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Characterisation of stress response and antibiotic resistance factors of Gram negative bacteria
thesisposted on 23.02.2017, 23:04 by Narayanan, Shalini
When the stress response occurs in Neisseria gonorrhoeae, the molecular chaperone DnaK is activated. DnaK is a heat shock protein. During heat shock, the genes encoding the DnaK chaperone machine (DnaK/DnaJ/GrpE) in N. gonorrhoeae are transcribed from RpoH (σ³²)-dependent promoters. In contrast to Escherichia coli, the molecular mechanisms associated with this stress response signal in N. gonorrhoeae is not well understood. Failure to isolate viable rpoH null mutants in N. gonorrhoeae implies the gene encoding gonococcal RpoH is essential. As part of this study, we report the cloning, purification and biochemical characterisation of N. gonorrhoeae DnaK (NgDnaK) and RpoH. We observed that the sequences of DnaK and RpoH from N. gonorrhoeae are 73% and 50% identical to the sequences of the E. coli homologues respectively. In solution, NgDnaK exists as an assortment of monomers, dimers and other high order oligomers. Following the addition of ATP, NgDnaK dissociates into monomeric species, just as observed in E. coli DnaK (EcDnaK). Nucleotide-free NgDnaK displays micromolar affinity for a short model substrate, the NR-peptide, which is close to that reported for EcDnaK. In contrast to EcDnaK, the intrinsic ATPase activity of purified NgDnaK revealed to be significantly higher with a Vmax of 193 pmol phosphate released per minute per microgram DnaK in the absence of co-chaperones, with the turnover number 0.4 minˉ¹ against ATP under assay conditions. Biochemical analysis on N. gonorrhoeae heat shock proteins, revealed that association between these NgRpoH and NgDnaK to be non-specific and ATP-dependent, in wide contrast to EcDnaK which binds σ³² specifically in a monomeric fashion even without the presence of ATP. These findings together with the absence of E. coli σ³² DnaK binding site, in NgRpoH, propose that the mechanism of NgDnaK/NgRpoH interaction is different from that observed in E. coli. The induction of bacterial resistance to conventional antibiotics is emerging at an alarming rate. This highlights the clinical requirement for the development of novel types of antimicrobial agents. Short proline-rich antibacterial peptides (PR-AMPs), isolated from insects and mammals, have been suggested to be ideal targets against evolving antibacterial resistant pathogens. These peptides, with good serum stability and lack of toxicity towards mammalian cells, were shown to kill responsive bacteria by deactivating intracellular biomolecular targets via non-lytic mechanism. Several proline-rich bactericidal peptides are predicted to function by inhibiting the molecular chaperone, DnaK, in a stereospecific manner. Extensive studies on this class of peptides, led to the proposal that PR-AMPs possess multiple targets within bacterial cells, and that the development of bacterial resistance to be significantly lower. The work presented as part of the thesis, refutes this hypothesis by demonstrating that resistance to pyrrhocoricin occurs spontaneously. The generation of spontaneous E. coli mutants that are resistant to pyrrhocoricin occurs at a relatively high frequency (6x10ˉ⁷) via the deletion of a non-essential gene that encodes the putative permease, SbmA. Moreover, sequencing of dnaK in pyrrhocoricin-resistant mutants, isolated from several biologically-independent experiments, revealed that dnaK did not contribute to pyrrhocoricin resistance. This finding questions the sustainability of the developing insect derived PR-AMPs as potential antimicrobial therapeutics.