Protein aggregation and glycation in Escherichia coli exposed to desiccation-rehydration stress

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Abstract

In natural environments, bacteria often enter a state of anhydrobiosis due to water loss. Multiple studies have demonstrated that desiccation may lead to protein aggregation and glycation both in vivo and in vitro. However, the exact effects of water-loss-induced proteotoxic stress and the interplay between protein glycation and aggregation in bacteria remain elusive. Our studies revealed that protein aggregates formation in Escherichia coli started during desiccation and continued during the rehydration stage. The aggregates were enriched in proteins prone to liquid-liquid phase separation. Although it is known that glycation may induce protein aggregation in vitro, the aggregates formed in E. coli contained low levels of glycation products compared to the soluble protein fraction. Carnosine, glycine betaine and trehalose diminished the formation of protein aggregates and glycation products, resulting in increased E. coli viability. Notably, although high concentrations of glycine-betaine and trehalose significantly enhanced protein aggregation, glycation was still inhibited and E. coli cells survived desiccation better than bacteria grown without osmolytes. Taken together, our results suggest that the aggregates might play protective functions during early desiccation-rehydration stress. Moreover, it seems glycation rather than protein aggregation is the main cause of E. coli death upon 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).

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