Identification of a negative regulator for the pathogenicity island of enterohemorrhagic<Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7

EnterohemorrhagicEscherichia coli (EHEC) forms histological lesions termed attaching and effacing lesions (A/E lesions) on infected large intestine tissue. The major virulence factors involved in A/E lesions reside on a locus of enterocyte effacement (LEE), a pathogenicity island. The LEE comprises 41 specific open reading frames, of which most are organized in 5 major operons,LEE1,LEE2,LEE3,LEE4, andtir (LEE5). The expression of LEE genes is regulated in a complicated manner by environmental factors such as temperature, osmolarity, and quorum sensing. Current knowledge is that regulation is hierarchical: a pivotal positive regulator,ler, is first stimulated, which in turn activates the expression of other operons. Herein, we report on the presence of a negative regulation protein located within the LEE.L0044 is 372 bp in length and is located outside of the 5 major operons. An isogenicL0044 deletion mutant displayed loss of the repression phenotype and increased synthesis of several LEE proteins when bacteria were cultured under repressive conditions that disfavor expression of LEE proteins. Reciprocally,trans expression ofL0044 suppressed the expression of the LEE. Furthermore, mRNA ofler increased as a result of deletingL0044, and disruptingler in aL0044-deleted background reversed the loss of the repression phenotype. Thus,L0044 plays a role in regulating the expression of virulence genes in EHEC by modulating the activation ofler.     

Effects of Psidium guajava leaf extract on secretion systems of gram‐negative enteropathogenic bacteria

In this study, 672 plant‐tissue extracts were screened for phytochemicals that inhibit the function of the type III secretion system (T3SS) of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC). Among candidates examined, an extract from the leaves of Psidium guajava (guava) was found to inhibit secretion of EPEC‐secreted protein B (EspB) from EPEC and EHEC without affecting bacterial growth. Guava extract (GE) also inhibited EPEC and EHEC from adhering to, and injecting EspB into, HEp‐2 cells. GE seemed to block translocation of EspB from the bacterial cells to the culture medium. In addition, GE also inhibited the T3SS of Yersinia pseudotuberculosis and Salmonella enterica serovar Typhimurium. After exposure to GE, Y. pseudotuberculosis stopped secreting Yersinia outer proteins and was unable to induce apoptosis of mouse bone marrow‐derived macrophages. S. typhimurium exposed to GE stopped secreting Sip proteins and was unable to invade HEp‐2 cells. GE inhibited secretion of EspC, the type V secretion protein of EPEC, but not secretion of Shiga toxin 2 from EHEC. Thus, our results suggest that guava leaves contain a novel type of antimicrobial compound that could be used to treat and prevent gram‐negative enteropathogenic bacterial infections.

     

Identification and characterization of NleA, a non-LEE-encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 uses a specialized protein translocation apparatus, the type III secretion system (TTSS), to deliver bacterial effector proteins into host cells. These effectors interfere with host cytoskeletal pathways and signalling cascades to facilitate bacterial survival and replication and promote disease. The genes encoding the TTSS and all known type III secreted effectors in EHEC are localized in a single pathogenicity island on the bacterial chromosome known as the locus for enterocyte effacement (LEE). In this study, we performed a proteomic analysis of proteins secreted by the LEE-encoded TTSS of EHEC. In addition to known LEE-encoded type III secreted proteins, such as EspA, EspB and Tir, a novel protein, NleA (non-LEE-encoded effector A), was identified. NleA is encoded in a prophage-associated pathogenicity island within the EHEC genome, distinct from the LEE. The LEE-encoded TTSS directs translocation of NleA into host cells, where it localizes to the Golgi apparatus. In a panel of strains examined by Southern blot and database analyses, nleA was found to be present in all other LEE-containing pathogens examined, including enteropathogenic E. coli and Citrobacter rodentium, and was absent from non-pathogenic strains of E. coli and non-LEE-containing pathogens. NleA was determined to play a key role in virulence of C. rodentium in a mouse infection model.     

