scholarly journals Recruitment and membrane interactions of host cell proteins during attachment of enteropathogenic and enterohaemorrhagic Escherichia coli

2012 ◽  
Vol 445 (3) ◽  
pp. 383-392 ◽  
Author(s):  
Diana Munera ◽  
Eric Martinez ◽  
Svetlana Varyukhina ◽  
Arvind Mahajan ◽  
Jesus Ayala-Sanmartin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Actin Polymerization ◽  
Attachment Site ◽  
Bacterial Attachment ◽  
Dependent Manner ◽  
Cellular Processes ◽  
Host Cell Proteins ◽  
Enterohaemorrhagic Escherichia Coli ◽  
Attachment Sites ◽  
Infected Cells

EPEC (enteropathogenic Escherichia coli) and EHEC (enterohaemorrhagic Escherichia coli) are attaching and effacing pathogens frequently associated with infectious diarrhoea. EPEC and EHEC use a T3SS (type III secretion system) to translocate effectors that subvert different cellular processes to sustain colonization and multiplication. The eukaryotic proteins NHERF2 (Na+/H+ exchanger regulatory factor 2) and AnxA2 (annexin A2), which are involved in regulation of intestinal ion channels, are recruited to the bacterial attachment sites. Using a stable HeLa-NHERF2 cell line, we found partial co-localization of AnxA2 and NHERF2; in EPEC-infected cells, AnxA2 and NHERF2 were extensively recruited to the site of bacterial attachment. We confirmed that NHERF2 dimerizes and found that NHERF2 interacts with AnxA2. Moreover, we found that AnxA2 also binds both the N- and C-terminal domains of the bacterial effector Tir through its C-terminal domain. Immunofluorescence of HeLa cells infected with EPEC showed that AnxA2 is recruited to the site of bacterial attachment in a Tir-dependent manner, but independently of Tir-induced actin polymerization. Our results suggest that AnxA2 and NHERF2 form a scaffold complex that links adjacent Tir molecules at the plasma membrane forming a lattice that could be involved in retention and dissemination of other effectors at the bacterial attachment site.

scholarly journals Functional analysis of EspB from enterohaemorrhagic Escherichia coli

Microbiology ◽  
2005 ◽  
Vol 151 (10) ◽  
pp. 3277-3286 ◽  
Author(s):  
Hao-Jie Chiu ◽  
Wan-Jr Syu
Keyword(s):  
Escherichia Coli ◽  
Type Iii Secretion ◽  
Pore Formation ◽  
Transmembrane Domain ◽  
Attachment Site ◽  
Bacterial Attachment ◽  
The Other ◽  
Host Cells ◽  
Type Iii ◽  
Enterohaemorrhagic Escherichia Coli

In enterohaemorrhagic Escherichia coli (EHEC), the type III secretion protein EspB is translocated into the host cells and plays an important role in adherence, pore formation and effector translocation during infection. The secretion domain of EspB has been mapped previously. To define the other functional determinants of EspB, several plasmids encoding different fragments of EspB were created and analysed to see which of them lost the functions of the full-length molecule. One finding was that residues 118–190 of EspB were required for both efficient translocation of EspB and interaction of EspB with EspA. Additionally, the segment consisting of residues 217–312 was necessary for bacterial adherence. Furthermore, a predicted transmembrane domain (residues 99–118) was found to be critical for EHEC to cause red blood cell haemolysis, presumably by forming pores in the cell membrane. The same segment was also important for actin accumulation induced beneath the bacterial-attachment site. Taken together, these data indicate that the EspB protein (312 residues in total) has functions associated with its different regions. These regions may interact with each other or with other components of the type III system to orchestrate the intricate actions of EHEC during infection.

scholarly journals SepZ/EspZ Is Secreted and Translocated into HeLa Cells by the Enteropathogenic Escherichia coli Type III Secretion System

2005 ◽  
Vol 73 (7) ◽  
pp. 4327-4337 ◽  
Author(s):  
Kristen J. Kanack ◽  
J. Adam Crawford ◽  
Ichiro Tatsuno ◽  
Mohamed A. Karmali ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
Hela Cells ◽  
Type Iii Secretion ◽  
Type Iii Secretion System ◽  
Secretion System ◽  
Bacterial Attachment ◽  
Host Cells ◽  
Dependent Manner ◽  
Cell Infection ◽  
Type Iii

