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Whole human saliva proteins were untreated or StcE treated and separated by SDS-PAGE. (A) Coomassie-stained gel. Two large-molecular-weight protein bands that changed as a result of rStcE′ treatment are indicated by arrowheads. (B and C) Immunoblots probed with antiserum against MUC7 (B) and antibody against gp340 (C). Lanes in all three panels: 1, rStcE′; 2, untreated saliva; 3, rStcE′-treated saliva.
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Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a diarrheal pathogen that causes attaching and effacing (A/E) lesions on intestinal epithelial cells. Strains
of the O157 serogroup carry the large virulence plasmid pO157, which encodes the etp type II secretion system that secretes the genetically linked zinc metalloprotease StcE. The Ler regul...
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Escherichia coli O157:H7 is a source of foodborne illness, causing diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome. E. coli O157:H7 secretes, via the etp type II secretion system, a metalloprotease, StcE, that specifically cleaves the serpin C1 esterase inhibitor. We determined
by hybridization techniques the prevalence of stcE and etp...
Citations
... Mucinases are important for penetrating mucus barriers so that bacteria reach underlying epithelia and execute virulence activities 26,56 . A prime example for this is the StcE enzyme initially found in EHEC O157:H7 49 . ...
Microbes utilize polysaccharides to protect their surfaces and build biofilms, whereas metazoans employ large mucins densely decorated with O-glycans to protect surfaces and keep microbes at a distance. However, gut microbes in mucus also feed on host mucins, thus imposing a need for continuous renewal to maintain protection, clearance and mucus homeostasis. Glycopeptidases that can cleave mucins are known, but mucinases that specifically cleave mucins are not. Here we report the discovery of such microbial mucinases that cleave mucins with trimmed glycans, recognize dense clusters of O-glycans, and employ a structural fold and catalytic machinery reminiscent of glycan hydrolases and peptidases. These di-glutamate mucinases are also found in eukaryotes, and we propose that they are designed to clear mucins following scavenging of O-glycans to promote healthy gut–microbiome homeostasis.
... Beyond all YenTc components, a set of other proteins showed a similar fold change (FC) (log 2 FC < −3 and false discovery rate (FDR) < 1%; Supplementary Table 1) when compared with the cell culture grown at 25 °C. These included potential virulence factors, such as Chi3 (chitinase) 31 , Cbp (chitin-binding protein) 31 , PirA (insect-related toxin) 32 , Pil36 (pilin), NucA (nuclease) 33 , Tlh (hemolysin) 34 , M66/StcE (metalloprotease) [35][36][37][38][39] and a regulator of the lysis cassette (OmpR/PhoB-containing protein) 30 . WT cells showed the same trend of the aforementioned proteomic change (log 2 FC < −3, FDR < 1%) upon temperature decrease, but in addition also revealed the upregulation of the endolysin (A0A3S6F4L4) from the LC (log 2 < −2 and FDR < 10%; Supplementary Table 2 and Extended Data Fig. 9). ...
... Altogether, we show that filaments are composed of the protein A0A3S6EYX4, having similarities to metalloprotease M66. Interestingly, the homologue StcE represents a secreted virulence factor in enterohemorrhagic Escherichia coli (EHEC) [35][36][37][38]46 . ...
... Alternative to the processing of bacterial proteins, the target of M66 could be components of the host. In fact, M66/StcE homologues function as a virulence factor in EHEC [35][36][37][38][39] and are also found in Vibrio 58 , Enterovibrio, Aliivibrio, Pseudomonas, Pectobacterium, Shewanella and Aeromonas species. In EHEC, StcE is secreted via the Type II Secretion System and supports penetration into the host and adherence to epithelial gut cells 36,37,39,46 by proteolytic remodelling of the mucosal lining 35,38 . ...
Tc toxins are virulence factors of bacterial pathogens. Although their structure and intoxication mechanism are well understood, it remains elusive where this large macromolecular complex is assembled and how it is released. Here we show by an integrative multiscale imaging approach that Yersinia entomophaga Tc (YenTc) toxin components are expressed only in a subpopulation of cells that are ‘primed’ with several other potential virulence factors, including filaments of the protease M66/StcE. A phage-like lysis cassette is required for YenTc release; however, before resulting in complete cell lysis, the lysis cassette generates intermediate ‘ghost’ cells, which may serve as assembly compartments and become packed with assembled YenTc holotoxins. We hypothesize that this stepwise mechanism evolved to minimize the number of cells that need to be killed. The occurrence of similar lysis cassettes in diverse organisms indicates a conserved mechanism for Tc toxin release that may apply to other extracellular macromolecular machines.
