Flagellum Is Responsible for Promoting Effects of Viable Escherichia coli on Calcium Oxalate Crystallization, Crystal Growth, and Crystal Aggregation

Abstract

Urease-producing bacteria (especially Proteus mirabilis) can cause infection kidney stone. However, recent studies have shown that intact viable non-urease-producing bacteria such as Escherichia coli might also promote calcium oxalate (CaOx) kidney stone formation but with unclear mechanism. We thus hypothesized that some relevant bacterial components might be responsible for such promoting effects of the intact viable E. coli. Flagella, capsule, lipopolysaccharide (LPS), and outer membrane vesicles (OMVs) were isolated/purified and their stone modulatory activities were evaluated using CaOx crystallization, crystal growth, and crystal aggregation assays. Among these, flagella had the most potent promoting effects on CaOx crystallization, crystal growth, and crystal aggregation. Validation was performed by deflagellation demonstrating that the deflagellated intact viable E. coli had markedly reduced CaOx crystal modulatory activities in all aspects (comparable to those of the negative controls). Similarly, neutralization of the isolated/purified flagella using a specific anti-flagellin antibody, not an isotype control, could abolish the promoting effects of flagella. These findings provide direct evidence indicating that flagellum is responsible for the promoting effects of the viable E. coli on CaOx crystallization, crystal growth and aggregation.

Escherichia coli Aggravates Calcium Oxalate Stone Formation via PPK1/Flagellin‐Mediated Renal Oxidative Injury and Inflammation

“…Flic mutations lead to a significant reduction in the pathogenic capacity of bacteria [33]. In this study, flagellin was found to directly promote the aggregation of COM crystals, a finding that is also consistent with the study by Rattiyaporn et al [34] Other 13 Oxidative Medicine and Cellular Longevity mouse model. The formation of urinary tract stones involves biomineralization, and bacterial biofilm participates in this process [36].…”

“…This may be related to other virulence factors secreted by E. coli, which are less influenced by PPK1 (like LPS) [43,44]. However, the results of us supported LPS may not a leading element in the aggregation of CaOx crystals, which is also consistent with Rattiyaporn's study [34].…”

A Perspective on the Metabolic Potential for Microbial Contributions to Urolithiasis

“…Additionally, in vitro studies have shown that the flagella from strains of E. coli in the urinary tract promote CaOx crystallization (22). These data suggest a complex, direct involvement of kidney bacteria in lithogenesis.…”

Oxidative Modifications Switch Modulatory Activities of Urinary Proteins From Inhibiting to Promoting Calcium Oxalate Crystallization, Growth, and Aggregation

“…CaOx crystal aggregation assay was performed as previously described ( 46 , 47 ). Briefly, CaOx crystals were generated as described for the crystallization assay but with larger volume.…”

“…CaOx crystals (dry weight of 1000 μg) were resuspended in 1 ml of the crystallization buffer in each well of the 6-well plate (Corning, Inc). An equal volume (4 μl) of the artificial urine (control) or the tested proteins (1 μg/μl in the artificial urine) was added into each well, and the plate was continuously shaken in a shaking incubator (Zhicheng) at 150 rpm and 25 °C for 1 h. Thereafter, formation of CaOx crystal aggregate (defined as an assembly of three or more individual CaOx crystals that tightly joined together) ( 46 , 47 ) was observed and imaged under the Eclipse Ti-S inverted phase-contrast light microscope. Number of CaOx crystal aggregates was counted from 15 randomized HPFs per well.…”