Lack of intracellular trehalose affects formation of Escherichia coli persister cells

Kuczyńska-Wiśnik, Dorota; Stojowska, Karolina; Matuszewska, Ewelina; Leszczyńska, Daria; Algara, María Moruno; Augustynowicz, Mateusz; Laskowska, Ewa

doi:10.1099/mic.0.000012

Cited by 28 publications

(32 citation statements)

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“…These monosaccharides are central metabolites that can be degraded to obtain energy and biosynthetic building blocks or stored as glycogen (Wilson et al , ; Ruhal et al , ). This is consistent with the earlier studies demonstrating that the Δ otsA mutant exhibited an approximately twofold higher level of UDP‐glucose than the WT strain (Böhringer et al , ) and with our previous study showing that early stationary phase Δ otsA cells accumulated more glycogen than WT cells (Kuczyńska‐Wiśnik et al , ). In the absence of trehalose synthesis in Δ otsA cells, the excess glucose‐6‐phosphate could be converted to pyruvate and then to acetyl‐CoA and AcP (Enjalbert et al , ) that is engaged in nonenzymatic lysine acetylation, which is the dominant form of acetylation in E. coli cultures in stationary phase (Kuhn et al , ; Wolfe, ).…”

Section: Resultssupporting

confidence: 93%

“…Although it has been well documented that protein aggregation is influenced by trehalose, our previous studies revealed that the formation of protein aggregates was not affected in Δ otsA cells that are unable to synthesize trehalose. The levels and densities of E. coli protein aggregates isolated by two‐step sucrose gradient ultracentrifugation were comparable in WT and Δ otsA cells (Kuczyńska‐Wiśnik et al , ). It is known that sucrose, similar to trehalose, can stabilize proteins (Ferreira et al , ).…”

Section: Resultsmentioning

confidence: 99%

“…Several studies have suggested that the Δ otsA mutation affects the aggregation of endogenous proteins in E. coli (Singer and Lindquist, ; Schultz et al , ; Leszczynska et al , ) . However, our previous study found that the levels of protein aggregates isolated by sucrose gradient ultracentrifugation were comparable in both wild type (WT) and Δ otsA strains (Kuczyńska‐Wiśnik et al , ). In the present study, using a sucrose‐free alternative method of aggregate isolation, we showed that the lack of trehalose synthesis in Δ otsA cells affects N‐lysine acetylation and leads to enhanced protein aggregation.…”

Section: Introductionmentioning

confidence: 98%

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

Algara

Kuczyńska-Wiśnik

Dębski

et al. 2019

Molecular Microbiology

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Summary 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.

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Section: Resultssupporting

confidence: 93%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

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

Algara

Kuczyńska-Wiśnik

Dębski

et al. 2019

Molecular Microbiology

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“…Clinically, persisters have been found to be important to the pathogenesis of infections, including persistent tuberculosis, reoccurring uropathogenic E. coli infections, and Pseudomonas aeruginosa infections in cystic fibrosis patients (10)(11)(12)(13)(14). The rate of persister formation is much higher in stationary-phase cells than in actively growing cells (10,(15)(16)(17)(18)(19). In addition to increasing rates of persister formation, stationary phase can also increase rates of resistance and tolerance to antibiotics (18,(20)(21)(22)(23).…”

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confidence: 99%

Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase

2017

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Gram-negative bacteria have effective methods of excluding toxic compounds, including a largely impermeable outer membrane (OM) and a range of efflux pumps. Furthermore, when cells become nutrient limited, RpoS enacts a global expression change providing cross-protection against many stresses. Here, we utilized sensitivity to an anionic detergent (sodium dodecyl sulfate [SDS]) to probe changes occurring to the cell's permeability barrier during nutrient limitation. Escherichia coli is resistant to SDS whether cells are actively growing, carbon limited, or nitrogen limited. In actively growing cells, this resistance depends on the AcrAB-TolC efflux pump; however, this pump is not necessary for protection under either carbon-limiting or nitrogen-limiting conditions, suggesting an alternative mechanism(s) of SDS resistance. In carbon-limited cells, RpoS-dependent pathways lessen the permeability of the OM, preventing the necessity for efflux. In nitrogen-limited but not carbon-limited cells, the loss of rpoS can be completely compensated for by the AcrAB-TolC efflux pump. We suggest that this difference simply reflects the fact that nitrogen-limited cells have access to a metabolizable energy (carbon) source that can efficiently power the efflux pump. Using a transposon mutant pool sequencing (Tn-Seq) approach, we identified three genes, sanA, dacA, and yhdP, that are necessary for RpoS-dependent SDS resistance in carbon-limited stationary phase. Using genetic analysis, we determined that these genes are involved in two different envelope-strengthening pathways. These genes have not previously been implicated in stationary-phase stress responses. A third novel RpoS-dependent pathway appears to strengthen the cell's permeability barrier in nitrogen-limited cells. Thus, though cells remain phenotypically SDS resistant, SDS resistance mechanisms differ significantly between growth states. IMPORTANCE Gram-negative bacteria are intrinsically resistant to detergents and many antibiotics due to synergistic activities of a strong outer membrane (OM) permeability barrier and efflux pumps that capture and expel toxic molecules eluding the barrier. When the bacteria are depleted of an essential nutrient, a program of gene expression providing cross-protection against many stresses is induced. Whether this program alters the OM to further strengthen the barrier is unknown. Here, we identify novel pathways dependent on the master regulator of stationary phase that further strengthen the OM permeability barrier during nutrient limitation, circumventing the need for efflux pumps. Decreased permeability of nutrient-limited cells to toxic compounds has important implications for designing new antibiotics capable of targeting Gram-negative bacteria that may be in a growth-limited state.KEYWORDS Escherichia coli, RpoS, efflux pumps, outer membrane, permeability, stationary phase