ABSTRACT Enteropathogenic Escherichia coli (EPEC) is a major bacterial cause of infantile diarrhea in developing countries and is the prototype for a group of gastrointestinal pathogens causing characteristic attaching and effacing (A/E) histopathology on intestinal epithelia. A/E pathogens utilize a type III secretion system (TTSS), encoded by the locus of enterocyte effacement (LEE) pathogenicity island, to deliver effector proteins into host cells. Here, we investigate sequence divergence of the LEE-encoded SepZ protein and identify it as a TTSS-secreted and -translocated molecule. SepZ is hypervariable among A/E pathogens, with sequences sharing between 60 to 81% amino acid identity with SepZ of EPEC. A SepZ-CyaA fusion was secreted and translocated into HeLa cells in a TTSS-dependent manner. Additionally, we determined that the first 20 amino acids of SepZ were sufficient to direct its translocation. In contrast to previous studies suggesting a role in invasion and the structure and/or regulation of the TTSS, we found that SepZ does not mediate uptake of EPEC into host cells or affect translocation and tyrosine phosphorylation of the translocated intimin receptor. Immunohistochemistry reveals that, after an extended HeLa cell infection, accumulated SepZ can be detected beneath the site of bacterial attachment in a subset of pedestal regions. To indicate its newly identified status as a translocated effector protein, we propose to rename SepZ as EspZ.

scholarly journals Covert Cross-Feeding Revealed by Genome-Wide Analysis of Fitness Determinants in a Synthetic Bacterial Mutualism

2020 ◽  
Vol 86 (13) ◽  
Author(s):  
Breah LaSarre ◽  
Adam M. Deutschbauer ◽  
Crystal E. Love ◽  
James B. McKinlay
Keyword(s):  
Escherichia Coli ◽  
Microbial Communities ◽  
Rhodopseudomonas Palustris ◽  
Microbial Interactions ◽  
Dependent Manner ◽  
Fermentation Products ◽  
Content Type ◽  
E Coli ◽  
Cellular Processes ◽  
Genome Wide

ABSTRACT Microbial interactions abound in natural ecosystems and shape community structure and function. Substantial attention has been given to cataloging mechanisms by which microbes interact, but there is a limited understanding of the genetic landscapes that promote or hinder microbial interactions. We previously developed a mutualistic coculture pairing Escherichia coli and Rhodopseudomonas palustris, wherein E. coli provides carbon to R. palustris in the form of glucose fermentation products and R. palustris fixes N2 gas and provides nitrogen to E. coli in the form of NH4+. The stable coexistence and reproducible trends exhibited by this coculture make it ideal for interrogating the genetic underpinnings of a cross-feeding mutualism. Here, we used random barcode transposon sequencing (RB-TnSeq) to conduct a genome-wide search for E. coli genes that influence fitness during cooperative growth with R. palustris. RB-TnSeq revealed hundreds of genes that increased or decreased E. coli fitness in a mutualism-dependent manner. Some identified genes were involved in nitrogen sensing and assimilation, as expected given the coculture design. The other identified genes were involved in diverse cellular processes, including energy production and cell wall and membrane biogenesis. In addition, we discovered unexpected purine cross-feeding from R. palustris to E. coli, with coculture rescuing growth of an E. coli purine auxotroph. Our data provide insight into the genes and gene networks that can influence a cross-feeding mutualism and underscore that microbial interactions are not necessarily predictable a priori. IMPORTANCE Microbial communities impact life on Earth in profound ways, including driving global nutrient cycles and influencing human health and disease. These community functions depend on the interactions that resident microbes have with the environment and each other. Thus, identifying genes that influence these interactions will aid the management of natural communities and the use of microbial consortia as biotechnology. Here, we identified genes that influenced Escherichia coli fitness during cooperative growth with a mutualistic partner, Rhodopseudomonas palustris. Although this mutualism centers on the bidirectional exchange of essential carbon and nitrogen, E. coli fitness was positively and negatively affected by genes involved in diverse cellular processes. Furthermore, we discovered an unexpected purine cross-feeding interaction. These results contribute knowledge on the genetic foundation of a microbial cross-feeding interaction and highlight that unanticipated interactions can occur even within engineered microbial communities.

scholarly journals Repetitive, Marker-Free, Site-Specific Integration as a Novel Tool for Multiple Chromosomal Integration of DNA

2013 ◽  
Vol 79 (12) ◽  
pp. 3563-3569 ◽  
Author(s):  
Kia Vest Petersen ◽  
Jan Martinussen ◽  
Peter Ruhdal Jensen ◽  
Christian Solem
Keyword(s):  
Low Frequency ◽  
Attachment Site ◽  
Plasmid Vector ◽  
Bacterial Attachment ◽  
Xylose Utilization ◽  
Site Specific ◽  
Content Type ◽  
Site Specific Integration ◽  
Attachment Sites ◽  
Marker Free