Microaerobic conditions enhance type III secretion and adherence of enterohaemorrhagic Escherichia coli to polarized human intestinal epithelial cells

Advances in the understanding of the pathogenesis of enterohaemorrhagic Escherichia coli (EHEC) have greatly benefited from the use of human epithelial cell lines under aerobic conditions. However, in the target site of EHEC infection, the human intestine, conditions are microaerobic. In our study we used polarized human colon carcinoma cells in a vertical diffusion chamber system to investigate the influence of reduced apical oxygen levels on EHEC colonization. While apical microaerobiosis did not affect cell integrity and barrier function, numbers of adherent bacteria were significantly increased under low compared with high apical oxygen concentrations. In addition, expression and translocation of EHEC type III secreted (T3S) effector proteins was considerably enhanced under microaerobic conditions and dependent on the presence of host cells. Increased colonization was mainly mediated via EspA as adherence levels of an isogenic deletion mutant were not influenced by low oxygen levels. Other potential adherence factors (E. coli common pilus and flagella) were only minimally expressed under high and low oxygen levels. Addition of nitrate and trimethylamine N‐oxide as terminal electron acceptors for anaerobic respiration failed to further increase bacterial colonization or T3S under microaerobiosis. This study indicates that EHEC T3S and colonization are enhanced by the microaerobic environment in the gut and therefore might be underestimated in conventional aerobic cell culture systems.     

Presence and Characterization of a Mosaic Genomic Island Which Distinguishes Sorbitol-Fermenting Enterohemorrhagic Escherichia coli O157:H? from E. coli O157:H7

A mosaic genomic island comprising Shigella resistance locus (SRL) sequences flanked by segments of Escherichia coli O157:H7 strain EDL933 O islands 43, 81, and 82 was identified in sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H⁻ strain 493/89. This mosaic island is absent from strain EDL933. PCR targeting the SRL-related sequence is a useful tool to distinguish SF EHEC O157:H⁻ from EHEC O157:H7.     

Serology and immunochemistry ofStreptococcus MG andStreptococcus salivarius

Precipitation and cross adsorption show thatStreptococcus NCTC 8037/50 contains the group antigen F of Lancefield and the type antigen III of Ottens. Sera prepared against this strain contain either anti-F and anti-III antibodies or anti-III antibodies only.The qualitative chemical analysis of the formamide extract ofStreptococcus NCTC 8037/50 and the inhibition of its quantitative precipitation reaction with simple sugars are the same as for a formamide extract of an F III strain.Nineteen strains, classified by Dr. de Moor (Utrecht) or by Dr. Seeleman (Hamburg) asStreptococcus MG, were serologically identical with either F III, L III, or 0 III (meaning zero III) streptococci. Some of these 0 III strains gave slimy colonies on saccharose agar according to Chapman, and on this property could be regarded asStreptococcus salivarius.About 70% of 42 freshly isolatedStr. salivarius strains were shown to contain type III antigen. With one of the strains without type III antigen (strain 51) an antiserum could be prepared which gave precipitation reactions with all non typableStr. salivarius strains and also with some type III positive strains. This was confirmed by cross adsorption tests.Qualitative chemical analyses of the hydrolysates of formamide extracts of F III and bothStreptococcus salivarius strains were compared with each other.The inhibition reaction of the quantitative precipitation of formamide extracts of F III,Str. salivarius N.C.T.C. 8606 andStr. salivarius 51 with the homologous and heterologous sera revealed a-glucosidic endgroup of the determinant groups of the III-andStr. salivarius 8606-antigen to be present. Slight differences in the inhibition pattern showed that these determinant groups were not identical. The determinant group ofStr. salivarius 51 is quite different; the-glucosidic linked sugars gave no inhibition at all. The best inhibitor was in this case rhamnose, although the inhibition was weak.     

Type III Secretion Homologs Are Present in <Emphasis Type="Italic">Brucella melitensis</Emphasis>, <Emphasis Type="Italic">B. ovis</Emphasis>, and <Emphasis Type="Italic">B. suis</Emphasis> biovars 1, 2, and 3

Protein sequences from characterized type III secretion (TTS) systems were used as probes in silico to identify several TTS gene homologs in the genome sequence of Brucella suis biovar 1 strain 1330. Four of the genes, named flhB, fliP, fliR, and fliF on the basis of greatest homologies to known flagellar apparatus proteins, were targeted in PCR and hybridization assays to determine their distribution among other Brucella nomen species and biovars. The results indicated that flhB, fliP, fliR and fliF are present in Brucella melitensis, Brucella ovis, and Brucella suis biovars 1, 2 and 3. Similar homologos have been reported previously in Brucella abortus. Using RT-PCR assays, we were unable to detect any expression of these genes. It is not yet known whether the genes are the cryptic remnants of a flagellar system or are actively involved in a process contributing to pathogenicity or previously undetected motility, but they are distributed widely in Brucella and merit further study to determine their role. Received: 11 February 2002 / Accepted: 13 June 2002     

