scholarly journals Structural and Functional Variation in Outer Membrane Polysaccharide Export (OPX) Proteins from the Two Major Capsule Assembly Pathways Present inEscherichia coli

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Caitlin Sande ◽  
Catrien Bouwman ◽  
Elisabeth Kell ◽  
Nicholas N. Nickerson ◽  
Sharookh B. Kapadia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Outer Membrane ◽  
Abc Transporter ◽  
Capsular Polysaccharides ◽  
Content Type ◽  
E Coli ◽  
Capsule Production ◽  
Group 2 ◽  
Group 1

ABSTRACTCapsular polysaccharides (CPSs) are virulence factors for many important pathogens. InEscherichia coli, CPSs are synthesized via two distinct pathways, but both require proteins from the outer membrane polysaccharide export (OPX) family to complete CPS export from the periplasm to the cell surface. In this study, we compare the properties of the OPX proteins from the prototypical group 1 (Wzy-dependent) and group 2 (ABC transporter-dependent) pathways inE. coliK30 (Wza) andE. coliK2 (KpsD), respectively. In addition, we compare an OPX fromSalmonella entericaserovar Typhi (VexA), which shares structural properties with Wza, while operating in an ABC transporter-dependent pathway. These proteins differ in distribution in the cell envelope and formation of stable multimers, but these properties do not align with acylation or the interfacing biosynthetic pathway. InE. coliK2, murein lipoprotein (Lpp) plays a role in peptidoglycan association of KpsD, and loss of this interaction correlates with impaired group 2 capsule production. VexA also depends on Lpp for peptidoglycan association, but CPS production is unaffected in anlppmutant. In contrast, Wza and group 1 capsule production is unaffected by the absence of Lpp. These results point to complex structure-function relationships between different OPX proteins.IMPORTANCECapsules are protective layers of polysaccharides that surround the cell surface of many bacteria, including that ofEscherichia coliisolates andSalmonella entericaserovar Typhi. Capsular polysaccharides (CPSs) are often essential for virulence because they facilitate evasion of host immune responses. The attenuation of unencapsulated mutants in animal models and the involvement of protein families with conserved features make the CPS export pathway a novel candidate for therapeutic strategies. However, appropriate “antivirulence” strategies require a fundamental understanding of the underpinning cellular processes. Investigating export proteins that are conserved across different biosynthesis strategies will give important insight into how CPS is transported to the cell surface.

scholarly journals Preparation of Sticky Escherichia coli through Surface Display of an Adhesive Catecholamine Moiety

2013 ◽  
Vol 80 (1) ◽  
pp. 43-53 ◽  
Author(s):  
Joseph P. Park ◽  
Min-Jung Choi ◽  
Se Hun Kim ◽  
Seung Hwan Lee ◽  
Haeshin Lee
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Communication Systems ◽  
Cell Attachment ◽  
Surface Display ◽  
Content Type ◽  
E Coli ◽  
Material Surfaces ◽  
Mussel Adhesive ◽  
Adhesive Proteins

ABSTRACTMussels attach to virtually all types of inorganic and organic surfaces in aqueous environments, and catecholamines composed of 3,4-dihydroxy-l-phenylalanine (DOPA), lysine, and histidine in mussel adhesive proteins play a key role in the robust adhesion. DOPA is an unusual catecholic amino acid, and its side chain is called catechol. In this study, we displayed the adhesive moiety of DOPA-histidine onEscherichia colisurfaces using outer membrane protein W as an anchoring motif for the first time. Localization of catecholamines on the cell surface was confirmed by Western blot and immunofluorescence microscopy. Furthermore, cell-to-cell cohesion (i.e., cellular aggregation) induced by the displayed catecholamine and synthesis of gold nanoparticles on the cell surface support functional display of adhesive catecholamines. The engineeredE. coliexhibited significant adhesion onto various material surfaces, including silica and glass microparticles, gold, titanium, silicon, poly(ethylene terephthalate), poly(urethane), and poly(dimethylsiloxane). The uniqueness of this approach utilizing the engineered stickyE. coliis that no chemistry for cell attachment are necessary, and the ability of spontaneousE. coliattachment allows one to immobilize the cells on challenging material surfaces such as synthetic polymers. Therefore, we envision that mussel-inspired catecholamine yielded stickyE. colithat can be used as a new type of engineered microbe for various emerging fields, such as whole living cell attachment on versatile material surfaces, cell-to-cell communication systems, and many others.

