Intracellular Protective Functions and Therapeutical Potential of Trehalose.

Trehalose is a naturally occurring, non-reducing saccharide widely distributed in nature. Over the years, research on trehalose has revealed that this initially thought simple storage molecule is a multifunctional and multitasking compound protecting cells against various stress factors. This review presents data on the role of trehalose in maintaining cellular homeostasis under stress conditions and in the virulence of bacteria and fungi. Numerous studies have demonstrated that trehalose acts in the cell as an osmoprotectant, chemical chaperone, free radical scavenger, carbon source, virulence factor, and metabolic regulator. The increasingly researched medical and therapeutic applications of trehalose are also discussed.

Influence of Nε-Lysine Acetylation on the Formation of Protein Aggregates and Antibiotic Persistence in E. coli.

Numerous studies indicate that reversible Nε-lysine acetylation in bacteria may play a key role in the regulation of metabolic processes, transcription and translation, biofilm formation, virulence, and drug resistance. Using appropriate mutant strains deficient in non-enzymatic acetylation and enzymatic acetylation or deacetylation pathways, we investigated the influence of protein acetylation on cell viability, protein aggregation, and persister formation in Escherichia coli. Lysine acetylation was found to increase protein aggregation and cell viability under the late stationary phase. Moreover, increased lysine acetylation stimulated the formation of persisters. These results suggest that acetylation-dependent aggregation may improve the survival of bacteria under adverse conditions (such as the late stationary phase) and during antibiotic treatment. Further experiments revealed that acetylation-favorable conditions may increase persister formation in Klebsiella pneumoniae clinical isolate. However, the exact mechanisms underlying the relationship between acetylation and persistence in this pathogen remain to be elucidated.

Liquid-Liquid Phase Separation and Protective Protein Aggregates in Bacteria.

Liquid-liquid phase separation (LLPS) and the formation of membraneless organelles (MLOs) contribute to the spatiotemporal organization of various physiological processes in the cell. These phenomena have been studied and characterized mainly in eukaryotic cells. However, increasing evidence indicates that LLPS-driven protein condensation may also occur in prokaryotes. Recent studies indicate that aggregates formed during proteotoxic stresses may also play the role of MLOs and increase the fitness of bacteria under stress. The beneficial effect of aggregates may result from the sequestration and protection of proteins against irreversible inactivation or degradation, activation of the protein quality control system and induction of dormancy. The most common stress that bacteria encounter in the natural environment is water loss. Therefore, in this review, we focus on protein aggregates formed in E. coli upon desiccation-rehydration stress. In silico analyses suggest that various mechanisms and interactions are responsible for their formation, including LLPS, disordered sequences and aggregation-prone regions. These data support findings that intrinsically disordered proteins and LLPS may contribute to desiccation tolerance not only in eukaryotic cells but also in bacteria. LLPS-driven aggregation may be a strategy used by pathogens to survive antibiotic treatment and desiccation stress in the hospital environment.

New Strategies to Kill Metabolically-Dormant Cells Directly Bypassing the Need for Active Cellular Processes.

Antibiotic therapy failure is often caused by the presence of persister cells, which are metabolically-dormant bacteria capable of surviving exposure to antimicrobials. Under favorable conditions, persisters can resume growth leading to recurrent infections. Moreover, several studies have indicated that persisters may promote the evolution of antimicrobial resistance and facilitate the selection of specific resistant mutants; therefore, in light of the increasing numbers of multidrug-resistant infections worldwide, developing efficient strategies against dormant cells is of paramount importance. In this review, we present and discuss the efficacy of various agents whose antimicrobial activity is independent of the metabolic status of the bacteria as they target cell envelope structures. Since the biofilm-environment is favorable for the formation of dormant subpopulations, anti-persister strategies should also include agents that destroy the biofilm matrix or inhibit biofilm development. This article reviews examples of selected cell wall hydrolases, polysaccharide depolymerases and antimicrobial peptides. Their combination with standard antibiotics seems to be the most promising approach in combating persistent infections.

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

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.

Antibiotic Heteroresistance in Klebsiella pneumoniae.