The bacterial virulence factor NleA undergoes host-mediated O-linked glycosylation

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC) are gastrointestinal pathogens responsible for severe diarrheal illness. EHEC and EPEC form “attaching and effacing” lesions during colonization and, upon adherence, inject proteins directly into host intestinal cells via the type III secretion system (T3SS). Injected bacterial proteins have a variety of functions but generally alter host cell biology to favor survival and/or replication of the pathogen. Non-LEE-encoded effector A (NleA) is a T3SS-injected effector of EHEC, EPEC, and the related mouse pathogen Citrobacter rodentium. Studies in mouse models indicate that NleA has an important role in bacterial virulence. However, the mechanism by which NleA contributes to disease remains unknown. We have determined that the following translocation into host cells, a serine and threonine-rich region of NleA is modified by host-mediated mucin-type O-linked glycosylation. Surprisingly, this region was not present in several clinical EHEC isolates. When expressed in C. rodentium, a non-modifiable variant of NleA was indistinguishable from wildtype NleA in an acute mortality model but conferred a modest increase in persistence over the course of infection in mixed infections in C57BL/6J mice. This is the first known example of a bacterial effector being modified by host-mediated O-linked glycosylation. Our data also suggests that this modification may confer a selective disadvantage to the bacteria during in vivo infection.     

Pas, a Novel Protein Required for Protein Secretion and Attaching and Effacing Activities of Enterohemorrhagic Escherichia coli

Enterohemorrhagic Escherichia coli (EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological phenotype collectively known as the attaching and effacing lesion. The genes encoding the products responsible for this phenotype are located on a 35-kb pathogenicity island designated the locus of enterocyte effacement, which is also shared by enteropathogenic E. coli. We have identified an open reading frame (ORF) which is located upstream of the espA, espB, and espD genes on the complementary strand and which exhibits high homology to the genes spiB from Salmonella, yscD from Yersinia, and pscD from Pseudomonas. Localization studies showed that the encoded product is present in the cytoplasmic and inner membrane fractions of EHEC. The construction and characterization of a recombinant clone containing an in-frame deletion of this ORF demonstrated that the encoded product is a putative member of a type III system required for protein secretion. Disruption of this ORF, designated pas (protein associated with secretion), abolished the secretion of Esp proteins. The mutant adhered only poorly and lost its capacities to trigger attaching and effacing activity and to invade HeLa cells. These results demonstrate that Pas is a virulence-associated factor that plays an essential role in EHEC pathogenesis.     

The N-Terminal Domain of EspF Induces Host Cell Apoptosis after Infection with Enterohaemorrhagic Escherichia coli O157:H7

Enterohemorrhagic Escherichia coli (EHEC) employs a type III secretion system (TTSS) to export the translocator and effector proteins required for mucosal colonization. As an important bacterial effector protein in locus of enterocyte effacement four, the EspF protein causes F-actin filament aggregations to form attaching and effacing (A/E) lesions, and induces the destruction of brush-border microvilli and cytoskeletal rearrangements to form pedestals. However, the molecular pathogenesis of A/E lesions due to EHEC O157:H7 infection is unclear. In this study, we constructed an espF-deficient mutant (ΔespF) with a 162-bp deletion in the N-terminal domain by using overlap extension PCR. The results showed that EHEC EspF translocated into intestinal epithelial cells, targeted mitochondria and induced apoptosis. The ΔespF mutant, compared to EHEC prototype Guangzhou strain, had lower cell attachment and effacement abilities, lower caspase-9/3 and lactate dehydrogenase levels, lower bacterial adhesion, weaker mitochondria apoptosis, and a higher mouse survival rate. Our results demonstrate the probable function of the EspF N-terminal domain, which targets mitochondria and binds mitochondria heat shock protein 70 to induce cell apoptosis via A/E lesions. These findings may be invaluable in clarifying the molecular pathogenesis of EspF of EHEC O157:H7.     

Collagen Binding of Bifidobacterium adolescentis

In this study, 13 bifidobacterial strains were tested for their ability to adhere to immobilized extracellular matrix (ECM) proteins. Only two Bifidobacterium adolescentis strains adhered to immobilized type I and type V collagens, but not to laminin, fibronectin, and type III and IV collagens. The adhesion of B. adolescentis BB-119 to type V collagen was inhibited by type I and V collagens and gelatin, and was diminished after protease treatment of the cells. Periodate treatment of immobilized collagen and the presence of galactose inhibited the adhesion of strain BB-119 to type V collagen. Two cell surface proteins with molecular masses of 36 kDa and 52 kDa from strain BB-119 were found to bind to horseradish peroxidase-conjugated type V collagen by ligand blotting. We concluded that B. adolescentis BB-119 binds to type V collagen at galactose chains as target via these two cell surface proteins by their lectin-like activity. Received: 15 October 1996 / Accepted: 20 November 1996     

Evolutionary and functional diversity of the Pseudomonas type IVa pilin island

In Pseudomonas aeruginosa, most proteins involved in type IVa pilus (T4aP) biogenesis are highly conserved except for the major pilin PilA and the minor pilins involved in pilus assembly. Here we show that each of the five major pilin alleles is associated with a specific set of minor pilins, and unrelated strains with the same major pilin type have identical minor pilin genes. The sequences of the minor pilin genes of strains with group III and V pilins are identical, suggesting that these groups diverged recently through further evolution of the major pilin cluster. Both gene clusters are localized on a single ‘pilin island’ containing putative tRNA recombinational hotspots, and a similar organization of pilin genes was identified in other Pseudomonas species. To address the biological significance of group‐specific differences, cross‐complementation studies using group II (PAO1) and group III (PA14) minor pilins were performed. Heterologous minor pilins complemented twitching motility to various extents except in the case of PilX, which was non‐functional in non‐native backgrounds. A recombinant PA14 strain expressing the PAO1 minor pilins regained motility only upon co‐introduction of the PA14 pilX gene. Comparison of PilX and PilQ secretin sequences from group II, III and V genomes revealed discrete regions of sequence that co‐varied between groups. Our data suggest that changes in PilX sequence have led to compensatory changes in the PilQ secretin monomer such that heterologous PilX proteins are no longer able to promote opening of the secretin to allow pili to appear on the cell surface.     

The Gut Bacterium Bacteroides thetaiotaomicron Influences the Virulence Potential of the Enterohemorrhagic Escherichia coli O103:H25

Enterohemorrhagic E. coli (EHEC) is associated with severe gastrointestinal disease. Upon entering the gastrointestinal tract, EHEC is exposed to a fluctuating environment and a myriad of other bacterial species. To establish an infection, EHEC strains have to modulate their gene expression according to the GI tract environment. In order to explore the interspecies interactions between EHEC and an human intestinal commensal, the global gene expression profile was determined of EHEC O103:H25 (EHEC NIPH-11060424) co-cultured with B. thetaiotaomicron (CCUG 10774) or grown in the presence of spent medium from B. thetaiotaomicron. Microarray analysis revealed that approximately 1% of the EHEC NIPH-11060424 genes were significantly up-regulated both in co-culture (30 genes) and in the presence of spent medium (44 genes), and that the affected genes differed between the two conditions. In co-culture, genes encoding structural components of the type three secretion system were among the most affected genes with an almost 4-fold up-regulation, while the most affected genes in spent medium were involved in chemotaxis and were more than 3-fold up-regulated. The operons for type three secretion system (TTSS) are located on the Locus of enterocyte effacement (LEE) pathogenicity island, and qPCR showed that genes of all five operons (LEE1-LEE5) were up-regulated. Moreover, an increased adherence to HeLa cells was observed in EHEC NIPH-11060424 exposed to B. thetaiotaomicron. Expression of stx2 genes, encoding the main virulence factor of EHEC, was down-regulated in both conditions (co-culture/spent medium). These results show that expression of EHEC genes involved in colonization and virulence is modulated in response to direct interspecies contact between cells, or to diffusible factors released from B. thetaiotaomicron. Such interspecies interactions could allow the pathogen to recognize its predilection site and modulate its behaviour accordingly, thus increasing the efficiency of colonization of the colon mucosa, facilitating its persistence and increasing its virulence potential.     

Spa15 of Shigella flexneri, a third type of chaperone in the type III secretion pathway

The type III secretion (TTS) pathway is used by numerous Gram-negative pathogens to inject virulence factors into eukaryotic cells. In addition to a functional TTS apparatus, secretion of effector proteins depends upon specific chaperones. Using a two-hybrid screen in yeast and a co-purification assay in Shigella flexneri, we demonstrated that Spa15, which is encoded by an operon for components of the TTS apparatus, is associated in the cytoplasm with three proteins that are secreted by the TTS pathway, IpaA, IpgB1 and OspC3. Spa15 was found to be necessary for stability of IpgB1 but not IpaA, and for secretion of IpaA molecules that were stored in the cytoplasm but not those that were synthesized while the secretion apparatus was active. The ability of Spa15 to associate with several non-homologous secreted proteins, the presence of Spa15 homologues in other TTS systems and the location of the corresponding genes within operons for components of the TTS apparatus suggest that Spa15 belongs to a new class of TTS chaperones.     