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“…Recent work on the mechanisms of persister formation suggests that the effects of TA systems are controlled by guanosine pentaphosphate [(p)ppGpp] (11,15), placing persister formation under the control of the stringent response as a result of nutrient exhaustion (reviewed in reference 16). However, protein aggregation (17), trehalose deficiency (18), acid stress (19), biofilm formation (9), the SOS response (20), and quorum sensing (21) are among the additional factors implicated in persister formation. It therefore seems likely that persisters can arise by multiple interconnected pathways and may have selective value in response to stresses or other environmental conditions.…”

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confidence: 99%

(p)ppGpp-Dependent Persisters Increase the Fitness of Escherichia coli Bacteria Deficient in Isoaspartyl Protein Repair

VandenBerg

Ahn

Visick

2016

Appl Environ Microbiol

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The L-isoaspartyl protein carboxyl methyltransferase (PCM) repairs protein damage resulting from spontaneous conversion of aspartyl or asparaginyl residues to isoaspartate and increases long-term stationary-phase survival of Escherichia coli under stress. In the course of studies intended to examine PCM function in metabolically inactive cells, we identified pcm as a gene whose mutation influences the formation of ofloxacin-tolerant persisters. Specifically, a ⌬pcm mutant produced persisters for an extended period in stationary phase, and a ⌬glpD mutation drastically increased persisters in a ⌬pcm background, reaching 23% of viable cells. The high-persister double mutant showed much higher competitive fitness than the pcm mutant in competition with wild type during long-term stationary phase, suggesting a link between persistence and the mitigation of unrepaired protein damage. We hypothesized that reduced metabolism in the high-persister strain might retard protein damage but observed no gross differences in metabolism relative to wild-type or single-mutant strains. However, methylglyoxal, which accumulates in glpD mutants, also increased fitness, suggesting a possible mechanism. High-level persister formation in the ⌬pcm ⌬glpD mutant was dependent on guanosine pentaphosphate [(p)ppGpp] and polyphosphate. In contrast, persister formation in the ⌬pcm mutant was (p)ppGpp independent and thus may occur by a distinct pathway. We also observed an increase in conformationally unstable proteins in the high-persister strain and discuss this as a possible trigger for persistence as a response to unrepaired protein damage. IMPORTANCEProtein damage is an important factor in the survival and function of cells and organisms. One specific form of protein damage, the formation of the abnormal amino acid isoaspartate, can be repaired by a nearly universally conserved enzyme, PCM. PCMdirected repair is associated with stress survival and longevity in bacteria, insects, worms, plants, mice, and humans, but much remains to be learned about the specific effects of protein damage and repair. This paper identifies an unexpected connection between isoaspartyl protein damage and persisters, subpopulations in bacterial cultures showing increased tolerance to antibiotics. In the absence of PCM, the persister population in Escherichia coli bacteria increased, especially if the metabolic gene glpD was also mutated. High levels of persisters in pcm glpD double mutants correlated with increased fitness of the bacteria in a competition assay, and the fitness was dependent on the signal molecule (p)ppGpp; this may represent an alternative pathway for responding to protein damage. I soaspartyl damage to proteins occurs when aspartyl or asparaginyl residues spontaneously isomerize via a succinimide intermediate (1) (Fig. 1), adversely affecting protein function (2). The damage can be repaired when the abnormal isoaspartyl (isoAsp) residue is recognized and methylated by the L-isoaspartyl protein carboxyl methyltransferase (PCM) (Fig. 1)...

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Lack of intracellular trehalose affects formation of Escherichia coli persister cells

Cited by 28 publications

References 65 publications

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

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

Novel RpoS-Dependent Mechanisms Strengthen the Envelope Permeability Barrier during Stationary Phase

(p)ppGpp-Dependent Persisters Increase the Fitness of Escherichia coli Bacteria Deficient in Isoaspartyl Protein Repair

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