Abstract:
Chronic infections that can relapse after recurrent courses of antibiotic therapy are a major biomedical problem. Bacteria are able to survive prolonged antibiotic treatments not only by acquiring resistance through genetic mutations, but also by exhibiting persistent phenotype in a small subpopulation of genetically uniform cells. These rare phenotypically distinct cells, known as persisters, become transiently tolerant to antibiotics by restraining their growth and entering a dormant-like physiological state. After antibiotic removal, persister cells can sense improved conditions and resume their growth to produce the same phenotypically heterogeneous population that contains a small fraction of drug-tolerant cells. The emergence of persisters is triggered by the activation of so-called toxin-antitoxin (TA) genetic modules, composed of two genes: one encoding a toxin that interferes with essential cellular processes, and another encoding an antitoxin that inhibits toxin activity. One of Escherichia coli toxins is a serine/threonine protein kinase HipA that promotes antibiotic tolerance through phosphorylation of the glutamate-tRNA ligase (GltX), causing a halt in translation, inhibition of growth and induction of persistence. Toxic activity of HipA can be counteracted by the corresponding antitoxin protein HipB through a HipA-HipB interaction and by transcriptional repression of the hipBA operon. The first gene associated to bacterial persistence was identified by the isolation of a gain-of-function allele hipA7 from E. coli. The hipA7 gene encodes a HipA variant, HipA7, that increases persistence markedly compared to the wild-type HipA due to two amino acid substitutions (G22S and D291A) and has therefore been widely used as a model for studying persistence. Whereas ectopically expressed hipA inhibits growth and induces persistence, hipA7 expression does not inhibit growth, yet leads to equal increase in persistence as the wild-type HipA. Based on this, it was suggested that growth inhibition and persistence are separate phenotypes caused by two distinct functions of HipA, which could be explained by different substrate specificities of the two kinase variants. Moreover, because HipA affects multiple cellular functions, primarily protein and RNA synthesis, it is likely that this kinase has more than one substrate. Such relaxed substrate specificity is often seen for other bacterial kinases. To investigate the differences in HipA- and HipA7-mediated growth inhibition and persistence in vivo, I employed a stable isotope labeling by amino acids in cell culture (SILAC)-based quantitative phosphoproteomic workflow in combination with high-resolution mass spectrometry. When mildly produced from plasmids, both HipA and HipA7 phosphorylated GltX as the main substrate, which is likely the primary determinant of persistence. Unlike HipA7, HipA phosphorylated several additional substrates involved in translation, transcription, and replication, such as ribosomal protein L11 (RplK) and the negative modulator of replication initiation SeqA. Conversely, HipA7 exhibited reduced kinase activity in vitro and phosphorylated only a few additional substrates in vivo only when it was highly overproduced from the plasmid. Furthermore, in the model where hipA7 was expressed from the chromosome, it lead to the increase in GltX phosphorylation compared to wild-type hipA, providing a direct evidence that HipA7 targets GltX in vivo. Under these conditions, phage shock protein PspA was detected as an additional potential substrate of HipA7. To determine the influence of novel phosphorylation events detected in this study on persistence, the function of RplK site-specific phosphorylation was further investigated. However, initial testing did not reveal connection between phosphorylated RplK and persistence dependent on the activity of the GTP pyrophosphokinase RelA. Taken together, this study shows that HipA and HipA7 differ substantially in their kinase activities and substrate pools, which may contribute to their distinct phenotypes. Moreover, these results contribute to understanding of molecular mechanisms of HipA and HipA7. In addition, the phosphoproteome data obtained here yielded a comprehensive collection of phosphorylation events in E. coli and can thus serve as a valuable resource for further studies of phosphoregulation in bacteria. Except for investigating signaling events related to HipA-mediated growth inhibition, the second aim of this work was to establish a method for studying turnover of individual proteins during persistence and resuscitation on a system-wide scale. To that end, I performed a time-resolved analysis of protein abundances during HipA-induced persistence and HipB-mediated resuscitation by implementing a dynamic SILAC pulse-labeling approach in conjunction with MS-based proteomic analysis. This method enabled to selectively label persister cells during antibiotic treatment and determine half-lives of several hundred proteins synthesized under growth-inhibited conditions. Accordingly, analysis of newly synthesized proteins revealed that persistence was characterized by reduced metabolism, cell division and cellular respiration. Conversely, proteins involved in general stress response and translation exhibited higher abundance and turnover. The same methodology was then applied to persistence induced by another toxin, mRNase RelE, in order to investigate common signature of toxin regulation. Indeed, a high overlap between two experiments was observed, yielding a set of proteins that are actively produced during persistence and therefore likely involved in the maintenance of the persistent state. Notably, proteins involved in stress response, protein folding, protein degradation, RNA production and ribosome biogenesis were newly synthesized during persistence in both models. Although mechanisms of persister formation are relatively well understood, much less is known about molecular processes that provoke their resuscitation, a state that is greatly responsible for the reactivation of the disease. Therefore, pulse-labeling was applied to resuscitating persister cells and the resulting data set revealed a number of proteins, the synthesis of which was triggered at the early stage of the wake-up process, including a positive control, antitoxin HipB. In contrast to persistence, resuscitation was characterized by increased metabolism orientated towards energy production and biosynthesis of amino acids. Altogether, this study shows that the dynamic SILAC approach applied to the model of toxin and antitoxin expression could be used as a general strategy for studying newly synthesized proteins in the context of persistence and resuscitation.
bacterial persistence
Print-on-Demand