Characterization and Functional Study of Escherichia Coli Outer Membrane Vesicle

Abstract

With increasing antibiotic overuse and misuse, nosocomial infections caused by multidrug-resistant bacteria have become emerging health issues throughout the world. Escherichia coli (E. coli) producing a variety of β-lactamases including CTX-M-14, carbapenemase and New Delhi metallo-β-lactamase (NDM) which irreversibly bind to β-lactam antibiotics, maintaining the integrity of the cell membrane. As the dissemination of β-lactamases resistance genes bla is associated with outer membrane vesicles (OMVs), characterization and functional study of OMVs from antibiotic resistance E. coli will provide new insight for control and treatment of complicated infection.

OMVs are 20 - 300 nm nanoparticles released from the surfaces of Gram-negative bacteria, consisting of DNA, RNA, proteins, lipopolysaccharide, and phospholipids. Due to continuous vesiculation, real-time selective cargos define the crucial role of OMVs in intercellular communication. OMVs also contribute to horizontal gene transfers apart from transformation, conjugation and transfection. Counter-transcribed RNA (ctRNA) is one of the plasmid-encoded antisense RNAs that regulates plasmid replication and incompatibility. Here, OMVs were extracted from E. coli carrying ctRNA-gene. After OMV treatment, the multidrug-resistant (MDR) plasmid in each bacterium was removed. This approach can be potentially utilized to eradicate resistance plasmids in individuals vulnerable to severe sepsis, enabling subsequent usage of less toxic and more effective antibiotics.

Urinary tract infections (UTIs) caused by MDR urinary pathogenic E. coli (UPEC) are among the major threatens to elderly health. The development of a vaccine for UTIs would be the ideal approach, especially for recurrent UTIs with MDR UPEC. Pathogen-specific OMVs have been used but the characteristics of UPEC OMVs have not yet been fully investigated, leaving a blank in the UPEC virulence mechanism. Here, virulome profiles of 6 OMVs derived from Hong Kong clinical isolates UPEC together with laboratory E. coli J53 OMV were characterized by PCR and the corresponding proteomic repertoires were profiled by tandem mass spectrometry. Virulence-associated adhesion protein-coding genes and siderophores coding genes were found in the vesicle cargos. 79 core proteins were identified from OMVs of all E. coli strains, whereas 30 OMV-specific proteins of UPEC were found. Proteins specific to UPEC-OMV are associated with virulence, osmolarity stress response, oxidative phosphorylation, ATP synthesis and membrane protein synthesis, potentially benefiting the colonization, invasiveness and survival of the recipient bacteria. Two of the Hong Kong clinical isolate ST131 E. coli strains, namely PMH and QMH, secrete OMVs with aromatic amino acid biosynthesis protein AroB which functions in bacterial motility.

The proteomic profile of UPEC-OMVs suggested that MDR plasmids disseminated via regulating OMV cargo selection. To investigate MDR-plasmids-regulated vesicle biogenesis and/or vesicle cargo selection, we compared the proteomic profiles between OMVs derived from bacteria in the presence and absence of MDR plasmids. Although over 75% of proteins overlap in OMVs from bacteria with and without MDR plasmids, an array of proteins was differentially packed in each OMV. Tra proteins associated with trans-conjugation and pili synthesis were ubiquitously identified in the OMVs of original bacteria but not in OMVs from plasmid-free strains. This result indicates that OMVs derived from clinical isolate UPEC are associated with bacterial virulence, and plasmid-regulated vesiculation would be another promising direction to develop new treatments for MDR-UPEC.

In all, this thesis demonstrates that OMVs from drug resistance bacteria carrying virulence factors and plasmid-encoded proteins play an essential role in horizontal gene transfers. On one hand, these OMVs could be utilized as a delivery tool for the ctRNA gene transfer and remove MDR plasmids from the recipient bacteria, facilitating the subsequent application of an accessible antibiotic with fewer side effects. On the other hand, UPEC-derived OMVs carrying pathogen-specific antigens could be developed as a vaccine that prevents recurrent UTIs. This work provides new insights into the biological characteristics of OMVs and will be beneficial for the development of OMVs as therapeutics and prophylactic nanoparticles for clinical applications.

Date of Award	17 Dec 2020
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Chi Kong Terrence LAU (Supervisor)