\u03b1-Hemolysin of uropathogenic E. coli regulates NLRP3 inflammasome activation and mitochondrial dysfunction in THP-1 macrophages

Vivek Verma,Rakesh Singh Dhanda,Henrik Thorlacius,Manisha Yadav,Sonal Yadav,Parveen Kumar,Manisha Yadav,Surbhi Gupta,Henrik Thorlacius

doi:10.1038/s41598-020-69501-1

Abstract

Hemolysin expressing UPEC strains have been associated with severe advanced kidney pathologies, such as cystitis and pyelonephritis, which are associated with an inflammatory response. Macrophages play an important role in regulating an inflammatory response during a urinary tract infection. We have studied the role of purified recombinant α-hemolysin in inducing inflammatory responses and cell death in macrophages. Acylation at lysine residues through HlyC is known to activate proHlyA into a fully functional pore-forming toxin, HlyA. It was observed that active α-hemolysin (HlyA) induced cleavage of caspase-1 leading to the maturation of IL-1β, while inactive α-hemolysin (proHlyA) failed to do so in THP-1 derived macrophages. HlyA also promotes deubiquitination, oligomerization, and activation of the NLRP3 inflammasome, which was found to be dependent on potassium efflux. We have also observed the co-localization of NLRP3 within mitochondria during HlyA stimulations. Moreover, blocking of potassium efflux improved the mitochondrial health in addition to a decreased inflammatory response. Our study demonstrates that HlyA stimulation caused perturbance in potassium homeostasis, which led to the mitochondrial dysfunction followed by an acute inflammatory response, resulting in cell death. However, the repletion of intracellular potassium stores could avoid HlyA induced macrophage cell death. The findings of this study will help to understand the mechanism of α-hemolysin induced inflammatory response and cell death.

Highlights

A urinary tract infection (UTI) is one of the most common bacterial infections and is the second most common nosocomial infection[1]
An insignificant amount of endotoxin contamination was found in our preparations of HlyA and proHlyA, unable to produce a synergistic effect during stimulations
We found that the levels of proIL-1β and procaspase-1 were not significantly different after stimulation and an effect was seen on the cleavage of both proteins (Fig. 1A) (Supplementary Information 1)

Summary

Introduction

A urinary tract infection (UTI) is one of the most common bacterial infections and is the second most common nosocomial infection[1]. Zhou et al reported the presence of NLRP3 in mitochondria and its activation during mitochondrial dysfunction, leading to cell death in response to various stimulators, such as nigericin, monosodium urate crystals and alum[40]. It was found that mtDNA copies were significantly (p = 0.0125) increased during HlyA stimulation of THP-1m, which was not seen on the inhibition of K+ efflux (Fig. 6), indicating a possible role of potassium efflux in the regulation of mitochondrial biogenesis.

Results

Conclusion