Klebsiella pneumoniae is one of the most common pathogens responsible for infections, including pneumonia, urinary tract infections, and bacteremias. The increasing prevalence of multidrug-resistant K. pneumoniae was recognized in 2017 by the World Health Organization as a critical public health threat. Heteroresistance, defined as the presence of a subpopulation of cells with a higher MIC than the dominant population, is a frequent phenotype in many pathogens. Numerous reports on heteroresistant K. pneumoniae isolates have been published in the last few years. Heteroresistance is difficult to detect and study due to its phenotypic and genetic instability. Recent findings provide strong evidence that heteroresistance may be associated with an increased risk of recurrent infections and antibiotic treatment failure. This review focuses on antibiotic heteroresistance mechanisms in K. pneumoniae and potential therapeutic strategies against antibiotic heteroresistant isolates.

Mechanisms Protecting Acinetobacter baumannii against Multiple Stresses Triggered by the Host Immune Response, Antibiotics and Outside-Host Environment.

Acinetobacter baumannii is considered one of the most persistent pathogens responsible for nosocomial infections. Due to the emergence of multidrug resistant strains, as well as high morbidity and mortality caused by this pathogen, A. baumannii was placed on the World Health Organization (WHO) drug-resistant bacteria and antimicrobial resistance research priority list. This review summarizes current studies on mechanisms that protect A. baumannii against multiple stresses caused by the host immune response, outside host environment, and antibiotic treatment. We particularly focus on the ability of A. baumannii to survive long-term desiccation on abiotic surfaces and the population heterogeneity in A. baumannii biofilms. Insight into these protective mechanisms may provide clues for the development of new strategies to fight multidrug resistant strains of A. baumannii.

New insight into the mechanisms protecting bacteria during desiccation.

Desiccation is a common stress that bacteria face in the natural environment, and thus, they have developed a variety of protective mechanisms to mitigate the damage caused by water loss. The formation of biofilms and the accumulation of trehalose and sporulation are well-known strategies used by bacteria to survive desiccation. Other mechanisms, including intrinsically disordered proteins and the anti-glycation defence, have been mainly studied in eukaryotic cells, and their role in bacteria remains unclear. We have recently shown that the impairment of trehalose synthesis results in higher glucose availability, leading to the accumulation of acetyl phosphate and enhanced protein acetylation, which in turn stimulates protein aggregation. In the absence of trehalose synthesis, excess glucose may stimulate non-enzymatic glycosylation and the formation of advanced glycation end products (AGEs) bound to proteins. Therefore, we propose that trehalose may prevent protein damage, not only as a chemical chaperone but also as a metabolite that indirectly counteracts detrimental protein acetylation and glycation.

Trehalose protects Escherichia coli against carbon stress manifested by protein acetylation and aggregation.

The disaccharide trehalose is widely distributed in nature and can serve as a carbon reservoir, a signaling molecule for controlling glucose metabolism and a stress protectant. We demonstrated that in Escherichia coli ΔotsA cells, which are unable to synthesize trehalose, the aggregation of endogenous proteins during the stationary phase was increased in comparison to wild-type cells. The lack of trehalose synthesis boosted Nε-lysine acetylation of proteins, which in turn enhanced their hydrophobicity and aggregation. This increased Nε-lysine acetylation could result from carbon overflow and the accumulation of acetyl phosphate caused by the ΔotsA mutation. These findings provide a better understanding of the previously reported protective functions of trehalose in protein stabilization and the prevention of protein aggregation. Our results indicate that trehalose may participate in proteostasis not only as a chemical chaperone but also as a metabolite that indirectly counteracts detrimental protein acetylation. We propose that trehalose protects E. coli against carbon stress - the synthesis and storage of trehalose can prevent carbon overflow, which otherwise is manifested by protein acetylation and aggregation.

Proteomic analysis of protein homeostasis and aggregation.

