Biofilm Persisters
Chaminda Jayampath Seneviratne in Microbial Biofilms, 2017
Biofilms demonstrate extensive structural, chemical and biological heterogeneity, containing cells in various physiological states. In response to local environmental conditions, biofilms enrich differentiation of specific phenotypes with increased adaptability [43]. In biofilms, cells within the internal regions often encounter limited access to nutrients and enter into a dormant state [44]. In addition, bacteria trigger a stringent response that promotes cell survival under nutrient-limited conditions. This response is coordinated by RelA- and SpoT-mediated synthesis of the alarmone guanosine tetraphosphate (ppGpp) that massively reprogrammes gene expression via direct interaction with RNA polymerase or indirect σ-factor competition [45]. Indeed, antibiotic tolerance of bacterial biofilm persisters has been closely linked to TA operons, dormancy and stringent response. It has been reported that overexpression of the TA gene yafQ induces multidrug tolerance in E. coli biofilms, and disruption of yafQ reduces the level of persisters in the biofilms, but not in stationary-phase planktonic cells [46]. Inactivation of this stringent response by deletion of RelA and SpoT in P. aeruginosa resulted in a dramatic decrease of persistence in stationary phase and biofilms, and the reduced susceptibility was restored via complementation of the two genes [18]. The requirement of ppGpp for persistence has also been observed in E. coli biofilms, and it is demonstrated that ppGpp induces slow growth and antibiotic tolerance via activation of TA systems through inorganic polyphosphate- and Lon protease-dependent degradation of antitoxins [47].
Eukaryotic Dna-Dependent Rna Polymerases: An Evaluation of Their Role in the Regulation of Gene Expression
Gerald M. Kolodny in Eukaryotic Gene Regulation, 2018
The ease with which yeast cells can be grown in large amounts has encouraged their use as a model for many aspects of eukaryotic biochemistry. Included among these, the regulation of gene expression and the stringent response of yeast have been well characterized. Gross and Pogo129 showed that starvation or cycloheximide treatment had no effects on the levels of extractable enzymes but reduced a-amanitin-resistant and -sensitive nuclear activities by 67% and 50%, respectively. Cycloheximide added after a period of starvation relaxed the inhibition of rRNA synthesis in vivo but not the depressed nuclear RNA polymerase activities. Later work130,131 used a mutant temperature-sensitive with respect to protein synthesis together with starvation and translation inhibitors. Fluctuations (as opposed to labeling kinetics) of nucleotide pools were not extensively investigated although the authors discovered the importance of using uridine rather than uracil in vivo (due to the effects on nucleotide metabolism during stringency). As a result, the quantitative nature of in vivo changes was not clearly demonstrated. The RNA polymerases were not temperature-sensitive in themselves, but the endogenous nuclear activities were; shifting starved cells to the nonper-missive temperature again released rRNA inhibition as did cycloheximide, but again the release was not observed in isolated nuclei in vitro. The interpretation of these observations was that two factors were involved in rRNA synthesis: one switching on and the other switching off. Thus, starvation reduces the production of a positive factor, and complete shutdown of protein synthesis eliminates a negative factor also. No evidence is available concerning the nature of these factors or their sites of action, and their implied involvement is only a preliminary attempt to explain a rather complex series of events. Recent studies using methyl-labeling of RNA has indicated more accurately the degree of inhibition observed at the level of RNA metabolism in vivo,132 and the yeast system continues to offer great promise in the elucidation of stringency in eukaryotic cells.
