Dataset Information

Polysaccharide length affects mycobacterial cell shape and antibiotic susceptibility.

ABSTRACT: Bacteria control the length of their polysaccharides, which can control cell viability, physiology, virulence, and immune evasion. Polysaccharide chain length affects immunomodulation, but its impact on bacterial physiology and antibiotic susceptibility was unclear. We probed the consequences of truncating the mycobacterial galactan, an essential linear polysaccharide of about 30 residues. Galactan covalently bridges cell envelope layers, with the outermost cell wall linkage point occurring at residue 12. Reducing galactan chain length by approximately half compromises fitness, alters cell morphology, and increases the potency of hydrophobic antibiotics. Systematic variation of the galactan chain length revealed that it determines periplasm size. Thus, glycan chain length can directly affect cellular physiology and antibiotic activity, and mycobacterial glycans, not proteins, regulate periplasm size.

SUBMITTER: Justen AM

PROVIDER: S-EPMC7494350 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Polysaccharide length affects mycobacterial cell shape and antibiotic susceptibility.

Justen Alexander M AM Hodges Heather L HL Kim Lili M LM Sadecki Patric W PW Porfirio Sara S Ultee Eveline E Black Ian I Chung Grace S GS Briegel Ariane A Azadi Parastoo P Kiessling Laura L LL

Science advances 20200916 38

Bacteria control the length of their polysaccharides, which can control cell viability, physiology, virulence, and immune evasion. Polysaccharide chain length affects immunomodulation, but its impact on bacterial physiology and antibiotic susceptibility was unclear. We probed the consequences of truncating the mycobacterial galactan, an essential linear polysaccharide of about 30 residues. Galactan covalently bridges cell envelope layers, with the outermost cell wall linkage point occurring at r ...[more]

PMID: 32938674

Similar Datasets

Project description:ATP is a key molecule of cell physiology, but despite its importance, there are currently no methods for monitoring single-cell ATP fluctuations in live bacteria. This is a major obstacle in studies of bacterial energy metabolism, because there is a growing awareness that bacteria respond to stressors such as antibiotics in a highly individualistic manner. Here, we present a method for long-term single-cell tracking of ATP levels in Mycobacterium smegmatis based on a combination of microfluidics, time-lapse microscopy, and Förster resonance energy transfer (FRET)-based ATP biosensors. Upon treating cells with antibiotics, we observed that individual cells undergo an abrupt and irreversible switch from high to low intracellular ATP levels. The kinetics and extent of ATP switching clearly discriminate between an inhibitor of ATP synthesis and other classes of antibiotics. Cells that resume growth after 24 h of antibiotic treatment maintain high ATP levels throughout the exposure period. In contrast, antibiotic-treated cells that switch from ATP-high to ATP-low states never resume growth after antibiotic washout. Surprisingly, only a subset of these nongrowing ATP-low cells stains with propidium iodide (PI), a widely used live/dead cell marker. These experiments also reveal a cryptic subset of cells that do not resume growth after antibiotic washout despite remaining ATP high and PI negative. We conclude that ATP tracking is a more dynamic, sensitive, reliable, and discriminating marker of cell viability than staining with PI. This method could be used in studies to evaluate antimicrobial effectiveness and mechanism of action, as well as for high-throughput screening.New antimicrobials are urgently needed to stem the rising tide of antibiotic-resistant bacteria. All antibiotics are expected to affect bacterial energy metabolism, directly or indirectly, yet tools to assess the impact of antibiotics on the ATP content of individual bacterial cells are lacking. The method described here for single-cell tracking of intracellular ATP in live bacteria has many advantages compared to conventional ensemble-averaged assays. It provides a continuous real-time readout of bacterial ATP content, cell vitality, and antimicrobial mechanism of action with high temporal resolution at the single-cell level. In combination with high-throughput microfluidic devices and automated microscopy, this method also has the potential to serve as a novel screening tool in antimicrobial drug discovery.

Dataset Information

Polysaccharide length affects mycobacterial cell shape and antibiotic susceptibility.

Publications

Polysaccharide length affects mycobacterial cell shape and antibiotic susceptibility.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure