Recent studies have pointed out the preventive role of beetroot extracts against cancers and their cytotoxic activity on cancer cells. Among many different natural compounds, these extracts contained betanin and its stereoisomer isobetanin, which belongs to the betalain group of highly bioavailable antioxidants. However, a precise identification of the molecules responsible for this tumor-inhibitory effect was still required. We isolated a betanin/isobetanin concentrate from fresh beetroots, corresponding to the highest purified betanin extract used for studying anticancer activities of these molecules. The cytotoxicity of this betanin-enriched extract was then characterized on cancer and normal cells and we highlighted the death signalling pathways involved. Betanin/isobetanin concentrate significantly decreased cancer cell proliferation and viability. Particularly in MCF-7-treated cells, the expressions of apoptosis-related proteins (Bad, TRAILR4, FAS, p53) were strongly increased and the mitochondrial membrane potential was altered, demonstrating the involvement of both intrinsic and extrinsic apoptotic pathways. Autophagosome vesicles in MCF-7-treated cells were observed, also suggesting autophagic cell death upon betanin/isobetanin treatment. Importantly, the betanin-enriched extract had no obvious effect towards normal cell lines. Our data bring new insight to consider the betanin/isobetanin mix as therapeutic anticancer compound, alone or in combination with classical chemotherapeutic drugs, especially in functional p53 tumors.

The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.

The plasma membrane acts as one of the first lines of defense by establishing a physical barrier against microbes. Nevertheless, bacteria have developed a range of strategies to invade the host tissues efficiently. In this chapter, we focus on this understudied area and describe how bacteria target or redirect host membrane

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC to host tissues is the first step enabling bacterial colonization. Adhesins such as fimbriae and flagella mediate this process. Here, we studied the interaction of the bacterial flagellum with the host cell’s plasma membrane using giant unilamellar vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components, including proteins and glycoproteins. In contrast, with GUVs, we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains, or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild-type EHEC but not of a nonflagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagellum-mediated adhesion to plasma membranes has broad implications for host-pathogen interactions. IMPORTANCE Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues, and form biofilm. Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagellum-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagellar adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts, are important in bacterial adhesion. These findings demonstrate that plasma membrane adhesion via bacterial flagella play a significant role for an important human pathogen. This mechanism represents a promising target for the development of novel antiadhesion therapies.

35The flagellum is the motility device of many bacteria and the long external filament is 36 made of several thousand copies of a single protein, flagellin. While posttranslational 37 modifications of flagellin are common among bacterial pathogens, the role of lysine 38 methylation remained unknown. Here, we show that both flagellins of Salmonella 39 enterica, FliC and FljB, are methylated at surface-exposed lysine residues. A 40 Salmonella mutant deficient in flagellin methylation was outcompeted for gut 41 colonization in a gastroenteritis mouse model. In support, methylation of flagellin 42 promoted invasion of epithelial cells in vitro. Lysine methylation increased the surface 43 hydrophobicity of flagellin and enhanced flagella-dependent adhesion of Salmonella to 44 phosphatidylcholine vesicles and epithelial cells. In summary, posttranslational flagellin 45 methylation constitutes a novel mechanism how flagellated bacteria facilitate adhesion 46 to hydrophobic host cell surfaces and thereby contributes to efficient gut colonization 47 and successful infection of the host. 48 49 50 51 52 53 54 Introduction: 57 The Gram-negative enteropathogen Salmonella enterica uses a variety of strategies to 58 successfully enter and replicate within a host. In this respect, bacterial motility enables 59 the directed movement of the bacteria towards nutrients or the target site of infection. A 60 rotary nanomachine, the flagellum, mediates motility of many bacteria, including 61 Salmonella enterica 1 . Flagella also play a central role in other infection processes, 62 involving biofilm formation, immune system modulation and adhesion 2-4 . 63 The eukaryotic plasma membrane plays an important role in the interaction of 64 flagellated bacteria with host cells during the early stages of infection 5 . The flagella of S. 65 enterica, Escherichia coli and Pseudomonas aeruginosa can function as adhesion 66 molecules 6-8 mediating the contact to various lipidic plasma membrane components, 67 including cholesterol, phospholipids, sulfolipids and the gangliosides GM1 and aGM1 9-68 12 . 69 Structurally, the flagellum consists out of three main parts: the basal body embedded 70 within the inner and outer membranes of the bacterium, a flexible linking structure -the 71 hook, and the long, external filament, which functions as the propeller of the motility 72 device 13 . The filament is formed by more than 20,000 subunits of a single protein, 73 flagellin. Many S. enterica serovars express either of two distinct flagellins, FliC or FljB, 74 in a process called flagellar phase variation 14,15 . FliC-expressing bacteria display a 75 distinct motility behavior on host cell surfaces and a competitive advantage in 76 colonization of the intestinal epithelia compared to FljB-expressing bacteria 16 . However, 77while the structure of FliC has been determined previously 17 , the structure of FljB 78 remained unknown. 130We next aligned the amino acid sequences of FljB and FliC up-and downstream of the 131 identified ɛ-N-methyl-lysine residues (...

The plasma membrane acts as one of the first lines of defense by establishing a physical barrier against microbes. Nevertheless, bacteria have developed a range of strategies to invade the host tissues efficiently. In this chapter, we focus on this understudied area and describe how bacteria target or redirect host membrane

AbstractEnterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC on host tissues is the first step enabling bacterial colonization. Adhesins like fimbriae and flagella mediate this mechanism. Here, we studied the interaction of the bacterial flagellum with the host cell’s plasma membrane using Giant Unilamellar Vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components including proteins and glycoproteins. In contrast, with GUVs we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild type EHEC but not of a non-flagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagella-mediated adhesion to plasma membranes has broad implications to host-pathogen interactions.ImportanceBacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues and form biofilm. Enterohemorrhagic E. coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagella-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagella adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts are important in bacterial adhesion. These findings significantly strengthen plasma membrane adhesion via bacterial flagella in an important human pathogen. This mechanism represents a promising target for the development of novel anti-adhesion therapies.