Structural characterization of caseinolytic protease (ClpP) from Klebsiella pneumonia.
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Date
2023
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Abstract
Klebsiella pneumonia (K. pneumoniae) is a non-motile bacterium that is characterized as an
opportunistic pathogen known to cause hospital-acquired infections (HAI) in
immunocompromised patients. It is part of a group of pathogens referred to as ESKAPE
pathogens. Other pathogens that form a part of this group include; Enterococcus faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas
aeruginosa and Enterobacter. These pathogens are known to “ESKAPE” the effect of
antibiotics. Moreover, Klebsiella pneumoniae is amongst the top 8 most critical pathogens in
hospitals therefore, this pathogen poses a great global concern and is life threatening in lowand
middle-income countries. Caseinolytic proteases (ClpP) are thermostable proteins which
play an important role in protein homeostasis by degrading aggregated and misfolded proteins
and allows the pathogen to survive in different environmental conditions. To our knowledge,
there are no studies which have focused on the diversity of clpp genes in the Klebsiella species
as well the expression and purification K. pneumoniae (Kp) ClpP, to date. Therefore, this study
aimed to address this knowledge gap. Bioinformatic analysis was used to investigate the
diversity of ClpP in the Klebsiella species. ClpP was found to be present in all the investigated
Klebsiella strains with each strain containing more than one ClpP and 17 different ClpP
isoforms were identified across the species. Homology modelling of the hypothetical Kp ClpP
structure and molecular dynamic simulations showed that this protein was mainly alpha
helical, highly dynamic, stable and flexible. The gene encoding for Kp ClpP was cloned into a
pColdI vector. This was followed by successful expression Kp ClpP with a band size of 25
kDa, this was slightly higher than the expected size of 21 kDa. Western blot and liquid
chromatography mass spectrometry (LC-MS) analysis was used to confirm that the 25 kDa
protein was indeed Kp ClpP. This protein was then purified to homogeneity using affinity
chromatography.
Description
Masters Degree. University of KwaZulu-Natal, Pietermaritzburg.