... 72 Finally, the zinc metalloproteases StcE and SslE secreted by E. coli EHEC and EPEC/ETEC respectively, contribute to intimate adherence of these bacteria to host cells, a process that is essential for colonization. [73][74][75][76][77][78] Other proteases have been described as virulence factors in gastroenteritis. As H. pylori, Salmonella typhimurium and Campylobacter jejuni, a bacterium responsible for foodborne infections, interact with the host cell epithelium and establish infection by HtrA. ...
... 10,68,69,71 StcE and SslE, metalloproteases from diarrheagenic E. coli strains, also cleave mucin glycoproteins, which may help the pathogen to reach the epithelium. [74][75][76] In addition, the M60like protease family cleaves the mucin glycoprotein backbone in a manner that is dependent on the presence of specific glycan sidechain structures. 125,126 Many pathogens express this family of proteases for host invasion. ...
Proteases are an evolutionarily conserved family of enzymes that degrade peptide bonds and have been implicated in several common gastrointestinal (GI) diseases. Although luminal proteolytic activity is important for maintenance of homeostasis and health, the current review describes recent advances in our understanding of how overactivity of luminal proteases contributes to the pathophysiology of celiac disease, irritable bowel syndrome, inflammatory bowel disease and GI infections. Luminal proteases, many of which are produced by the microbiota, can modulate the immunogenicity of dietary antigens, reduce mucosal barrier function and activate pro-inflammatory and pro-nociceptive host signaling. Increased proteolytic activity has been ascribed to both increases in protease production and decreases in inhibitors of luminal proteases. With the identification of strains of bacteria that are important sources of proteases and their inhibitors, the stage is set to develop drug or microbial therapies to restore protease balance and alleviate disease.
... Such is the case for the enterohemorrhagic Escherichia coli (EHEC) that expresses the powerful protease C1 esterase inhibitor, better known as StcE, which strongly degrades mucin glycans (mucinase; Malaker et al., 2019). StcE mediates EHEC pathogenesis by harshly degrading mucins in the mucosal lining of the human gut, exposing the epithelia to the pathogen (Grys et al., 2005). Other pathogens express similar virulent mucinases that contribute to disease states (Sauvaitre et al., 2022). ...
In the human gastrointestinal tract, the gut mucosa and the bacterial component of the microbiota interact and modulate each other to accomplish a variety of critical functions. These include digestion aid, maintenance of the mucosal barrier, immune regulation, and production of vitamins, hormones, and other metabolites that are important for our health. The mucus lining of the gut is primarily composed of mucins, large glycosylated proteins with glycosylation patterns that vary depending on factors including location in the digestive tract and the local microbial population. Many gut bacteria have evolved to reside within the mucus layer and thus encode mucus-adhering and -degrading proteins. By doing so, they can influence the integrity of the mucus barrier and therefore promote either health maintenance or the onset and progression of some diseases. The viral members of the gut – mostly composed of bacteriophages – have also been shown to have mucus-interacting capabilities, but their mechanisms and effects remain largely unexplored. In this review, we discuss the role of bacteriophages in influencing mucosal integrity, indirectly via interactions with other members of the gut microbiota, or directly with the gut mucus via phage-encoded carbohydrate-interacting proteins. We additionally discuss how these phage-mucus interactions may influence health and disease states.
... Ler regulates transcription of LEE2, LEE3, LEE4, and LEE5 operons (Figs. 1 and 2) (Elliott et al., 2000). In addition, Ler regulates the non-LEE stcE gene that encodes a metalloprotease (Elliott et al., 2000;Lathem et al., 2002;Li et al., 2004;Grys et al., 2005;Hews et al., 2017) that facilitates intimate adherence of EHEC to the host cell (Grys et al., 2005). Mutation of ler blocks the expression of T3SS proteins, whereas complementation restores it, suggesting that Ler regulates all vital genes essential to form AE lesions; thus, Ler protein is a global regulator of virulence genes in EHECs LEE (Figs. 1 and 2) (Elliott et al., 2000). ...
... Ler regulates transcription of LEE2, LEE3, LEE4, and LEE5 operons (Figs. 1 and 2) (Elliott et al., 2000). In addition, Ler regulates the non-LEE stcE gene that encodes a metalloprotease (Elliott et al., 2000;Lathem et al., 2002;Li et al., 2004;Grys et al., 2005;Hews et al., 2017) that facilitates intimate adherence of EHEC to the host cell (Grys et al., 2005). Mutation of ler blocks the expression of T3SS proteins, whereas complementation restores it, suggesting that Ler regulates all vital genes essential to form AE lesions; thus, Ler protein is a global regulator of virulence genes in EHECs LEE (Figs. 1 and 2) (Elliott et al., 2000). ...