scholarly journals Antibiotic-Mediated Modulations of Outer Membrane Vesicles in Enterohemorrhagic Escherichia coli O104:H4 and O157:H7

2017 ◽  
Vol 61 (9) ◽  
Author(s):  
Andreas Bauwens ◽  
Lisa Kunsmann ◽  
Helge Karch ◽  
Alexander Mellmann ◽  
Martina Bielaszewska
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Shiga Toxin ◽  
Membrane Vesicles ◽  
Polymyxin B ◽  
Outer Membrane Vesicles ◽  
Enterohemorrhagic Escherichia Coli ◽  
Content Type ◽  
E Coli ◽  
Major Virulence Factor

ABSTRACT Ciprofloxacin, meropenem, fosfomycin, and polymyxin B strongly increase production of outer membrane vesicles (OMVs) in Escherichia coli O104:H4 and O157:H7. Ciprofloxacin also upregulates OMV-associated Shiga toxin 2a, the major virulence factor of these pathogens, whereas the other antibiotics increase OMV production without the toxin. These two effects might worsen the clinical outcome of infections caused by Shiga toxin-producing E. coli. Our data support the existing recommendations to avoid antibiotics for treatment of these infections.

scholarly journals Bioinformatic and Molecular Analysis of Inverse Autotransporters from Escherichia coli

mSphere ◽  
2019 ◽  
Vol 4 (4) ◽  
Author(s):  
Kelvin G. K. Goh ◽  
Danilo G. Moriel ◽  
Steven J. Hancock ◽  
Minh-Duy Phan ◽  
Mark A. Schembri
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Biofilm Formation ◽  
Secretion System ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Type V Secretion ◽  
K 12 ◽  
Type V

ABSTRACT Proteins secreted by the type V secretion system possess multiple functions, including the capacity to mediate adhesion, aggregation, and biolfilm formation. The type V secretion system can be divided into five subclasses, one of which is the type Ve system. Proteins of the type Ve secretion system are also referred to as inverse autotransporters (IATs). In this study, we performed an in silico analysis of 126 completely sequenced Escherichia coli genomes available in the NCBI database and identified several distinct IAT-encoding gene families whose distribution varied throughout the E. coli phylogeny. The genes included three characterized IATs (intimin, fdeC, and yeeJ) and four uncharacterized IATs (here named iatA, iatB, iatC, and iatD). The four iat genes were cloned from the completely sequenced environmental E. coli strain SMS-3-5 and characterized. Three of these IAT proteins (IatB, IatC, and IatD) were expressed at the cell surface and possessed the capacity to mediate biofilm formation in a recombinant E. coli K-12 strain. Further analysis of the iatB gene, which showed a unique association with extraintestinal E. coli strains, suggested that its regulation is controlled by the LeuO global regulator. Overall, this study provides new data describing the prevalence, sequence variation, domain structure, function, and regulation of IATs found in E. coli. IMPORTANCE Escherichia coli is one of the most prevalent facultative anaerobes of the human gut. E. coli normally exists as a harmless commensal but can also cause disease following the acquisition of genes that enhance its pathogenicity. Adhesion is an important first step in colonization of the host and is mediated by an array of cell surface components. In E. coli, these include a family of adhesins secreted by the type V secretion system. Here, we identified and characterized new proteins from an emerging subclass of the type V secretion system known as the inverse autotransporters (IATs). We found that IAT-encoding genes are present in a wide range of strains and showed that three novel IATs were localized on the E. coli cell surface and mediated biofilm formation. Overall, this study provides new insight into the prevalence, function, and regulation of IATs in E. coli.

