Regrowth-delay body as a bacterial subcellular structure marking multidrug-tolerant persisters

Abstract

Bacteria have long been recognized to be capable of entering a phenotypically non-growing persister state, in which the cells exhibit an extended regrowth lag and a multidrug tolerance, thus posing a great challenge in treating infectious diseases. Owing to their non-inheritability, low abundance of existence, lack of metabolic activities, and high heterogeneity, properties of persisters remain poorly understood. Here, we report our accidental discovery of a subcellular structure that we term the regrowth-delay body, which is formed only in non-growing bacterial cells and sequesters multiple key proteins. This structure, that dissolves when the cell resumes growth, is able to be viewed as a marker of persisters. Our studies also indicate that persisters exhibit different depth of persistence, as determined by the status of their regrowth-delay bodies. Our findings imply that suppressing the formation and/or promoting the dissolution of regrowth-delay bodies could be viable strategies for eradicating persisters.

Protein Aggregation is Associated with Acinetobacter baumannii Desiccation Tolerance

“…In yeast, research has shown protein aggregation to be reversible and protect proteins from stresses [54,56]. Recent studies in E. coli suggested potential roles of protein aggregates in stress responses, although it is still unclear whether aggregates are dissolved or passed down to offspring, or if both could happen depending on the situations [72,73]. Based on our results, protein aggregation might protect some of the A. baumannii proteome from damage during desiccation and provide functional proteins for recovery once rehydrated.…”

High-throughput living-cell protein crosslinking analysis uncovers the physiological relevance of forming the “inserted” state of the ATP synthase ε-subunit

“…Protein photocrosslinking coupled with scarless genome-targeted site-directed mutagenesis reveal that the "inserted" state of the ATP synthase ε-subunit could be detected in living bacterial cells overnight cultured in common LB rich medium 5 In an attempt to find out whether the "inserted" state of the ε-subunit of the ATP synthase is actually formed in living cells, we focused on the interactions involving the C-terminal helix (i.e., the α-helix at the C-terminal end) of the ε-subunit. For this purpose, we made use of the photoactivatable unnatural amino acid p-benzoyl-L-phenylalanine (Bpa), which after being genetically incorporated into a target protein and upon UV irradiation would be able to effectively mediate covalent photocrosslinking with the interacting proteins in cells living under any particular experimental conditions (Chin et al, 2002;Zhang et al, 2011;Fu et al, 2013;Wang et al, 2016;Yu et al, 2019;.…”

“…S1b), in which the functioning of the ATP synthase was indispensable for the cells to grow. In addition, for an effective introduction of Bpa into target proteins, here we made use of the E. coli bacterial strain LY928 that we generated by inserting the optimized DNA sequences encoding the orthogonal Bpa-tRNA synthetase and the orthogonal tRNA Bpa at a specifically selected site on the genomic DNA causing hardly any disruption on cellular activities (Wang et al, 2016;Yu et al, 2019).…”

“…S1a). The LY928 bacterial strain utilized here was constructed by integrating the genes encoding the orthogonal amino-acyl tRNA synthetase (aaRS) and orthogonal tRNA, both needed for incorporating the unnatural amino acid Bpa, into its genomic DNA (Wang et al, 2016;Yu et al, 2019). An irradiation of the cells by UV light (for 30 sec to 10 min) would allow Bpa-mediated crosslinking to occur in living cells.…”

“…In this regard, we decided to test our luck by performing in vivo protein photocrosslinking analysis on this C-terminal helix as mediated by the unnatural amino acid Bpa (p-benzoyl-L-phenylalanine), an approach that was initially developed by Schultz and colleagues (Chin et al, 2002), and has been extensively applied in our lab with a few technical modifications and improvements (Fu et al, , 2013Wang et al, 2016;Zhang et al, 2011). This approach recently allowed us to unveil how the FtsZ protein, a key cell division protein that has been extensively studied under in vitro conditions (Matsui et al, 2012;Li et al, 2013), assembles into the dynamic Z-ring structure via novel lateral interactions of its protofilaments in actively dividing living bacterial cells (Guan et al, 2018) but is sequestered in an reversible subcellular structure that we named as the regrowth-delay bodies in non-dividing/non-growing cells (Yu et al, 2019).…”