High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography

Desmarais, Samantha M.; Tropini, Carolina; Miguel, Amanda; Cava, Felipe; Monds, Russell D.; Pedro, Miguel A. de; Huang, Kerwyn Casey

doi:10.1074/jbc.m115.661660

Cited by 33 publications

(32 citation statements)

References 71 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Since we are concerned with the asymmetry between cell faces, we determined the initial lengths and the proportional growth constants for outer faces separately from inner faces. Assuming that PG density remains constant (Desmarais et al, 2015), the amount of old PG along each face serves as a proxy for its length at the time of fluorophore transition. Likewise, the amount of new PG represents the increase in length following this transition.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

A Periplasmic Polymer Curves Vibrio cholerae and Promotes Pathogenesis

Bartlett

Bratton

Duvshani

et al. 2017

Cell

Self Cite

124

View full text Add to dashboard Cite

Summary Pathogenic Vibrio cholerae remains a major human health concern. V. cholerae has a characteristic curved rod morphology, with a longer outer face and a shorter inner face. Previously, the mechanism and function of this curvature were unknown. Here we identify and characterize CrvA, the first curvature determinant in V. cholerae. CrvA self-assembles into filaments at the inner face of cell curvature. Unlike traditional cytoskeletons, CrvA localizes to the periplasm, and thus could be considered a periskeletal element. To quantify how curvature forms, we developed QuASAR (Quantitative Analysis of Sacculus Architecture Remodeling), which measures subcellular peptidoglycan dynamics. QuASAR reveals that CrvA asymmetrically patterns peptidoglycan insertion rather than removal, causing more material insertion into the outer face than the inner face. Furthermore, crvA is quorum regulated and CrvA-dependent curvature increases at high cell density. Finally, we demonstrate that CrvA promotes motility in hydrogels and confers an advantage in host colonization and pathogenesis.

show abstract

Section: Resultsmentioning

confidence: 99%

“…UPLC samples were prepared as described previously (Desmarais et al, 2015). V. cholerae C6706 wildtype and Δ crvA mutants were streaked on Sm500 LB plates.…”

Section: Star Methodsmentioning

confidence: 99%

A Periplasmic Polymer Curves Vibrio cholerae and Promotes Pathogenesis

Bartlett

Bratton

Duvshani

et al. 2017

Cell

Self Cite

124

View full text Add to dashboard Cite

show abstract

“…It is worth noting that our nano-LC strategy uses only 300 ng of PG material, about 30 times less material than a recently published “highly sensitive” UPLC strategy [6]. The relatively short run time (about 50 min) is much faster than traditional offline fractionation (140 min for C. difficile ), and comparable to the UPLC strategy [6], which employs no mass spectrometry and is hence more suited to comparison than to discovery.…”

Section: Resultsmentioning

confidence: 99%

“…The relatively short run time (about 50 min) is much faster than traditional offline fractionation (140 min for C. difficile ), and comparable to the UPLC strategy [6], which employs no mass spectrometry and is hence more suited to comparison than to discovery. Nano-LC-ESI-MS/MS thus appears to be the most effect data acquisition strategy yet devised for PG structural analyses.…”

Section: Resultsmentioning

confidence: 99%

“…Individual fractions are collected, desalted and analysed by MS and MS/MS. Improvement of chromatography equipment has increased the throughput of PG analyses, allowing the separation of several micrograms of muropeptides in less than 30 min [5, 6]. Recent studies have reported PG structural analysis using online LC-ESI-MS [7] or LC-ESI-MS/MS [8–10] for the identification of muropeptides.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Towards an automated analysis of bacterial peptidoglycan structure

2016

View full text Add to dashboard Cite

Peptidoglycan (PG) is an essential component of the bacterial cell envelope. This macromolecule consists of glycan chains alternating N-acetylglucosamine and N-acetylmuramic acid, cross-linked by short peptides containing nonstandard amino acids. Structural analysis of PG usually involves enzymatic digestion of glycan strands and separation of disaccharide peptides by reversed-phase HPLC followed by collection of individual peaks for MALDI-TOF and/or tandem mass spectrometry. Here, we report a novel strategy using shotgun proteomics techniques for a systematic and unbiased structural analysis of PG using high-resolution mass spectrometry and automated analysis of HCD and ETD fragmentation spectra with the Byonic software. Using the PG of the nosocomial pathogen Clostridium difficile as a proof of concept, we show that this high-throughput approach allows the identification of all PG monomers and dimers previously described, leaving only disambiguation of 3–3 and 4–3 cross-linking as a manual step. Our analysis confirms previous findings that C. difficile peptidoglycans include mainly deacetylated N-acetylglucosamine residues and 3–3 cross-links. The analysis also revealed a number of low abundance muropeptides with peptide sequences not previously reported. Graphical AbstractThe bacterial cell envelope includes plasma membrane, peptidoglycan, and surface layer. Peptidoglycan is unique to bacteria and the target of the most important antibiotics; here it is analyzed by mass spectrometry. Electronic supplementary materialThe online version of this article (doi:10.1007/s00216-016-9857-5) contains supplementary material, which is available to authorized users.

show abstract

Bacillus licheniformis peptidoglycan characterization by CZE–MS: Assessment with the benchmark RP‐HPLC‐MS method

et al. 2019

View full text Add to dashboard Cite

Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non‐soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N‐acetyl‐muramidase, cleaving the β1→4 bound between N‐acetylglucosamine and N‐acetylmuramic acid. Here, we report the use of CZE–MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID–MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated.

show abstract

High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography

Cited by 33 publications

References 71 publications

A Periplasmic Polymer Curves Vibrio cholerae and Promotes Pathogenesis

A Periplasmic Polymer Curves Vibrio cholerae and Promotes Pathogenesis

Towards an automated analysis of bacterial peptidoglycan structure

Bacillus licheniformis peptidoglycan characterization by CZE–MS: Assessment with the benchmark RP‐HPLC‐MS method

Contact Info

Product

Resources

About