Biology subjects

Quintero-Yanes, A.

Publications and source records attributed to Quintero-Yanes, A..

3 recordsLinked to original sources

bioRxiv2024

Multiplexing bacteriocin synthesis to kill and prevent antimicrobial resistance

Antibiotic resistance represents an emergency for global public health. This calls for using alternative drugs and developing innovative therapies based on a clear understanding of their mechanisms of action and resistance in bacteria. Bacteriocins represent a unique class of natural molecules selectively eliminating bacteria. These secreted proteins exhibit a narrower spectrum of activity compared to conventional broad-spectrum antimicrobials by interacting with specific protein and lipid receptors on bacterial cell envelopes. Despite their diverse molecular structures, the commonality of being genetically encoded makes bacteriocins amenable to synthetic biology design. In using cell-free gene expression (CFE) and continuous-exchange CFE (CECFE), we produced controlled combinations (cocktails) of bacteriocins in single synthesis reactions for the first time. A first set of bacteriocin cocktails comprising both linear and circular proteins allowed the targeting of different bacterial species. Other cocktails were designed to target one bacterial species and considering bacteriocins pathways to cross the cell-envelope. Such combinations demonstrated efficient bacterial eradication and prevention of resistance. We illustrate the effectiveness of these bacteriocin mixtures in eradicating various human pathogenic-multiresistant--isolates. Finally, we highlight their potential as targeted and versatile tools in antimicrobial therapy by testing a combination of bacteriocins for treatment in vivo in the animal model Galleria mellonella.

synthetic biology↗

bioRxiv2023

Regulation of potassium homeostasis in Caulobacter crescentus

Potassium (K+) is an essential physiological element determining membrane potential, intracellular pH, osmotic/turgor pressure, and protein synthesis in cells. Nevertheless, K+ homeostasis remains poorly studied in bacteria. Here we describe the regulation of potassium uptake systems in the oligotrophic -proteobacterium Caulobacter crescentus known as a model for asymmetric cell division. We show that C. crescentus can grow in concentrations from the micromolar to the millimolar range by essentially using two K+ transporters to maintain potassium homeostasis, the low affinity Kup and the high affinity Kdp uptake systems. When K+ is not limiting, we found that the kup gene is essential while kdp inactivation does not impact the growth. In contrast, kdp becomes critical but not essential and kup dispensable for growth in K+-limited environments. However, in the absence of kdp, mutations in kup were selected to improve growth in K+-depleted conditions, likely by improving the affinity of Kup for K+. In addition, mutations in the KdpDE two-component system, which regulates kdpABC expression, suggest that the inner membrane sensor regulatory component KdpD works as a kinase in early stages of growth and as a phosphatase to regulate transition into stationary phase. Our data also show that KdpE is not only phosphorylated by KdpD but also by another non-cognate histidine kinase. On top of this, we determined the KdpE-dependent and independent K+ transcriptome and identified the direct targets of KdpE. Together, our work illustrates how an oligotrophic bacterium responds to fluctuation in K+ availability. ImportancePotassium (K+) is a key metal ion involved in many essential cellular processes. Its transport and regulation have been mainly studied in the bacterial model species Escherichia coli and Bacillus subtilis. Here we show that the oligotroph Caulobacter crescentus can support growth at lower K+ concentrations by mainly using two K+ uptake systems, the low-affinity Kup and the high-affinity Kdp. Interestingly, in the absence of Kdp, point mutations in Kup was selected to increase affinity for K+, which improved growth in K+-depleted conditions. Using genome-wide approaches, we also determined the entire set of genes required for C crescentus to survive at low K+ concentration as well as the full K+-dependent regulon. Finally, we found that the transcriptional regulation mediated by the KdpDE two-component system is unconventional since unlike E. coli, the inner membrane sensor regulatory component KdpD works rather as a phosphatase on the phosphorylated response regulator KdpE[~]P. To our knowledge, this is the first comprehensive study of K+ homeostasis in bacteria.

microbiology↗

bioRxiv2022

The two-component system ChvGI maintains cell envelope homeostasis in Caulobacter crescentus

Two-component systems (TCS) are often used by bacteria to rapidly assess and respond to environmental changes. The ChvG/ChvI (ChvGI) TCS conserved in -proteobacteria is known for regulating expression of genes related to exopolysaccharide production, virulence and growth. The sensor kinase ChvG autophosphorylates upon yet unknown signals and phosphorylates the response regulator ChvI to regulate transcription. Recent studies in Caulobacter crescentus showed that chv mutants are sensitive to vancomycin treatment and fail to grow in synthetic minimal media. In this work, we identified the osmotic imbalance as the main cause of growth impairment in synthetic minimal media. We also determined the ChvI regulon and found that ChvI regulates cell envelope architecture by controlling outer membrane, peptidoglycan assembly/recycling and inner membrane proteins. In addition, we found that ChvI phosphorylation is also activated upon antibiotic treatment with vancomycin. We also challenged chv mutants with other cell envelope related stress and found that treatment with antibiotics targeting transpeptidation of peptidoglycan during cell elongation impairs growth of the mutant. Moreover, these antibiotics activate expression of the chvIG-hprK operon. Finally, we observed that the sensor kinase ChvG relocates from a patchy-spotty distribution to distinctive foci after transition from complex to synthetic minimal media. Interestingly, this pattern of (re)location has been described for proteins involved in cell growth control and peptidoglycan synthesis upon osmotic shock. Overall, our data support that the ChvGI TCS is mainly used to monitor and respond to osmotic imbalances and damages in the peptidoglycan layer to maintain cell envelope homeostasis. ImportanceThe cell envelope is the first barrier protecting cells from harsh environmental conditions, such as temperature, pH, oxidative and osmotic imbalances. It is also an obstacle for the intake of antibiotics targeting essential cellular processes. Therefore, molecular components and systems responding to cell envelope stress and maintaining cell envelope homeostasis are important targets for drug therapy. Here we show that the two-component system ChvGI, highly conserved in free-living and pathogenic -proteobacteria, is activated upon osmotic upshift and treatment with antibiotics targeting peptidoglycan synthesis to activate the transcription of multiple genes involved in cell envelope homeostasis in Caulobacter crescentus. We also show that the kinase sensor ChvG displays a dynamic localisation pattern that changes depending on osmotic imbalance. To our knowledge this is the first two-component system reported to change his cellular localisation upon environmental stress.

microbiology↗