Association of Enterohemorrhagic Escherichia coliHemolysin with Serotypes of Shiga-Like-Toxin-ProducingEscherichia coli of Human and Bovine Origins

1998 ◽  
Vol 64 (11) ◽  
pp. 4134-4141 ◽  
Author(s):  
Carlton Gyles ◽  
Roger Johnson ◽  
Anli Gao ◽  
Kim Ziebell ◽  
Denis Pierard ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Severe Disease ◽  
Sheep Erythrocyte ◽  
Pcr Amplification ◽  
E Coli ◽  
Hemolysin Gene ◽  
Group 3 ◽  
Group 2 ◽  
Bovine Feces ◽  
Group 1

ABSTRACT In this study we investigated whether the enterohemorrhagicEscherichia coli (EHEC) hemolysin gene ehxAcould be used as an indicator of pathogenicity in Shiga-like-toxin-producing Escherichia coli (SLTEC) isolates. The isolates in a collection of 770 SLTEC strains of human and bovine origins were assigned to group 1 (230 human and 138 bovine SLTEC isolates belonging to serotypes frequently implicated in human disease), group 2 (85 human and 183 bovine isolates belonging to serotypes less frequently implicated in disease), and group 3 (134 bovine isolates belonging to serotypes not implicated in disease). PCR amplification was used to examine all of the SLTEC isolates for the presence of ehxA and the virulence-associated geneseae, slt-I, and slt-II. The percentages of human isolates in groups 1 and 2 that were positive forehxA were 89 and 46%, respectively, and the percentages of bovine isolates in groups 1 to 3 that were positive forehxA were 89, 51, and 52%, respectively. The percentages of human isolates in groups 1 and 2 that were positive foreae were 92 and 27%, respectively, and the percentages of bovine isolates in groups 1 to 3 that were positive for eaewere 78, 15, and 19%, respectively. The frequencies of bothehxA and eae were significantly higher for group 1 isolates than for group 2 isolates. The presence of the ehxA gene was associated with serotype, as was the presence of the eae gene. Some serotypes, such as O117:H4, lacked both eae and ehxA and have been associated with severe disease, but only infrequently. Theslt-I genes were more frequent in group 1 isolates than in group 2 isolates, and the slt-II genes were more frequent in group 2 isolates than in group 1 isolates. In a second experiment we determined the occurrence of the ehxA andslt genes in E. coli isolated from bovine feces. Fecal samples from 175 animals were streaked onto washed sheep erythrocyte agar plates. Eight E. coli-like colonies representing all of the morphological types were transferred to MacConkey agar. A total of 1,080 E. coli isolates were examined, and the ehxA gene was detected in 12 independent strains, only 3 of which were positive for slt. We concluded that the ehxA gene was less correlated with virulence than the eae gene was and that EHEC hemolysin alone has limited value for screening bovine feces for pathogenic SLTEC because of presence of the ehxA gene in bovine isolates that are not SLTEC.

scholarly journals Initial Steps of Colicin E1 Import across the Outer Membrane of Escherichia coli

2007 ◽  
Vol 189 (7) ◽  
pp. 2667-2676 ◽  
Author(s):  
Muriel Masi ◽  
Phu Vuong ◽  
Matthew Humbard ◽  
Karen Malone ◽  
Rajeev Misra
Keyword(s):  
Escherichia Coli ◽  
Cell Surface ◽  
Outer Membrane ◽  
Vitamin B ◽  
Whole Cells ◽  
E Coli ◽  
Colicin E1 ◽  
Unfolded State ◽  
Translocation Domain

ABSTRACT Data suggest a two-receptor model for colicin E1 (ColE1) translocation across the outer membrane of Escherichia coli. ColE1 initially binds to the vitamin B12 receptor BtuB and then translocates through the TolC channel-tunnel, presumably in a mostly unfolded state. Here, we studied the early events in the import of ColE1. Using in vivo approaches, we show that ColE1 is cleaved when added to whole cells. This cleavage requires the presence of the receptor BtuB and the protease OmpT, but not that of TolC. Strains expressing OmpT cleaved ColE1 at K84 and K95 in the N-terminal translocation domain, leading to the removal of the TolQA box, which is essential for ColE1's cytotoxicity. Supported by additional in vivo data, this suggests that a function of OmpT is to degrade colicin at the cell surface and thus protect sensitive E. coli cells from infection by E colicins. A genetic strategy for isolating tolC mutations that confer resistance to ColE1, without affecting other TolC functions, is also described. We provide further in vivo evidence of the multistep interaction between TolC and ColE1 by using cross-linking followed by copurification via histidine-tagged TolC. First, secondary binding of ColE1 to TolC is dependent on primary binding to BtuB. Second, alterations to a residue in the TolC channel interfere with the translocation of ColE1 across the TolC pore rather than with the binding of ColE1 to TolC. In contrast, a substitution at a residue exposed on the cell surface abolishes both binding and translocation of ColE1.

