Spatiotemporal Monitoring of the Antibiome Secreted by Bacillus Biofilms on Plant Roots Using MALDI Mass Spectrometry Imaging

Some soil <i>Bacilli</i> living in association with plant roots can protect their host from infection by pathogenic microbes and are therefore being developed as biological agents to control plant diseases. The plant-protective activity of these bacteria has been correlated with the potential to secrete a wide array of antibiotic compounds upon growth as planktonic cells in isolated cultures under laboratory conditions. However, in situ expression of these antibiotics in the rhizosphere where bacterial cells naturally colonize root tissues is still poorly understood. In this work, we used matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) to examine spatiotemporal changes in the secreted antibiome of Bacillus amyloliquefaciens developing as biofilms on roots. Nonribosomal lipopeptides such as the plant immunity elicitor surfactin or the highly fungitoxic iturins and fengycins were readily produced albeit in different time frames and quantities in the surrounding medium. Interestingly, tandem mass spectrometry (MS/MS) experiments performed directly from the gelified culture medium also allowed us to identify a new variant of surfactins released at later time points. However, no other bioactive compounds such as polyketides were detected at any time, strongly suggesting that the antibiome expressed in planta by B. amyloliquefaciens does not reflect the vast genetic arsenal devoted to the formation of such compounds. This first dynamic study reveals the power of MALDI MSI as tool to identify and map antibiotics synthesized by root-associated bacteria and, more generally, to investigate plant–microbe interactions at the molecular level

Spectroscopic analysis of non-steatotic hepatocytes on fatty liver.

<p>Spectroscopic analyses were performed on periportal hepatocytes on tissue section from normal or fatty liver. The video image is shown (left panel) with the corresponding averaged IR spectra (right panel) and the chemical imaging of the sum of DAG (middle panel).</p

Assignment of frequency to chemical functions.

<p>From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007408#pone.0007408-Dreissig1" target="_blank">[19]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007408#pone.0007408-Banyay1" target="_blank">[20]</a>.</p

Histological features of steatosis.

<p>Tissue sections of 6 µm thickness were performed on paraffin embedded biopsies from normal liver or from fatty liver and stained with HES (hematoxylin, eosin and safran). Normal hepatic lobule without steatosis (left panel) or fatty liver area exhibiting macrovacuolar and microvesicular steatosis (right panel) are shown. Upper panel: ×100, lower panel: ×400. PT: portal tract, BD: biliary duct, PV: portal vein, HA: hepatic artery, CLV: centrilobular vein, SV: steatotic vacuole.</p

History of patients and origin of samples.

<p>History of patients and origin of samples.</p

Analysis of steatosis using synchrotron FTIR microspectroscopy.

<p>A) Optical image of steatotic hepatocytes containing steatotic vesicles (white star) and non-steatotic hepatocytes (black star). B) Averaged IR spectra recorded inside steatotic vesicles (upper spectrum in blue) or on non-steatotic hepatocytes (lower spectrum in red). The band corresponding to olefin (3000–3060 cm⁻¹) is labelled by a black arrow. C) Chemical imaging of some bands on the tissue section.</p

Second derivatives of IR spectra.

<p>Spectra recorded on steatosis or non-steatotic hepatocytes were superimposed (upper panel). Second derivatives of the spectra were calculated and superimposed in the frequency domain 2600–3200 cm⁻¹ (lower panel).</p

Selected Protein Monitoring in Histological Sections by Targeted MALDI-FTICR In-Source Decay Imaging

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) is a rapidly growing method in biomedical research allowing molecular mapping of proteins on histological sections. The images can be analyzed in terms of spectral pattern to define regions of interest. However, the identification and the differential quantitative analysis of proteins require off line or in situ proteomic methods using enzymatic digestion. The rapid identification of biomarkers holds great promise for diagnostic research, but the major obstacle is the absence of a rapid and direct method to detect and identify with a sufficient dynamic range a set of specific biomarkers. In the current work, we present a proof of concept for a method allowing one to identify simultaneously a set of selected biomarkers on histological slices with minimal sample treatment using in-source decay (ISD) MSI and MALDI-Fourier transform ion cyclotron resonance (FTICR). In the proposed method, known biomarkers are spotted next to the tissue of interest, the whole MALDI plate being coated with 1,5-diaminonaphthalene (1,5-DAN) matrix. The latter enhances MALDI radical-induced ISD, providing large tags of the amino acid sequences. Comparative analysis of ISD fragments between the reference spots and the specimen in imaging mode allows for unambiguous identification of the selected biomarker while preserving full spatial resolution. Moreover, the high resolution/high mass accuracy provided by FTICR mass spectrometry allows the identification of proteins. Well-resolved peaks and precise measurements of masses and mass differences allow the construction of reliable sequence tags for protein identification. The method will allow the use of MALDI-FTICR MSI as a method for rapid targeted biomarker detection in complement to classical histology