Protein aggregation and glycation in Escherichia coli exposed to desiccation-rehydration stress
Introduction
Bacteria are often exposed to desiccation stress in the natural environment. Water loss leads to decreasing fluidity and concentration of intracellular metabolites and macromolecules in the cell (Esbelin et al., 2018, García, 2011, Grzyb and Skłodowska, 2022, Laskowska and Kuczyńska-Wiśnik, 2020, Lebre et al., 2017). Reduction of the hydration shell around proteins may lead to protein instability, denaturation and aggregation. Multiple studies in vitro indicate that aggregation may proceed during the rehydration of dried proteins (Chakrabortee et al., 2007, Fink, 1998, Prestrelski et al., 1993). Rehydration of lyophilized proteins may result in their aggregation due to the formation the partially folded intermediates during refolding (Fink, 1998). It has also been proposed that during desiccation, when proteins are unfolded, there is not enough time to form large aggregates; therefore, this process continues during rehydration (Chakrabortee et al., 2007). The formation of endogenous protein aggregates in bacteria after desiccation stress was observed in Escherichia coli (Moruno Algara et al., 2019) and Acinetobacter baumannii (Wang et al., 2020), but the fate of aggregates during in vivo rehydration remained unknown. Therefore, in this study, we analysed the effect of rehydration on protein aggregation in E. coli.
Protein aggregation during desiccation may result from irreversible oxidation and non-enzymatic glycosylation (glycation). Protein dysfunction impairs metabolism and repair pathways leading to the accumulation of reactive oxygen species (ROS) (Fredrickson et al., 2008; García, 2011; Harding et al., 2018). The loss of membrane integrity and disruption of the respiratory chain further enhance ROS formation and induce the production of reactive aldehydes involved in the oxidation and glycation of macromolecules. Oxidative stress initiated by desiccation induces the production of glyoxal and methylglyoxal, which are involved in the initial stage of glycation, the Maillard reaction (Lee and Park, 2017). Glycation targets amino acids at the protein N-terminus or those with an amino group in its side chain: lysine, arginine and histidine. The resulting adducts are transformed into Shiff's bases, Amadori products, and finally into advanced glycation end products (AGEs)-a heterogeneous group of products with intra- and intermolecular cross-links (Boteva and Mironova, 2019, Richarme et al., 2018). Protein glycation has been mainly linked to aging and human diseases (Fournet et al., 2018, Perrone et al., 2020, Rabbani and Thornalley, 2021). However, there is increasing evidence that bacteria, despite their short life span, can also accumulate glycated proteins, even under normal physiological conditions (Boteva and Mironova, 2019, Cohen-Or et al., 2013, Cohen-Or et al., 2011, Kram and Finkel, 2015, Mironova et al., 2005, Mironova et al., 2001, Potts et al., 2005). Numerous studies demonstrated that the formation of AGEs can cause proteotoxic effects and promote or accelerate protein unfolding and aggregation (Iannuzzi et al., 2014). Therefore, to further investigate the effects of desiccation-rehydration stress, we focused on glycation and its contribution to the formation of protein aggregates in E. coli.
Section snippets
Growth conditions
E. coli MC4100 [araD139 ∆(lacIPOZYA argF) U169 fla relA rpsL] was grown at 37 °C in 100 ml lysogeny broth (LB) medium in Erlenmeyer flasks with agitation. At an OD595 of 0.5, cells were pelleted, resuspended in 5 ml of spent medium, and desiccated in an open Petri dish at 21 °C and ∼40 % humidity. After complete drying (∼4 h), the bacteria were resuspended in 10 ml of 0.9 % NaCl, divided into two 5 ml aliquots and collected by centrifugation. One of the pellets was used as a sample containing
Rehydration of E. coli cells enhances endogenous protein aggregation
Protein aggregates were isolated from E. coli cultures exposed to desiccation and rehydration as described in the Material and methods Section (2.6.). Hyperosmotic stress, which bacteria encounter during drying (Vriezen et al., 2007), promotes protein aggregation. We found that in E. coli cells exposed to 4 h desiccation, ∼0.6 % of total cellular proteins formed aggregates (Fig. 1A). During the subsequent rehydration stage, the level of protein aggregates increased continuously, even after
Discussion
Our studies revealed that the formation of protein aggregates in E. coli exposed to desiccation-rehydration stress occurred mainly during rehydration (Fig. 1). The results are consistent with several other reports showing that rehydration of in vitro dried proteins often causes denaturation and aggregation (Chakrabortee et al., 2007, Prestrelski et al., 1993). The aggregates contained ribosomal proteins and other proteins belonging to different classes, including enzymes involved in the TCA
CRediT authorship contribution statement
Adrianna Łupkowska: Investigation, Validation, Visualization. Soroosh Monem: Investigation, Validation. Janusz Dębski: Investigation, Validation, Software. Karolina Stojowska-Swędrzyńska: Investigation, Validation, Visualization. Dorota Kuczyńska-Wiśnik: Conceptualization, Investigation, Writing – review & editing. Ewa Laskowska: Conceptualization, Writing – original draft, Writing – review & editing.
Conflicts of interest
The authors declare that they have no competing interests.
Acknowledgements
This work was supported by the University of Gdansk, Poland (task grant no. 531/D010-D241-22).
References (74)
- et al.
Crystal structure of osmoporin OmpC from E. coli at 2.0 Å
J. Mol. Biol.
(2006) - et al.
Global proteomic analysis of advanced glycation end products in the Arabidopsis proteome provides evidence for age-related glycation hot spots
J. Biol. Chem.
(2017) - et al.
Effects of sucrose, carnosine, and their mixture on the glass transition behavior and storage stability of freeze-dried lactic acid bacteria at various water activities
Cryobiology
(2022) - et al.
Desiccation: an environmental and food industry stress that bacteria commonly face
Food Microbiol.
(2018) - et al.
Formation of biological condensates via phase separation: characteristics, analytical methods, and physiological implications
J. Biol. Chem.
(2019) - et al.
Selectivity of aggregation-determining interactions
J. Mol. Biol.
(2015) - et al.
Identifying glycation hot-spots in bovine milk proteins during production and storage of skim milk powder
Int. Dairy J.
(2022) - et al.
Bacterial responses to glyoxal and methylglyoxal: reactive electrophilic species
Int. J. Mol. Sci.
(2017) - et al.
Role of protein-bound carbonyl groups in the formation of advanced glycation endproducts
Biochim. Biophys. Acta
(1997) - et al.
Diffprot - software for non-parametric statistical analysis of differential proteomics data
J. Proteom.
(2012)