ABSTRACTWe present a tool for repetitive, marker-free, site-specific integration inLactococcus lactis, in which a nonreplicating plasmid vector (pKV6) carrying a phage attachment site (attP) can be integrated into a bacterial attachment site (attB). The novelty of the tool described here is the inclusion of a minimal bacterial attachment site (attBmin), two mutatedloxPsequences (lox66andlox71) allowing for removal of undesirable vector elements (antibiotic resistance marker), and a counterselection marker (oroP) for selection ofloxPrecombination on the pKV6 vector. When transformed intoL. lactisexpressing the phage TP901-1 integrase, pKV6 integrates with high frequency into the chromosome, where it is flanked byattLandattRhybrid attachment sites. After expression of Cre recombinase from a plasmid that is not able to replicate inL. lactis,loxPrecombinants can be selected for by using 5-fluoroorotic acid. The introducedattBminsite can subsequently be used for a second round of integration. To examine ifattPrecombination was specific to theattBsite, integration was performed in strains containing theattB,attL, andattRsites or theattLandattRsites only. OnlyattP-attBrecombination was observed when all three sites were present. In the absence of theattBsite, a low frequency ofattP-attLrecombination was observed. To demonstrate the functionality of the system, the xylose utilization genes (xylABRandxylT) fromL. lactisstrain KF147 were integrated into the chromosome ofL. lactisstrain MG1363 in two steps.

Development of the Micromonospora carbonacea var. africana ATCC 39149 bacteriophage pMLP1 integrase for site-specific integration in Micromonospora spp.

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2443-2453 ◽  
Author(s):  
Dylan C. Alexander ◽  
David J. Devlin ◽  
Duane D. Hewitt ◽  
Ann C. Horan ◽  
Thomas J. Hosted
Keyword(s):  
Escherichia Coli ◽  
Sequence Analysis ◽  
Integration Site ◽  
Attachment Site ◽  
Bacterial Attachment ◽  
Temperate Bacteriophage ◽  
Site Specific ◽  
Sequence Identity ◽  
Site Specific Integration ◽  
Chromosome Sequence

Micromonospora carbonacea var. africana ATCC 39149 contains a temperate bacteriophage, pMLP1, that is present both as a replicative element and integrated into the chromosome. Sequence analysis of a 4·4 kb KpnI fragment revealed pMLP1 att/int functions consisting of an integrase, an excisionase and the phage attachment site (attP). Plasmids pSPRH840 and pSPRH910, containing the pMLP1 att/int region, were introduced into Micromonospora spp. by conjugation from Escherichia coli. Sequence analysis of DNA flanking the integration site confirmed site-specific integration into a tRNAHis gene in the chromosome. The pMLP1 attP element and chromosomal bacterial attachment (attB) site contain a 24 bp region of sequence identity located at the 3′ end of the tRNA. Integration of pMLP1-based plasmids in M. carbonacea var. africana caused a loss of the pMLP1 phage. Placement of an additional attB site into the chromosome allowed integration of pSPRH840 into the alternate attB site. Plasmids containing the site-specific att/int functions of pMLP1 can be used to integrate genes into the chromosome.

scholarly journals Genome-wide analysis of Escherichia coli fitness determinants in a cross-feeding mutualism with Rhodopseudomonas palustris

2020 ◽  
Author(s):  
Breah LaSarre ◽  
Adam M. Deutschbauer ◽  
Crystal E. Love ◽  
James B. McKinlay
Keyword(s):  
Escherichia Coli ◽  
Microbial Communities ◽  
Rhodopseudomonas Palustris ◽  
Microbial Interactions ◽  
Dependent Manner ◽  
Fermentation Products ◽  
E Coli ◽  
Cellular Processes ◽  
Genome Wide ◽  
A Genome

ABSTRACTMicrobial interactions abound in natural ecosystems and shape community structure and function. Substantial attention has been given to cataloging mechanisms by which microbes interact, but there is a limited understanding of the genetic landscapes that promote or hinder microbial interactions. We previously developed a mutualistic coculture pairing Escherichia coli and Rhodopseudomonas palustris, wherein E. coli provides carbon to R. palustris in the form of glucose fermentation products and R. palustris fixes N2 gas and provides nitrogen to E. coli in the form of NH4+. The stable coexistence and reproducible trends exhibited by this coculture make it ideal for interrogating the genetic underpinnings of a cross-feeding mutualism. Here, we used random barcode transposon sequencing (RB-TnSeq) to conduct a genome-wide search for E. coli genes that influence fitness during cooperative growth with R. palustris. RB-TnSeq revealed hundreds of genes that increased or decreased E. coli fitness in a mutualism-dependent manner. Some identified genes were involved in nitrogen sensing and assimilation, as expected given the coculture design. The other identified genes were involved in diverse cellular processes, including energy production and cell wall and membrane biogenesis. Additionally, we discovered unexpected purine cross-feeding from R. palustris to E. coli, with coculture rescuing growth of an E. coli purine auxotroph. Our data provide insight into the genes and gene networks that can influence a cross-feeding mutualism and underscore that microbial interactions are not necessarily predictable a priori.IMPORTANCEMicrobial communities impact life on earth in profound ways, including driving global nutrient cycles and influencing human health and disease. These community functions depend on the interactions that resident microbes have with the environment and each other. Thus, identifying genes that influence these interactions will aid the management of natural communities and the use of microbial consortia as biotechnology. Here, we identified genes that influenced Escherichia coli fitness during cooperative growth with a mutualistic partner, Rhodospeudomonas palustris. Although this mutualism centers on the bidirectional exchange of essential carbon and nitrogen, E. coli fitness was positively and negatively affected by genes involved in diverse cellular processes. Furthermore, we discovered an unexpected purine cross-feeding interaction. These results contribute knowledge on the genetic foundation of a microbial cross-feeding interaction and highlight that unanticipated interactions can occur even within engineered microbial communities.

