104 stx2/ehxA positive strains. Through RNASeq we determine the global transcriptome profile of one of these stx2 positive EAEC 0104:H4 strain (2087/15-G), which belongs to sequence type 678 (ST678) and a reference strain of E.coliATCC 25922. More than 500 genes and nearly 30 small regulatory RNAs were identified with highly significant differential expression between these strains of bacteria. According to Gene Ontology (GO) analysis ( Gene Ontology Consortium, 2008) these differentially expressed genes covers wide variety of biological processes including Transposition, DNA recombination xenobiotic catabolic process, cellular response to chemical stimulus, cell adhesion and others. . The differences obviously included the stx genes expression, which was completely absent in the reference strain ATCC 25922.and expressed in a in the EAEC 0104:H4. The biological functions of differentially expressed small regulatory RNAs are less defined, but the magnitude of differences (log2 Fold Change) for many of them is more than 5, which by itself indicates the importance of them for further studies. During the last decade, numerous studies have demonstrated that in addition to being potent protein synthesis inhibitors, stxs are also multifunctional proteins capable of activating multiple cell stress signaling pathways, which may result in apoptosis, autophagy or activation of the innate immune response. A intriguing hypothesis explaining how STEC might benefit from Shiga toxin production was suggested (?o?s et al., 2011; Mauro and Koudelka, 2011). The lysis of host eukaryotic cells allows for the effective release of Shiga toxins, which means that a bacterial cell producing Shiga toxin must die before large quantities of the toxin can act as a virulence factor. The logic question arises what is the benefit for a bacterium to produce Shiga toxin if its death is strongly coupled to toxin expression? The hypothesis is that if we assume that production of toxins occurs only in a small fraction of bacterial cells, thus, a few members of the population are sacrificed for the benefit of the rest and provides an example of “bacterial altruism.” (?os et al., 2011; Mauro and Koudelka, 2011) How the differentially expressed gene complement, individually or in combination alter Stx expression (and function), remains unknown. How they are involved in the virulence of bacteria? (It should be also emphasized that RNA-SEQ studies are not strongly quantitative and needs further validation experiments.)
Now we propose to get deeper into these preliminary data and to systematically investigate the molecular specificities/underlying molecular mechanism of altered? Stx production as key factor of E. coli virulence. We propose; to further validate the differences revealed by RNA-SEQ studies for a select number of selected genes and small regulatory RNAs by quantitative real-time PCR assays to design antisense oligonucleotides (ASO) against selected differentially expressed genes and study their influence on stxs expression in bacterial cultures; to study the influence of selected ASO on the complete proteome changes of bacteria; to study influence of selected ASOs on virulence of bacteria by in vitro cytotoxicity assay, which is based on bacterial ability to induce apoptosis in macrophage cultures.
Biosafety and Biosecurity are the areas for potential funding of the present project
As the STEC infections are characterized with broad geographical distributions the results of the project could lead to the intensive regional cooperation and further regional project.
Participating Institutions
PARTICIPATING
Ilia State University
COLLABORATOR
Norwegian Institute of Public Health Domain for Enviromental Health and Infectious Diseases Control
COLLABORATOR
University of Texas at San Antonio (UTSA)
LEADING
National Center for Disease Control and Public Health (NCDC)