... In addition to well-characterized LEE-encoded and non-LEE-encoded regulators that control AE phenotype, a total of 10 fimbrial genes and 13 non-fimbrial adhesins were found in EHEC (Hayashi et al., 2001;Perna et al., 2001). Of which, the long polar fimbriae ( Jordan et al., 2004;Farfan et al., 2011;Lloyd et al., 2012), E. coli common plus (Rendon et al., 2007), the autotransporter protein, EhaG (Totsika et al., 2012), and the pO157 virulence plasmid-encoded zinc metalloprotease (StcE) (Lathem et al., 2004;Grys et al., 2005) are involved in EHEC adhesion to epithelial cells and host colonization Figures 1 and 2). ...
Enterohemorrhagic Escherichia coli (EHEC) is one of the most common E. coli pathotypes reported to cause several outbreaks of foodborne illnesses. EHEC is a zoonotic pathogen, and ruminants, especially cattle, are considered important reservoirs for the most common EHEC serotype, E. coli O157:H7. Humans are infected indirectly through the consumption of food (milk, meat, leafy vegetables, and fruits) and water contaminated by animal feces or direct contact with carrier animals or humans. E. coli O157:H7 is one of the most frequently reported causes of foodborne illnesses in developed countries. It employs two essential virulence mechanisms to trigger damage to the host. These are the development of attaching and effacing (AE) phenotypes on the intestinal mucosa of the host and the production of Shiga toxin (Stx) that causes hemorrhagic colitis and hemolytic uremic syndrome. The AE phenotype is controlled by the pathogenicity island, the locus of enterocyte effacement (LEE). The induction of both AE and Stx is under strict and highly complex regulatory mechanisms. Thus, a good understanding of these mechanisms, major proteins expressed, and environmental cues involved in the regulation of the expression of the virulence genes is vital to finding a method to control the colonization of reservoir hosts, especially cattle, and disease development in humans. This review is a concise account of the current state of knowledge of virulence gene regulation in the LEE-positive EHEC.
... Degradation of mucins is important for the normal process of renewal and clearance of mucins in the mucus layers lining mucosal surfaces and essential for pathogenic bacteria like enterohemorrhagic Escherichia coli (EHEC) relying on the penetration of the protective mucus layers to reach the underlying epithelium 5,23,38,39 . While several mucinases have been reported and characterized so far, only two of these, StcE and ZmpC, derived from pathogenic bacteria, appear to be able to cleave mucins covered by complex elaborated O-glycan structures (Fig. 1b). ...
Mucinases of human gut bacteria cleave peptide bonds in mucins strictly depending on the presence of neighboring O-glycans. The Akkermansia muciniphila AM0627 mucinase cleaves specifically in between contiguous (bis) O-glycans of defined truncated structures, suggesting that this enzyme may recognize clustered O-glycan patches. Here, we report the structure and molecular mechanism of AM0627 in complex with a glycopeptide containing a bis-T (Galβ1-3GalNAcα1-O-Ser/Thr) O-glycan, revealing that AM0627 recognizes both the sugar moieties and the peptide sequence. AM0627 exhibits preference for bis-T over bis-Tn (GalNAcα1-O-Ser/Thr) O-glycopeptide substrates, with the first GalNAc residue being essential for cleavage. AM0627 follows a mechanism relying on a nucleophilic water molecule and a catalytic base Glu residue. Structural comparison among mucinases identifies a conserved Tyr engaged in sugar-π interactions in both AM0627 and the Bacteroides thetaiotaomicron BT4244 mucinase as responsible for the common activity of these two mucinases with bis-T/Tn substrates. Our work illustrates how mucinases through tremendous flexibility adapt to the diversity in distribution and patterns of O-glycans on mucins. AM0627 is a bis-O-glycan mucinase that might work in the final steps of mucus degradation, thereby providing a carbon and nitrogen source for Akkermansia muciniphila. Here, the authors provide molecular insights into AM0627 function from X-ray crystallography and computer simulations.
... Pathogens can secrete proteases to degrade Muc2, such as Trichuris muris nematodes (Henderson et al., 1999;Hasnain et al., 2012). Other pathogens secrete zinc metalloproteases that non-specifically cleave mucin-O-glycosylated proteins (Silva et al., 2003;Grys et al., 2005). Mucus degradation is not limited to pathogens. ...
The mucosal surfaces that form the boundary between the external environment and the underlying tissue are protected by a mucus barrier. Mucin glycoproteins, both secreted and cell surface mucins, are the major components of the barrier. They can exclude pathogens and toxins while hosting the commensal bacteria. In this review, we highlight the dynamic function of the mucins and mucus during infection, how this mucosal barrier is regulated, and how pathogens have evolved mechanisms to evade this defence system.
... Also, the activities of the LEE modulon were strongly correlated with the expression levels of ler (Pearson R = 0.79, P < 10 −10 ) ( Figure 1B), indicating that the LEE modulon primarily consisted of the Ler regulon. Furthermore, the LEE modulon contained lpxR, nleA, stcE, and etpC, which were not located in the LEE but known as the Ler regulon ( Figure 1A; Grys et al., 2005;Tobe et al., 2006;Roe et al., 2007;Ogawa et al., 2018). The activities of the LEE modulon were also highly correlated with the expression levels of these genes (Pearson R > 0.5, P < 10 −5 ) (Figure 1C), indicating that the modulon properly contained the genes located separately but co-regulated by Ler. ...
