Identification and imaging of peptides and proteins on Enterococcus faecalis biofilms by matrix assisted laser desorption ionization mass spectrometry

Blaze, M T Melvin; Aydın, Burcu; Carlson, Richard A.; Hanley, Luke

doi:10.1039/c2an35922g

Cited by 26 publications

(8 citation statements)

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“…Enolase has been described as a surface associated protein in most streptococci species and shows properties like plasminogen-binding. [ 35 ]. Furthermore, the presence of oxidoreductases and chaperone proteins in oral biofilms has been associated with inflammation [ 36 ].…”

Section: Discussionmentioning

confidence: 99%

Biochemical characterization of extracellular polymeric substances from endodontic biofilms

2018

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Apical periodontitis is frequently associated with the presence of bacteria biofilm, which has an indisputable impact on the prognosis of endodontic therapy due to the high resistance to adverse environmental conditions, chemicals, and antibiotic therapy that characterize bacteria within biofilm. The biofilm matrix acts as a protective shield over the encased microorganisms. The aim of this investigation was to identify the main biochemical components of biofilm matrix from endodontic mono- and dual-species biofilms. Enterococcus faecalis and Actinomyces naeslundii were cultured as mono- and dual-species biofilms for 14 days. Crude extracellular polymeric substances (EPSs) from biofilm matrices were extracted using chemical and physical methods. High-performance liquid chromatography, gas chromatography, and mass spectrometry were used to determine the carbohydrate, protein, and fatty acid components. Chemical analysis of the biofilm matrices revealed that they were mainly composed of stachyose, maltose, and mannose carbohydrates. The protein profile in all biofilm samples showed abundant oxidoreductases and chaperone proteins and some virulence- associated proteins mainly located in the membrane surface. High percentages of saturated and monounsaturated fatty acids were identified in all biofilm matrices, with a major prevalence of palmitic, stearic, and oleic acids. Based on the results, it was possible to obtain for the first time a general overview of the biochemical profile of endodontic biofilm matrices.

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Section: Discussionmentioning

confidence: 99%

Biochemical characterization of extracellular polymeric substances from endodontic biofilms

2018

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“…MALDI IMS has been used successfully for the analysis of small molecules and metabolites within bacterial communities [ 48 – 51 ]. To date, only one other study has utilized MALDI IMS for the direct analysis of protein species within a bacterial community [ 52 ]. M.T.…”

Section: Discussionmentioning

confidence: 99%

Adhesive Fiber Stratification in Uropathogenic Escherichia coli Biofilms Unveils Oxygen-Mediated Control of Type 1 Pili

et al. 2015

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Bacterial biofilms account for a significant number of hospital-acquired infections and complicate treatment options, because bacteria within biofilms are generally more tolerant to antibiotic treatment. This resilience is attributed to transient bacterial subpopulations that arise in response to variations in the microenvironment surrounding the biofilm. Here, we probed the spatial proteome of surface-associated single-species biofilms formed by uropathogenic Escherichia coli (UPEC), the major causative agent of community-acquired and catheter-associated urinary tract infections. We used matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) imaging mass spectrometry (IMS) to analyze the spatial proteome of intact biofilms in situ. MALDI-TOF IMS revealed protein species exhibiting distinct localizations within surface-associated UPEC biofilms, including two adhesive fibers critical for UPEC biofilm formation and virulence: type 1 pili (Fim) localized exclusively to the air-exposed region, while curli amyloid fibers localized to the air-liquid interface. Comparison of cells grown aerobically, fermentatively, or utilizing an alternative terminal electron acceptor showed that the phase-variable fim promoter switched to the “OFF” orientation under oxygen-deplete conditions, leading to marked reduction of type 1 pili on the bacterial cell surface. Conversely, S pili whose expression is inversely related to fim expression were up-regulated under anoxic conditions. Tethering the fim promoter in the “ON” orientation in anaerobically grown cells only restored type 1 pili production in the presence of an alternative terminal electron acceptor beyond oxygen. Together these data support the presence of at least two regulatory mechanisms controlling fim expression in response to oxygen availability and may contribute to the stratification of extracellular matrix components within the biofilm. MALDI IMS facilitated the discovery of these mechanisms, and we have demonstrated that this technology can be used to interrogate subpopulations within bacterial biofilms.

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“…We looked for the tentative sequence of the proteins/peptides from the common protein bands obtained from the biofilms of representative isolates of K. pneumoniae as described earlier for Enterococcus faecalis with modifications (Aydin et al, 2012). Briefly, the common protein bands of interest were excised from SDS-PAGE gel and destained thrice in 50% acetonitrile (ACN)/40 mM ammonium bicarbonate (pH 7.4), until the gel pieces became translucent white prior to digestion.…”

Section: Methodsmentioning

confidence: 99%

Classification of Clinical Isolates of Klebsiella pneumoniae Based on Their in vitro Biofilm Forming Capabilities and Elucidation of the Biofilm Matrix Chemistry With Special Reference to the Protein Content

et al. 2019

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Klebsiella pneumoniae is a human pathogen, capable of forming biofilms on abiotic and biotic surfaces. The limitations of the therapeutic options against Klebsiella pneumoniae is actually due to its innate capabilities to form biofilm and harboring determinants of multidrug resistance. We utilized a newer approach for classification of biofilm producing Klebsiella pneumoniae isolates and subsequently we evaluated the chemistry of its slime, more accurately its biofilm. We extracted and determined the amount of polysaccharides and proteins from representative bacterial biofilms. The spatial distribution of sugars and proteins were then investigated in the biofilm matrix using confocal laser scanning microscopy (CLSM). Thereafter, the extracted matrix components were subjected to sophisticated analysis incorporating Fourier transform infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, one-dimensional gel-based electrophoresis (SDS-PAGE), high performance liquid chromatography (HPLC), and MALDI MS/MS analysis. Besides, the quantification of its total proteins, total sugars, uronates, total acetyl content was also done. Results suggest sugars are not the only/major constituent of its biofilms. The proteins were harvested and subjected to SDS-PAGE which revealed various common and unique protein bands. The common band was excised and analyzed by HPLC. MALDI MS/MS results of this common protein band indicated the presence of different proteins within the biofilm. The 55 different proteins were identified including both cytosolic and membrane proteins. About 22 proteins were related to protein synthesis and processing while 15 proteins were identified related to virulence. Similarly, proteins related to energy and metabolism were 8 and those related to capsule and cell wall synthesis were 4. These results will improve our understanding of Klebsiella biofilm composition and will further help us design better strategies for controlling its biofilm such as techniques focused on weakening/targeting certain portions of the slime which is the most common building block of the biofilm matrix.

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Identification and imaging of peptides and proteins on Enterococcus faecalis biofilms by matrix assisted laser desorption ionization mass spectrometry

Cited by 26 publications

References 50 publications

Biochemical characterization of extracellular polymeric substances from endodontic biofilms

Biochemical characterization of extracellular polymeric substances from endodontic biofilms

Adhesive Fiber Stratification in Uropathogenic Escherichia coli Biofilms Unveils Oxygen-Mediated Control of Type 1 Pili

Classification of Clinical Isolates of Klebsiella pneumoniae Based on Their in vitro Biofilm Forming Capabilities and Elucidation of the Biofilm Matrix Chemistry With Special Reference to the Protein Content

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