Protein homeostasis (proteostasis) refers to the ability of cells to preserve the correct balance between protein synthesis, folding and degradation. Proteostasis is essential for optimal cell growth and survival under stressful conditions. Various extracellular and intracellular stresses including heat shock, oxidative stress, proteasome malfunction, mutations and aging-related modifications can result in disturbed proteostasis manifested by enhanced misfolding and aggregation of proteins. To limit protein misfolding and aggregation cells have evolved various strategies including molecular chaperones, proteasome system and autophagy. Molecular chaperones assist folding of proteins, protect them from denaturation and facilitate renaturation of the misfolded polypeptides, whereas proteasomes and autophagosomes remove the irreversibly damaged proteins. The impairment of proteostasis results in protein aggregation that is a major pathological hallmark of numerous age-related disorders, such as cataract, Alzheimer's, Parkinson's, Huntington's, and prion diseases. To discover protein markers and speed up diagnosis of neurodegenerative diseases accompanied by protein aggregation, proteomic tools have increasingly been used in recent years. Systematic and exhaustive analysis of the changes that occur in the proteomes of affected tissues and biofluids in humans or in model organisms is one of the most promising approaches to reveal mechanisms underlying protein aggregation diseases, improve their diagnosis and develop therapeutic strategies. Significance: In this review we outline the elements responsible for maintaining cellular proteostasis and present the overview of proteomic studies focused on protein-aggregation diseases. These studies provide insights into the mechanisms responsible for age-related disorders and reveal new potential biomarkers for Alzheimer's, Parkinson's, Huntigton's and prion diseases.

Physiologically distinct subpopulations formed in Escherichia coli cultures in response to heat shock.

Bacteria can form heterogeneous populations containing phenotypic variants of genetically identical cells. The heterogeneity of populations can be considered a bet-hedging strategy allowing adaptation to unknown environmental changes - at least some individual subpopulations or cells might be able to withstand future adverse conditions. Using Percoll gradient centrifugation, we demonstrated that in an Escherichia coli culture exposed to heat shock at 50 °C, two physiologically distinct subpopulations were formed. A high-density subpopulation (HD50) demonstrated continued growth immediately after its transfer to LB medium, whereas the growth of a low-density subpopulation (LD50) was considerably postponed. The LD50 subpopulation contained mainly viable but non-culturable bacteria and exhibited higher tolerance to sublethal concentrations of antibiotics or H2O2 than HD50 cells. The levels of aggregated proteins and main molecular chaperones were comparable in both subpopulations; however, a decreased number of ribosomes and a significant increase in protein oxidation were observed in the LD50 subpopulation as compared with the HD50 subpopulation. Interestingly, under anaerobic heat stress, the formation of the HD50 subpopulation was decreased and culturability of the LD50 subpopulation was significantly increased. In both subpopulations the level of protein aggregates formed under anaerobic and aerobic heat stress was comparable. We concluded that the formation of protein aggregates was independent of oxidative damage induced by heat stress, and that oxidative stress and not protein aggregation limited growth and caused loss of LD50 culturability. Our results indicate that heat stress induces the formation of distinct subpopulations differing in their ability to grow under standard and stress conditions.

Protein aggregation in Ehrlichia chaffeensis during infection of mammalian cells.

Ehrlichia chaffeensis is an obligatory intracellular pathogen transmitted through infected ticks to humans and other vertebrates. We investigated the extent of protein aggregation in E. chaffeensis during infection of canine macrophage cell line, DH82. We discovered that the size of the aggregated fraction of E. chaffeensis proteins increased during the first 48 h post infection. We also incubated the infected cells with guanidinium chloride (GuHCl), a known inhibitor of the protein-disaggregating molecular chaperone ClpB. Up to 0.5 mM GuHCl had no impact on the host cells, whereas the viability of the pathogen was reduced by ∼60% in the presence of the inhibitor. Furthermore, we found that the size of the aggregated protein fraction in E. chaffeensis increased significantly in cultures supplemented with 0.5 mM GuHCl, which also resulted in the preferential accumulation of ClpB with the aggregated proteins. Altogether, our results suggest that an exposure of E. chaffeensis to the stressful environment of a host cell results in an increased aggregation of the pathogen's proteins, which is exacerbated upon inhibition of ClpB. Our studies establish a link between protein quality control and pathogen survival during infection of a host.

The effect of protein acetylation on the formation and processing of inclusion bodies and endogenous protein aggregates in Escherichia coli cells.