... The activities of the LEE modulon were strongly dependent on the expression level of ler (Figure 1B), and thus the LEE modulon mostly consisted of the Ler regulon. Moreover, the LEE modulon contained additional genes such as lpxR, nleA, stcE, and etpC, which are not located in the LEE but regulated by Ler (Figures 1A,C; Grys et al., 2005;Roe et al., 2007;Ogawa et al., 2018), indicating that ICA can precisely identify the LEE modulon to contain the target genes of the Ler TRN even not located in LEE. The Stx modulon contained the genes of the Stx prophages: CP-933V and BP-933W ( Figure 1D). ...
The elucidation of the transcriptional regulatory networks (TRNs) of enterohemorrhagic Escherichia coli (EHEC) is critical to understand its pathogenesis and survival in the host. However, the analyses of current TRNs are still limited to comprehensively understand their target genes generally co-regulated under various conditions regardless of the genetic backgrounds. In this study, independent component analysis (ICA), a machine learning-based decomposition method, was used to decompose the large-scale transcriptome data of EHEC into the modulons, which contain the target genes of several TRNs. The locus of enterocyte effacement (LEE) and the Shiga toxin (Stx) modulons mainly consisted of the Ler regulon and the Stx prophage genes, respectively, confirming that ICA properly grouped the co-regulated major virulence genes of EHEC. Further investigation revealed that the LEE modulon contained the hypothetical Z0395 gene as a novel member of the Ler regulon, and the Stx modulon contained the thi and cus locus genes in addition to the Stx prophage genes. Correspondingly, the Stx prophage genes were also regulated by thiamine and copper ions known to control the thi and cus locus genes, respectively. The modulons effectively clustered the genes co-regulated regardless of the growth conditions and the genetic backgrounds of EHEC. The changed activities of the individual modulons successfully explained the differential expressions of the virulence and survival genes during the course of infection in bovines. Altogether, these results suggested that ICA of the large-scale transcriptome data can expand and enhance the current understanding of the TRNs of EHEC.
... Most genes required for AE lesion formation are clustered within a pathogenicity island (PAI) named locus of enterocyte effacement (LEE), which encodes a type III secretion system (T3SS) that injects many effector proteins into the host cell, leading to cytoskeleton rearrangement and effacement of the intestinal microvilli (10). In addition, EHEC can produce mucin-degrading enzymes such as the zinc metalloprotease StcE, which contributes to bacterial penetration of the mucus layer toward the colonic epithelium (11). ...
The interactions between the gut microbiota and pathogens are complex and can determine the outcome of an infection. Enterohemorrhagic Escherichia coli (EHEC) is a major human enteric pathogen that colonizes the colon through attaching and effacing (AE) lesions and uses microbiota-derived molecules as cues to control its virulence. Different gut commensals can modulate EHEC virulence. However, the lack of an animal model that recapitulates the human pathophysiology of EHEC infection makes it challenging to investigate how variations in microbiota composition could affect host susceptibility to this pathogen. Here, we addressed these interactions building from simple to more complex in vitro systems, culminating with the use of the physiological relevant human colonoids as a model to study the interactions between EHEC and different gut commensals. We demonstrated that Bacteroides thetaiotaomicron and Enterococcus faecalis enhance virulence expression and AE lesion formation in cultured epithelial cells, as well as on the colonic epithelium, while commensal E. coli did not affect these phenotypes. Importantly, in the presence of these three commensals together, virulence and AE lesion are enhanced. Moreover, we identified specific changes in the metabolic landscape promoted by different members of the gut microbiota and showed that soluble factors released by E. faecalis can increase EHEC virulence gene expression. Our study highlights the importance of interspecies bacterial interactions and chemical exchange in the modulation of EHEC virulence.
... ST11, the only representative of phylogroup E, from our study, was also observed to form a distinct cluster in the scatterplot of the top two axes of PCoA (Fig. 5) while also revealing peculiar trends concerning resistome (Fig. 1) and virulome (Fig. 2 and 3) profiles. The StcE protease, previously reported to help in the adherence of EHEC serotype O157:H7 to host cells, is secreted by the pO157-encoded etp type II secretion system (34). This gene was seen in 96.5% of the ST11 genomes, but was absent from the majority of the STs. ...
With the increase in the amounts of whole-genome data being generated, the application of relevant methods to mine biologically significant information from microbial genomes is of the utmost importance to public health genomics. Machine-learning methods have been used not just to predict or classify the data but also to identify the relevant features that could be linked with a particular class/target.