scholarly journals Molecular Characterization of the EhaG and UpaG Trimeric Autotransporter Proteins from Pathogenic Escherichia coli

2012 ◽  
Vol 78 (7) ◽  
pp. 2179-2189 ◽  
Author(s):  
Makrina Totsika ◽  
Timothy J. Wells ◽  
Christophe Beloin ◽  
Jaione Valle ◽  
Luke P. Allsopp ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Cell Surface ◽  
Specific Binding ◽  
Negative Regulator ◽  
Passenger Domain ◽  
Content Type ◽  
E Coli ◽  
Ecm Proteins

ABSTRACTTrimeric autotransporter proteins (TAAs) are important virulence factors of many Gram-negative bacterial pathogens. A common feature of most TAAs is the ability to mediate adherence to eukaryotic cells or extracellular matrix (ECM) proteins via a cell surface-exposed passenger domain. Here we describe the characterization of EhaG, a TAA identified from enterohemorrhagicEscherichia coli(EHEC) O157:H7. EhaG is a positional orthologue of the recently characterized UpaG TAA from uropathogenicE. coli(UPEC). Similarly to UpaG, EhaG localized at the bacterial cell surface and promoted cell aggregation, biofilm formation, and adherence to a range of ECM proteins. However, the two orthologues display differential cellular binding: EhaG mediates specific adhesion to colorectal epithelial cells while UpaG promotes specific binding to bladder epithelial cells. The EhaG and UpaG TAAs contain extensive sequence divergence in their respective passenger domains that could account for these differences. Indeed, sequence analyses of UpaG and EhaG homologues from severalE. coligenomes revealed grouping of the proteins in clades almost exclusively represented by distinctE. colipathotypes. The expression of EhaG (in EHEC) and UpaG (in UPEC) was also investigated and shown to be significantly enhanced in anhnsisogenic mutant, suggesting that H-NS acts as a negative regulator of both TAAs. Thus, while the EhaG and UpaG TAAs contain some conserved binding and regulatory features, they also possess important differences that correlate with the distinct pathogenic lifestyles of EHEC and UPEC.

scholarly journals NlpI Facilitates Deposition of C4bp on Escherichia coli by Blocking Classical Complement-Mediated Killing, Which Results in High-Level Bacteremia

2012 ◽  
Vol 80 (10) ◽  
pp. 3669-3678 ◽  
Author(s):  
Yu-ting Tseng ◽  
Shainn-Wei Wang ◽  
Kwang Sik Kim ◽  
Ying-Hsiang Wang ◽  
Yufeng Yao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Neonatal Meningitis ◽  
Content Type ◽  
E Coli ◽  
Classical Complement Pathway ◽  
High Level ◽  
Classical Complement

ABSTRACTNeonatal meningitisEscherichia coli(NMEC) is the most common Gram-negative organism that is associated with neonatal meningitis, which usually develops as a result of hematogenous spread of the bacteria. There are two key pathogenesis processes for NMEC to penetrate into the brain, the essential step for the development ofE. colimeningitis: a high-level bacteremia and traversal of the blood-brain barrier (BBB). Our previous study has shown that the bacterial outer membrane protein NlpI contributes to NMEC binding to and invasion of brain microvascular endothelial cells, the major component cells of the BBB, suggesting a role for NlpI in NMEC crossing of the BBB. In this study, we showed that NlpI is involved in inducing a high level of bacteremia. In addition, NlpI contributed to the recruitment of the complement regulator C4bp to the surface of NMEC to evade serum killing, which is mediated by the classical complement pathway. NlpI may be involved in the interaction between C4bp and OmpA, which is an outer membrane protein that directly interacts with C4bp on the bacterial surface. The involvement of NlpI in two key pathogenesis processes of NMEC meningitis may make this bacterial factor a potential target for prevention and therapy ofE. colimeningitis.

scholarly journals O-Antigen-Dependent Colicin Insensitivity of UropathogenicEscherichia coli

