Understanding the Small RNA-regulated Dissemination of Emerging blaCTX-M-14 Carrying Plasmid pHK01 in Hong Kong

理解小RNA調控的攜帶blaCTX-M-14基因的質粒pHK01在香港的傳播

Student thesis: Doctoral Thesis

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Award date20 Mar 2018

Abstract

The emerging dissemination of antibiotic resistance has become a major public health threat worldwide. And the dissemination of blaCTX-M is commonly achieved through plasmid conjugation. The CTX-M types of ESBL enzymes have emerged worldwide among Escherichia coli and become the most prevalent type of ESBLs in the world. Narrow host-range IncFII plasmids play important roles in the dissemination of blaCTX-M-14 throughout the world. Notably, blaCTX-M-14-carrying plasmid pHK01 and pHK01-like plasmids belong to IncFII group are widely disseminated in Hong Kong and mainland China. Studies of resistance genes in multidrug resistance plasmid were extensively performed but little is known about the bacterial host fitness upon plasmid acquisition.

To identify and characterize the novel chromosomal and plasmid-encoded sRNAs which involve in the dissemination of the CTX-M-encoding plasmid, pHK01, among bacteria in Hong Kong, and to determine the global changes of the RNA repertoire of E. coli upon introduction of pHK01. Next-generation sequencing and qRT-PCR were performed to study the transcriptome profile of E. coli J53 carrying pHK01 in different growth phases. Novel plasmid-encoded small RNAs (sRNAs) were predicted using bioinformatics methods and validated by Northern Blot. Putative sRNA targets were confirmed using qRT-PCR upon sRNA over-expression. Interestingly, one of these novel sRNAs AS-traI was able to shorten the time of lag phase in E. coli J53 carrying pHK01. The identification of plasmid-encoded sRNAs and their gene regulation of hosts will greatly improve the understanding of the effect of plasmid to host at the post-transcriptional level.

The physiological consequences induced by pHK01 in bacterial host E. coli J53 were also studied. Although no significant burden to host growth was introduced by pHK01, transcriptome sequencing followed by gene ontology analysis revealed that several pathways involving in metabolism and motility were changed. The reduction of host motility was further confirmed by motility assay. Moreover, this was further validated that the low motility of host was caused by the carriage of the plasmid using the curing experiment. In this thesis, a new, fast and incompatibility group-specific curing method that can remove the ESBL-plasmids from the bacteria host using countertranscribed RNA (ctRNA) was successfully developed and the method will be of general utility for studying plasmids-host interaction in clinical aspects. And the understanding of pHK01 induced motility change and novel plasmid-encoded sRNA will provide a new insight to the survival of mobile elements and their dissemination among bacteria.

In order to investigate the sRNA-regulated pathways involved in the plasmid dissemination, sRNAs knock-down experiments in J53/pHK01 were performed. Three plasmid-encoded sRNAs (CopA, FinP, AS-traI) were selected for further investigations. To test the effect of knock-down using decoy, sRNA and the decoy were probed by Northern blot analysis. The expression of FinP and AS-traI were reduced when their corresponding sRNA decoys were overexpressed, suggesting that the hybridization of decoys to sRNAs activates the sRNA degradation and hence, to silence the sRNAs. On the contrary, the amount of CopA sRNA significantly enhanced when the decoy was overexpressed. Notably, significant amount of decoy of CopA was also detected. One of the possible interpretation of this phenomenon was that the decoy sequestered CopA sRNA and restricted their cellular functions. Another possible reason was that the decoy doesn’t work in silencing the CopA sRNA.

By inducing the sRNA decoy which to either titrate away or silence the sRNA, an otherwise strongly inhibited target gene will be allowed to express subsequently. To further verify this speculation, qRT-PCR analysis was performed to measure the expression level of the sRNA target genes. CopA is the sRNA controls plasmid copy number by pairing with the target repA3 mRNA whereas FinP sRNA sequesters the Shine-Dalgarno sequence of traJ gene. Enhancement of the transcripts level of their target genes was expected if we silenced the sRNAs. The result indicated that the expression level of repA3 and traJ genes was significantly upregulated upon the silencing of CopA and FinP sRNAs respectively. Regarding the AS-traI sRNA which activates the expression of pHK01_031 and pHK01_029, their expression level were reduced significantly when AS-traI was silenced.

Taken together, the results described above could be summarized in four points. pHK01 did not cause any effect to the growth but reduced host motility. Based on the transcriptome analysis, the reduction of host motility was attributed to the downregulation of flagellar systems. Novel plasmid-encoded small RNAs in pHK01 were systematically identified and their expression at different stages of bacterial growth was characterized. Furthermore, one of the novel plasmid-encoded sRNAs, AS-traI, was found to be able to shorten the lag phase of the bacterial growth when overexpressed in J53/pHK01. Using ctRNA is a fast and incompatibility group’s plasmids-specific curing method to remove the ESBL-plasmids from the bacterial host. sRNA decoys can efficiently silence the sRNA either by activating the sRNA degradation or sequestering the sRNA. Moreover, these sRNAs were capable to control the expression of the genes that related to dissemination. All these results suggested the importance of plasmid-mediated host regulation in different perspectives which will be of general interest in the fields of microbial infectious disease.

    Research areas

  • drug resistance plasmid, pHK01, RNA-Seq, motility, sRNA, ESBL-plasmid curing, sRNA decoy