Dataset Information

Proteomic analysis of the exoproteome and surfaceome of Lactiplantibacillus plantarum CRL681: mechanisms of competitive exclusion against enterohemorrhagic Escherichia coli

ABSTRACT: The study focuses on the proteomic analysis of the exoproteome and surfaceome of Lactiplantibacillus plantarum CRL681, aiming to explore the molecular mechanisms underlying its antagonistic capacity against enterohemorrhagic Escherichia coli (EHEC). More than 600 proteins were identified by mass spectrometry across both proteomes, of which 285 were characterized as part of the secretome through a detailed analysis with bioinformatics tools and bibliographic references. The analysis of these proteins allowed us to conclude that some of them are involved in functions like energy metabolism, nutrient transport, cell adhesion, genetic information processing, and signaling. Additionally, moonlighting proteins were observed, which possess bacterial adhesion capabilities that may contribute to the L. plantarum CRL681 antagonistic actions against EHEC. This group included ribosomal proteins and peptidoglycan hydrolases, that would be responsible for the EHEC cell lysis observed under in vitro experimental conditions. Furthermore, the co-culture of the CRL681 strain with E. coli revealed differential expression of key proteins, such as the overexpression of glutathione reductase, involved in maintaining redox balance. This proteomic study is a step forward in the understanding of the molecular mechanisms underlying the antagonistic capacity of L. plantarum CRL681 against EHEC.

INSTRUMENT(S):

ORGANISM(S): Escherichia Coli Lactobacillus Plantarum Bacteria

SUBMITTER: Leonardo Albarracin

LAB HEAD: Julio Villena

PROVIDER: PXD059813 | Pride | 2026-02-02

REPOSITORIES: Pride

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Secretome Profiling of Lactiplantibacillus plantarum CRL681 Predicts Potential Molecular Mechanisms Involved in the Antimicrobial Activity Against Escherichia coli O157:H7.

Baillo Ayelen Antonella AA Albarracín Leonardo L Heredia Ojeda Eliana E Elean Mariano M Gong Weichen W Kitazawa Haruki H Villena Julio J Fadda Silvina S

Antibiotics (Basel, Switzerland) 20260117 1

Background/Objectives.Lactiplantibacillus plantarum CRL681 has previously demonstrated a strong antagonistic effect against Escherichia coli O157:H7 in food matrices; however, the molecular mechanisms underlying this activity remain poorly understood. Since initial interactions between beneficial bacteria and pathogens occur mainly at the cell surface and in the extracellular environment, the characterization of the bacterial secretome is essential for elucidating these mech ...[more]

PMID: 41594133

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Project description:EHEC is a food-borne pathogen that colonizes human GI tract and leads to infection. To understand the process of colonization and to decipher if any factors secreted by intestinal epithelial cells help EHEC during the infection process, we studied expression of EHEC virulence gene expression when exposed to intestinal epithelial cell conditioned medium. In our study, we observed that exposure to epithelial cell conditioned medium for 1 h and 3 h increases expression of 32 out of 41 EHEC LEE virulence genes. In addition, expression of the shiga toxin 1 (Stx1) gene is up-regulated at 1 h of exposure. Also, 17 genes encoded by prophage 933W, including those for Stx2, are also upregulated at both time-points. The increase in 933W prophage expression is mirrored by a 2.7-fold increase in intracellular Stx2 phage titers. Consistent with the increase in virulence gene expression, we observed a 5-fold increase in EHEC attachment to epithelial cells when exposed to conditioned medium, suggesting that EHEC utilizes host cell molecules to increase virulence and infectivity. The molecule(s) responsible for increased EHEC virulence is heat-sensitive as heating the conditioned medium to 95oC abolishes the increase in attachment to epithelial cells. A similar decrease was observed when the conditioned medium was treated with proteinase-K to degrade the proteins. The secreted molecule(s) was found to be larger than 3 kDa and strongly suggests that the HCT-8 secreted molecule that increases EHEC virulence and colonization is a protein-based molecule. Affymetrix E. coli Genome 2.0 Arrays were used to determine the changes in EHEC virulence gene expression on exposure to intestinal epithelial cell-secreted factors (through growth in conditioned medium). Overnight cultures of EHEC were diluted in LB medium to a turbidity of 0.1 at 600 nm. The cells were allowed to grow to a turbidity of 1.0 at 600 nm at 37Â°C and the EHEC cells were then resuspended in either fresh or conditioned medium. The cultures were then allowed to grow for 1 h or 3 h before cell pellets were collected by centrifugation and stored at -80Â°C. Total RNA was isolated from the cell pellets (173) and RNA quality was assessed using gel electrophoresis. Escherichia coli Genome 2.0 arrays (Affymetrix, Santa Clara, CA, USA) containing 10,208 probe sets for all 20,366 genes present in four strains of E. coli, including EHEC, were used to profile changes in gene expression using RNA samples for each treatment.

Project description:Two lineages of enterohemorrhagic (EHEC) Escherichia coli O157:H7 (EDL933, Stx1+ and Stx2+) and 86-24 (Stx2+) were investigated in regards to biofilm formation on an abiotic surface. Strikingly, EDL933 strain formed a robust biofilm while 86-24 strain formed no biofilm on either a polystyrene plate or a polyethylene tube. To identify the genetic mechanisms of different biofilm formation in two EHEC strains, DNA microarrays were first performed and phenotypic assays were followed. In the comparison of the EDL933 strain versus 86-24 strain, genes (csgBAC and csgDEFG) involved in curli biosynthesis were significantly induced while genes (trpLEDCB and mtr) involved in indole signaling were repressed. Additionally, a dozen of phage genes were differentially present between two strains. Curli assays using a Congo red plate and scanning electron microscopy corroborate the microarray data as the EDL 933 strain produces a large amount of curli, while 86-24 forms much less curli. Also, the indole production in the EDL933 was 2-times lower than that of 86-24. It was known that curli formation positively regulates and indole negatively regulates biofilm formation of EHEC. Hence, it appears that less curli formation and high indole production in the 86-24 strain are majorly responsible for no biofilm formation. For the microarray experiments, E. coli O157:H7 EDL933 and 86-24 were inoculated in 25 ml of LB in 250 ml shake flasks with overnight cultures that were diluted 1:100. Cells were shaken at 250 rpm and 37°C for an absorbance of 4.0 at 600 nm. Cells were immediately chilled with dry ice and 95% ethanol (to prevent RNA degradation) for 30 sec before centrifugation in 50 ml centrifuge tubes at 13,000 g for 2 min; cell pellets were frozen immediately with dry ice and stored -80°C. RNA was isolated using Qiagen RNeasy mini Kit (Valencia, CA, USA). RNA quality was assessed by Agilent 2100 bioanalyser using the RNA 6000 Nano Chip (Agilent Technologies, Amstelveen, The Netherlands), and quantity was determined by ND-1000 Spectrophotometer (NanoDrop Technologies, Inc., DE, USA).

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Dataset Information

Proteomic analysis of the exoproteome and surfaceome of Lactiplantibacillus plantarum CRL681: mechanisms of competitive exclusion against enterohemorrhagic Escherichia coli

Dataset's files

Publications

Secretome Profiling of <i>Lactiplantibacillus plantarum</i> CRL681 Predicts Potential Molecular Mechanisms Involved in the Antimicrobial Activity Against <i>Escherichia coli</i> O157:H7.

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