2018 ◽  
Vol 201 (4) ◽  
Author(s):  
Connor Sharp ◽  
Christine Boinett ◽  
Amy Cain ◽  
Nicholas G. Housden ◽  
Sandip Kumar ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Growth Conditions ◽  
Membrane Receptors ◽  
Cell Surface Receptor ◽  
Dominant Factor ◽  
Content Type ◽  
E Coli ◽  
O Antigen ◽  
K 12

ABSTRACTThe outer membrane of Gram-negative bacteria presents a significant barrier for molecules entering the cell. Nevertheless, colicins, which are antimicrobial proteins secreted byEscherichia coli, can target otherE. colicells by binding to cell surface receptor proteins and activating their import, resulting in cell death. Previous studies have documented high rates of nonspecific resistance (insensitivity) of variousE. colistrains toward colicins that is independent of colicin-specific immunity and is instead associated with lipopolysaccharide (LPS) in the outer membrane. This observation poses a contradiction: why doE. colistrains have colicin-expressing plasmids, which are energetically costly to retain, if cells around them are likely to be naturally insensitive to the colicin they produce? Here, using a combination of transposon sequencing and phenotypic microarrays, we show that colicin insensitivity of uropathogenicE. colisequence type 131 (ST131) is dependent on the production of its O-antigen but that minor changes in growth conditions render the organism sensitive toward colicins. The reintroduction of O-antigen intoE. coliK-12 demonstrated that it is the density of O-antigen that is the dominant factor governing colicin insensitivity. We also show, by microscopy of fluorescently labelled colicins, that growth conditions affect the degree of occlusion by O-antigen of outer membrane receptors but not the clustered organization of receptors. The result of our study demonstrate that environmental conditions play a critical role in sensitizingE. colitoward colicins and that O-antigen in LPS is central to this role.IMPORTANCEEscherichia coliinfections can be a major health burden, especially with the organism becoming increasingly resistant to “last-resort” antibiotics such as carbapenems. Although colicins are potent narrow-spectrum antimicrobials with potential as future antibiotics, high levels of naturally occurring colicin insensitivity have been documented which could limit their efficacy. We identify O-antigen-dependent colicin insensitivity in a clinically relevant uropathogenicE. colistrain and show that this insensitivity can be circumvented by minor changes to growth conditions. The results of our study suggest that colicin insensitivity amongE. coliorganisms has been greatly overestimated, and as a consequence, colicins could in fact be effective species-specific antimicrobials targeting pathogenicE. colisuch as uropathogenicE. coli(UPEC).

scholarly journals Flocculation of Escherichia coli Cells in Association with Enhanced Production of Outer Membrane Vesicles

2015 ◽  
Vol 81 (17) ◽  
pp. 5900-5906 ◽  
Author(s):  
Yoshihiro Ojima ◽  
Minh Hong Nguyen ◽  
Reiki Yajima ◽  
Masahito Taya
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Sodium Dodecyl ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Laser Scanning Microscopy ◽  
Content Type ◽  
E Coli ◽  
Enhanced Production ◽  
K 12

ABSTRACTMicrobial flocculation is a phenomenon of aggregation of dispersed bacterial cells in the form of flocs or flakes. In this study, the mechanism of spontaneous flocculation ofEscherichia colicells by overexpression of thebcsBgene was investigated. The flocculation induced by overexpression ofbcsBwas consistent among the variousE. colistrains examined, including the K-12, B, and O strains, with flocs that resembled paper scraps in structure being about 1 to 2 mm. The distribution of green fluorescent protein-labeledE. colicells within the floc structure was investigated by three-dimensional confocal laser scanning microscopy. Flocs were sensitive to proteinase K, indicating that the main component of the flocs was proteinous. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nano-liquid chromatography tandem mass spectrometry analyses of the flocs strongly suggested the involvement of outer membrane vesicles (OMVs) inE. coliflocculation. The involvement of OMVs in flocculation was supported by transmission electron microscopy observation of flocs. Furthermore,bcsB-inducedE. coliflocculation was greatly suppressed in strains with hypovesiculation phenotypes (ΔdsbAand ΔdsbBstrains). Thus, our results demonstrate the strong correlation between spontaneous flocculation and enhanced OMV production ofE. colicells.

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