Surface proteins of Shiga toxin-producing Escherichia coli mediate association with milk fat globules in raw milk.

Introduction: By adhering to host cells and colonizing tissues, bacterial pathogens can successfully establish infection. Adhesion is considered the first step of the infection process and bacterial adhesion to anti-adhesive compounds is now seen as a promising strategy to prevent infectious diseases. Among the natural sources of anti-adhesive molecules, the membrane of milk fat globules (MFGs) is of interest because of its compositional diversity of proteins and glycoconjugates. However, few studies have focused on the bacterial molecules involved in MFG- mediated inhibition of bacterial adhesion to enterocytes. Methods: We used three pathogenic Shiga toxin-producing Escherichia coli (STEC) strains (O26:H11 str. 21765, O157:H7 str. EDL933, and O103:H3 str. PMK5) as models to evaluate whether STEC surface proteins are involved in the affinity of STEC for MFG membrane proteins (MFGMPs). The affinity of STEC for MFGMPs was assessed both indirectly by a natural raw milk creaming test and directly by an adhesion test. Mass spectrometry was used to identify enriched STEC proteins within the protein fraction of MFGMs. Bacterial mutants were constructed and their affinity to MFGs were measured to confirm the role of the identified proteins. Results: We found that free STEC surface proteins inhibit the concentration of the pathogen in the MFG-enriched cream in a strain-dependent manner. Moreover, the OmpA and FliC proteins were identified within the protein fraction of MFGMs. Our results suggest that FliC protein participates in STEC adhesion to MFGMPs but other STEC molecules may also participate. Discussion: For the first time, this study highlighted, the involvement of STEC surface proteins in the affinity for MFGs. The mechanism of STEC-MFG association is still not fully understood but our results confirm the existence of receptor/ligand type interactions between the bacteria and MFGs. Further studies are needed to identify and specify the molecules involved in this interaction. These studies should consider the likely involvement of several factors, including adhesion molecules, and the diversity of each STEC strain.

Serotype-dependent adhesion of Shiga toxin-producing Escherichia coli to bovine milk fat globule membrane proteins.

Shiga toxin-producing Escherichia coli (STEC) are food-borne pathogens that can cause severe symptoms for humans. Raw milk products are often incriminated as vehicule for human STEC infection. However, raw milk naturally contains molecules, such as the milk fat globule membrane and associated proteins, that could inhibit pathogen adhesion by acting as mimetic ligands. This study aimed to: (i) evaluate the capability of STEC cells to adhere to bovine milk fat globule membrane proteins (MFGMPs), (ii) highlight STEC surface proteins associated with adhesion and (iii) evaluate the variation between different STEC serotypes. We evaluated the physicochemical interactions between STEC and milk fat globules (MFGs) by analyzing hydrophobic properties and measuring the ζ-potential. We used a plate adhesion assay to assess adhesion between MFGMPs and 15 Escherichia coli strains belonging to three key serotypes (O157:H7, O26:H11, and O103:H2). A relative quantitative proteomic approach was conducted by mass spectrometry to identify STEC surface proteins that may be involved in STEC-MFG adhesion. The majority of E. coli strains showed a hydrophilic profile. The ζ-potential values were between -3.7 and - 2.9 mV for the strains and between -12.2 ± 0.14 mV for MFGs. Our results suggest that non-specific interactions are not strongly involved in STEC-MFG association and that molecular bonds could form between STEC and MFGs. Plate adhesion assays showed a weak adhesion of O157:H7 E. coli strains to MFGMPs. In contrast, O26:H11 and O103:H2 serotypes attached more to MFGMPs. Relative quantitative proteomic analysis showed that the O26:H11 str. 21,765 differentially expressed five outer membrane-associated proteins or lipoproteins compared with the O157:H7 str. EDL933. This analysis also found strain-specific differentially expressed proteins, including four O26:H11 str. 21,765-specific proteins/lipoproteins and eight O103:H2 str. PMK5-specific proteins. For the first time, we demonstrated STEC adhesion to MFGMPs and discovered a serotype effect. Several outer membrane proteins-OmpC and homologous proteins, intimin, Type 1 Fimbriae, and AIDA-I-that may be involved in STEC-MFG adhesion were highlighted. More research on STEC's ability to adhere to MFGMs in diverse biological environments, such as raw milk cheeses and the human gastrointestinal tract, is needed to confirm the anti-adhesion properties of the STEC-MFG complex.

Shiga Toxin-Producing Escherichia coli and Milk Fat Globules.

Shiga toxin-producing Escherichia coli (STEC) are zoonotic Gram-negative bacteria. While raw milk cheese consumption is healthful, contamination with pathogens such as STEC can occur due to poor hygiene practices at the farm level. STEC infections cause mild to serious symptoms in humans. The raw milk cheese-making process concentrates certain milk macromolecules such as proteins and milk fat globules (MFGs), allowing the intrinsic beneficial and pathogenic microflora to continue to thrive. MFGs are surrounded by a biological membrane, the milk fat globule membrane (MFGM), which has a globally positive health effect, including inhibition of pathogen adhesion. In this review, we provide an update on the adhesion between STEC and raw MFGs and highlight the consequences of this interaction in terms of food safety, pathogen detection, and therapeutic development.