BACKGROUND: Acetylation of lysine residues is a reversible post-translational modification conserved from bacteria to humans. Several recent studies have revealed hundreds of lysine-acetylated proteins in various bacteria; however, the physiological role of these modifications remains largely unknown. Since lysine acetylation changes the size and charge of proteins and thereby may affect their conformation, we assumed that lysine acetylation can stimulate aggregation of proteins, especially for overproduced recombinant proteins that form inclusion bodies. RESULTS: To verify this assumption, we used Escherichia coli strains that overproduce aggregation-prone VP1GFP protein. We found that in ΔackA-pta cells, which display diminished protein acetylation, inclusion bodies were formed with a delay and processed faster than in the wild-type cells. Moreover, in ΔackA-pta cells, inclusion bodies exhibited significantly increased specific GFP fluorescence. In CobB deacetylase-deficient cells, in which protein acetylation was enhanced, the formation of inclusion bodies was increased and their processing was significantly inhibited. Similar results were obtained with regard to endogenous protein aggregates formed during the late stationary phase in ΔackA-pta and ΔcobB cells. CONCLUSIONS: Our studies revealed that protein acetylation affected the aggregation of endogenous E. coli proteins and the yield, solubility, and biological activity of a model recombinant protein. In general, decreased lysine acetylation inhibited the formation of protein aggregates, whereas increased lysine acetylation stabilized protein aggregates. These findings should be considered during the designing of efficient strategies for the production of recombinant proteins in E. coli cells.

Lack of intracellular trehalose affects formation of Escherichia coli persister cells.

Persisters are dormant antibiotic-tolerant cells that usually compose a small fraction of bacterial populations. In this work, we focused on the role of trehalose in persister formation. We found that the ΔotsA mutant, which is unable to synthesize trehalose, produced increased levels of persisters in the early stationary phase and under heat stress conditions. The lack of trehalose in the ΔotsA mutant resulted in oxidative stress, manifested by increased membrane lipid peroxidation after heat shock. Stationary ΔotsA cells additionally exhibited increased levels of oxidized proteins and apurinic/apyrimidinic sites in DNA as compared to WT cells. Oxidative stress caused by the lack of trehalose was accompanied by the overproduction of extracellular indole, a signal molecule that has been shown to stimulate persister formation. Our major conclusion is that intracellular trehalose protects E. coli cells against oxidative stress and limits indole synthesis, which in turn inhibits the formation of persisters.

The formation of persister cells in stationary-phase cultures of Escherichia coli is associated with the aggregation of endogenous proteins.

Persister cells (persisters) are transiently tolerant to antibiotics and usually constitute a small part of bacterial populations. Persisters remain dormant but are able to re-grow after antibiotic treatment. In this study we found that the frequency of persisters correlated to the level of protein aggregates accumulated in E. coli stationary-phase cultures. When 3-(N-morpholino) propanesulfonic acid or an osmolyte (trehalose, betaine, glycerol or glucose) were added to the growth medium at low concentrations, proteins were prevented from aggregation and persister formation was inhibited. On the other hand, acetate or high concentrations of osmolytes enhanced protein aggregation and the generation of persisters. We demonstrated that in the E. coli stationary-phase cultures supplemented with MOPS or a selected osmolyte, the level of protein aggregates and persister frequency were not correlated with such physiological parameters as the extent of protein oxidation, culturability, ATP level or membrane integrity. The results described here may help to understand the mechanisms underlying persister formation.

Antibiotics promoting oxidative stress inhibit formation of Escherichia coli biofilm via indole signalling.

Recent studies have revealed that antibiotics can promote the formation of reactive oxygen species which contribute to cell death. In this study, we report that five different antibiotics known to stimulate production of reactive oxygen species inhibited growth of Escherichia coli biofilm. We demonstrated that supression of biofilm formation was mainly a consequence of the increase in the extracellular concentration of indole, a signal molecule which suppresses growth of bacterial biofilm. Indole production was enhanced under antibiotic-mediated oxidative stress due to overexpression of tryptophanase (TnaA), which catalyzes synthesis of indole. We found that DMSO (dimethyl sulfoxide), a hydrogen peroxide scavenger, or the lack of trypthophanase, which catalyzes production of indole, partly restored formation of E. coli biofilm in the presence of antibiotics. In conclusion, these findings confirmed that antibiotics which promote formation of ROS (reactive oxygen species) can inhibit development of E. coli biofilm in an indole-dependent process.

IbpA the small heat shock protein from Escherichia coli forms fibrils in the absence of its cochaperone IbpB.