EspJ effector in enterohemorrhagic E. coli translocates into host mitochondria via an atypical mitochondrial targeting signal

EHEC is a bacterial pathogen causing diarrhea and hemorrhagic colitis in humans. To exert virulence, EHEC exploits a subset of effectors that are translocated into host cells via the type III secretion system. EspJ, which was recently identified as a type III secreted effector, is conserved in related pathogens such as EPEC and Citrobacter rodentium. However, the exact function of EspJ remains unclear. In the present study, we found that EspJ was unstable in host cells, which might be attributable to the N‐terminal part beginning from amino acid number 59. Using stable forms of EspJ derivatives, we demonstrated for the first time that EspJ has the ability to translocate into mitochondria via an atypical mitochondrial targeting signal at the N terminus (1–36 a.a.) of EspJ. It has been reported that a mitochondrial targeting effector, EspF, disrupts the mitochondrial membrane potential, resulting in an induction of host cell death. To further investigate EspJ function in mitochondria, HeLa cells were infected with wild‐type EPEC, an isogenic EspJ‐mutant or an EspJ‐overexpressing strain. The result of LDH release assay using an EspJ‐mutant showed that the EspJ effector appears not to be involved in cytotoxicity.     

Lysogeny with Shiga Toxin 2-Encoding Bacteriophages Represses Type III Secretion in Enterohemorrhagic Escherichia coli

Lytic or lysogenic infections by bacteriophages drive the evolution of enteric bacteria. Enterohemorrhagic Escherichia coli (EHEC) have recently emerged as a significant zoonotic infection of humans with the main serotypes carried by ruminants. Typical EHEC strains are defined by the expression of a type III secretion (T3S) system, the production of Shiga toxins (Stx) and association with specific clinical symptoms. The genes for Stx are present on lambdoid bacteriophages integrated into the E. coli genome. Phage type (PT) 21/28 is the most prevalent strain type linked with human EHEC infections in the United Kingdom and is more likely to be associated with cattle shedding high levels of the organism than PT32 strains. In this study we have demonstrated that the majority (90%) of PT 21/28 strains contain both Stx2 and Stx2c phages, irrespective of source. This is in contrast to PT 32 strains for which only a minority of strains contain both Stx2 and 2c phages (28%). PT21/28 strains had a lower median level of T3S compared to PT32 strains and so the relationship between Stx phage lysogeny and T3S was investigated. Deletion of Stx2 phages from EHEC strains increased the level of T3S whereas lysogeny decreased T3S. This regulation was confirmed in an E. coli K12 background transduced with a marked Stx2 phage followed by measurement of a T3S reporter controlled by induced levels of the LEE-encoded regulator (Ler). The presence of an integrated Stx2 phage was shown to repress Ler induction of LEE1 and this regulation involved the CII phage regulator. This repression could be relieved by ectopic expression of a cognate CI regulator. A model is proposed in which Stx2-encoding bacteriophages regulate T3S to co-ordinate epithelial cell colonisation that is promoted by Stx and secreted effector proteins.     

The pathogenic E. coli type III effector EspZ interacts with host CD98 and facilitates host cell prosurvival signalling

Enterohaemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC respectively) are diarrhoeal pathogens that cause the formation of attaching and effacing (A/E) lesions on infected host cells. These pathogens encode a type III secretion system (T3SS) used to inject effector proteins directly into host cells, an essential requirement for virulence. In this study, we identified a function for the type III secreted effector EspZ. Infection with EPEC ΔespZ caused increased cytotoxicity in HeLa and MDCK cells compared with wild‐type EPEC, and expressing espZ in cells abrogated this effect. Using yeast two‐hybrid, proteomics, immunofluorescence and co‐immunoprecipitation, it was demonstrated that EspZ interacts with the host protein CD98, which contributes to protection against EPEC‐mediated cytotoxicity. EspZ enhanced phosphorylation of focal adhesion kinase (FAK) and AKT during infection with EPEC, but CD98 only appeared to facilitate FAK phosphorylation. This study provides evidence that EspZ and CD98 promote host cell survival mechanisms involving FAK during A/E pathogen infection.