Biofilm and Quorum Sensing inhibitors: the road so far
Published in Expert Opinion on Therapeutic Patents, 2020
Simone Carradori, Noemi Di Giacomo, Martina Lobefalo, Grazia Luisi, Cristina Campestre, Francesca Sisto
Bacterial biofilms are ordered and structured aggregates described as ubiquitous forms of microbial communities occurring at solid-liquid, solid-air, liquid-liquid and liquid-air interfaces in different ecosystems. Their detection on mucosal linings of various organs, medical devices and wounds stimulated the researches toward these survival strategies employed by bacteria. Over 80% human infections can be related to biofilm presence [1]. They can consist of single microbial cells or co-cultures (10–15% of the total volume) embedded into a highly hydrated and self-produced exopolymeric matrix including microbial biopolymers (polysaccharides, proteins and glycoproteins, nucleic acids, lipids) as key components [2]. Polysaccharides, usually produced as structural elements of the bacterial cell wall and virulence factors, depend on the genetic profile of microorganisms involved and can be released into media (exopolysaccharides, EPS) [3]. The mechanism of resistance to antimicrobials and immune response in biofilm-related infections is different from plasmids, transposons and mutations [4], being strictly connected to (i) physical/chemical diffusion barriers for penetration; (ii) stress response activation; (iii) non-canonical growth and shape of the microorganisms; (iv) active metabolic resistance related to bacterial stringent response; and (v) emergence of new phenotypes (biofilm-related) as subpopulation of dormant cells (persisters). Genetically nonresistant planktonic cells can be killed by antibiotics, whereas when they grow up into a biofilm can be 1000 time more resistant to the same therapeutic arsenal [5].
Mycobacterial biofilms as players in human infections: a review
Published in Biofouling, 2021
Esmeralda Ivonne Niño-Padilla, Carlos Velazquez, Adriana Garibay-Escobar
Bacteria are also protected against environmental stress by a stringent response mediated by the hyperphosphorylated guanine nucleotides ppGpp and pppGpp, together known as (p)ppGpp (alarmone) (Gupta et al. 2015). (p)ppGpp is produced by the hydrolysis of c-di-GMP with HD-GYP PDE activity or through the transfer of pyrophosphate from ATP to GDP in a process mediated by Rel, an alarmone synthetase/hydrolase enzyme that also exhibits catalytic activity on (p)ppGpp to obtain GDP or GTP in return (Polkade et al. 2016; Prusa et al. 2018). The effect of knocking out one of Rel’s domains has been demonstrated, in which its synthetase activity was considered necessary for persistence during chronic infection and its hydrolase activity compromised M. tuberculosis survival in both acute and chronic stages, whereas the lack of the whole enzyme caused defective pellicles and biofilms (Weiss and Stallings 2013). Furthermore, its absence was found to limit the ability of mycobacteria to enter into a dormant state and weakened it against isoniazid treatment in infected mice (Dutta et al. 2019). Consistent with this finding, Rel was found to be involved in lung tubercle lesions, caseous granulomas, and dissemination in guinea pigs, a more accurate model for studying chronic TB infections (Klinkenberg et al. 2010).
The latest advances in β-lactam/β-lactamase inhibitor combinations for the treatment of Gram-negative bacterial infections
Published in Expert Opinion on Pharmacotherapy, 2019
Entasis Therapeutics is a pioneer in the development of antimicrobial niche therapy, their sulbactam-durlobactam (ETX2514) combination is slated to target MDR Acinetobacter spp. (Figure 2 and Table 2). This BL-BLI is in Phase 3 clinical trials for A. baumannii-calcoaceticus complex HABP, VABP, and bacteremia (clinicaltrials.gov identifier: NCT03894046). This is the only BL-BLI combination in development that demonstrates potent antimicrobial activity against Acinetobacter spp., a formidable threat to public health [85,104]. Sulbactam is traditionally known as a BLI, however due to sulbactam’s strong affinity for PBP3 in Acinetobacter spp., this BLI behaves as a BL [105]. Durlobactam inhibits class A, C, and D β-lactamases, thus is able to target the AmpC of Acinetobacter spp. (Acinetobacter-derived cephalosporinase, ADC) as well as the major groups of acquired oxacillinases (i.e., OXA-23-, OXA-24/40-, and OXA-58-families) in Acinetobacter spp [85,104]. Durlobactam also possesses β-lactam properties as it can inhibit PBP2 [85]. The sulbactam-durlobactam combination is effective in neutropenic murine thigh and lung infections models caused by MDR Acinetobacter spp. [85,104]. To identify potential resistance mechanisms to sulbactam-durlobactam, the combination and each drug alone were used to select for resistant mutants. With sulbactam, mutations in pbp3 were identified that result in amino acid substitutions to the PBP3 active site and affect sulbactam binding [106]. Moreover, alterations in the bacterial stringent response occurred, which were correlated with durlobactam exposure [106].
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