Small heat shock proteins (sHsps) associate with aggregated proteins, changing their physical properties in such a way that chaperone mediated disaggregation becomes much more efficient. In Escherichia coli two small Hsps, IbpA and IbpB, exist. They are 48% identical at the amino acid level, yet their roles in stabilisation of protein aggregates are quite distinct. Here we analysed the biochemical properties of IbpA. We found that IbpA assembles into protofilaments which in turn form mature fibrils. Such fibrils are atypical for sHsps. Interaction of IbpA with either its cochaperone IbpB or an aggregated substrate blocks IbpA fibril formation.

Small heat shock proteins and protein-misfolding diseases.

Small heat shock proteins (sHsps) are molecular chaperones ubiquitously distributed in numerous species, from bacteria to humans. A conserved C-terminal "alpha-crystallin" domain organized in a beta-sheet sandwich and oligomeric structure are common features of sHsps. sHsps protect cells against many kinds of stresses including heat shock, oxidative and osmotic stress. sHsps recognize unfolded proteins, prevent their irreversible aggregation and facilitate refolding of bound substrates in cooperation with ATP-dependent molecular chaperones (Hsp70/Hsp40). Mammalian sHsps (HSPBs) are multifunctional proteins involved in many cellular processes including those which are not directly related to protein folding and aggregation. HSPBs participate in cell development and cancerogenesis, regulate apoptosis and control cytoskeletal architecture. Recent data revealed that HSPBs also play an important role in membrane stabilization. Mutation in HSPB genes have been identified, which are responsible for the development of cataract, desmin related myopathy and neuropathies. HSPBs are often found as components of protein aggregates associated with protein-misfolding disorders, such as Parkinson's, Alzheimer's, Alexander's and prion diseases. It is supposed that the presence of HSPBs in intra- or extracellular protein deposits is a consequence of the chaperone activity of HSPBs, however more studies are needed to reveal the exact function of HSPBs during the formation (or removal) of disease-related aggregates.

Escherichia coli heat-shock proteins IbpA and IbpB affect biofilm formation by influencing the level of extracellular indole.

The development of Escherichia coli biofilm requires the differential expression of various genes implicated in cell signalling, stress responses, motility and the synthesis of structures responsible for cell attachment. The ibpAB operon is among the stress-response genes most induced during growth of the E. coli biofilm. In this study we demonstrated, to our knowledge for the first time, that the lack of IbpAB proteins in E. coli cells inhibited the formation of biofilm at the air-liquid interface, although it allowed normal planktonic growth. We showed that ibpAB mutant cells experienced endogenous oxidative stress, which might result from a decreased catalase activity. The endogenous oxidative stress in ibpAB cells led to increased expression of tryptophanase, an enzyme which catalyses the synthesis of indole. We demonstrated that the formation of biofilm by the ibpAB mutant was delayed due to the increase in the extracellular concentration of indole, which is known to play the role of a signal molecule, inhibiting biofilm growth.

Role of Escherichia coli heat shock proteins IbpA and IbpB in protection of alcohol dehydrogenase AdhE against heat inactivation in the presence of oxygen.

Escherichia coli small heat shock proteins IbpA and IbpB are molecular chaperones that bind denatured proteins and facilitate their subsequent refolding by the ATP-dependent chaperones DnaK/DnaJ/GrpE and ClpB. In vivo, the lack of IbpA and IbpB proteins results in increased protein aggregation under severe heat stress or delayed removal of aggregated proteins at recovery temperatures. In this report we followed the appearance and removal of aggregated alcohol dehydrogenase, AdhE, in E. coli submitted to heat stress in the presence of oxygen. During prolonged incubation of cells at 50 degrees C, when AdhE was progressively inactivated, we initially observed aggregation of AdhE and thereafter removal of aggregated AdhE. In contrast to previous studies, the lack of IbpA and IbpB did not influence the formation and removal of AdhE aggregates. However, in DeltaibpAB cells AdhE was inactivated and oxidized faster than in wild type strain. Our results demonstrate that IbpA and IbpB protected AdhE against thermal and oxidative inactivation, providing that the enzyme remained soluble. IbpA and IbpB were dispensable for the processing of irreversibly damaged and aggregated AdhE.