scholarly journals Septins suppress the release of vaccinia virus from infected cells

2018 ◽  
Vol 217 (8) ◽  
pp. 2911-2929 ◽  
Author(s):  
Julia Pfanzelter ◽  
Serge Mostowy ◽  
Michael Way
Keyword(s):  
Plasma Membrane ◽  
Vaccinia Virus ◽  
Membrane Trafficking ◽  
Live Cell Imaging ◽  
Actin Polymerization ◽  
Antiviral Effect ◽  
Cellular Processes ◽  
Viral Egress ◽  
Infected Cells ◽  
Cell Spread

Septins are conserved components of the cytoskeleton that play important roles in many fundamental cellular processes including division, migration, and membrane trafficking. Septins can also inhibit bacterial infection by forming cage-like structures around pathogens such as Shigella. We found that septins are recruited to vaccinia virus immediately after its fusion with the plasma membrane during viral egress. RNA interference–mediated depletion of septins increases virus release and cell-to-cell spread, as well as actin tail formation. Live cell imaging reveals that septins are displaced from the virus when it induces actin polymerization. Septin loss, however, depends on the recruitment of the SH2/SH3 adaptor Nck, but not the activity of the Arp2/3 complex. Moreover, it is the recruitment of dynamin by the third Nck SH3 domain that displaces septins from the virus in a formin-dependent fashion. Our study demonstrates that septins suppress vaccinia release by “entrapping” the virus at the plasma membrane. This antiviral effect is overcome by dynamin together with formin-mediated actin polymerization.

scholarly journals A SAC phosphoinositide phosphatase controls rice development via hydrolyzing phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate

2019 ◽  
Author(s):  
Tao Guo ◽  
Hua-Chang Chen ◽  
Zi-Qi Lu ◽  
Min Diao ◽  
Ke Chen ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Membrane Lipids ◽  
Dependent Manner ◽  
Cytoskeleton Organization ◽  
Cellular Processes ◽  
Phosphoinositide Phosphatase ◽  
Actin Cytoskeleton Organization ◽  
Phosphatidylinositol 4 ◽  
Related Proteins

AbstractPhosphoinositides (PIs) as regulatory membrane lipids play essential roles in multiple cellular processes. Although the exact molecular targets of PIs-dependent modulation remain largely elusive, the effects of disturbed PIs metabolism could be employed to propose regulatory modules associated with particular downstream targets of PIs. Here, we identified the role of GRAIN NUMBER AND PLANT HEIGHT 1 (GH1), which encodes a suppressor of actin (SAC) domain-containing phosphatase with unknown function in rice. Endoplasmic reticulum-localized GH1 specifically dephosphorylated and hydrolyzed phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Inactivation of GH1 resulted in massive accumulation of both PI4P and PI(4,5)P2, while excessive GH1 caused their depletion. Notably, superabundant PI4P and PI(4,5)P2 could both disrupt actin cytoskeleton organization and suppress cell elongation. Interestingly, both PI4P and PI(4,5)P2 inhibited actin-related proteins 2 and 3 (Arp2/3) complex-nucleated actin branching networks in vitro, whereas PI(4,5)P2 showed more dramatic effect in a dose-dependent manner. Overall, the overaccumulation of PI(4,5)P2 resulted from dysfunction of SAC phosphatase possibly perturbs Arp2/3 complex-mediated actin polymerization, thereby disordering the cell development. These findings imply that Arp2/3 complex might be the potential molecular target of PI(4,5)P2-dependent modulation in eukaryotes, thereby providing new insights into the relationship between PIs homeostasis and plants growth and development.

scholarly journals Differences in adherence and virulence gene expression between two outbreak strains of enterohaemorrhagic Escherichia coli O157 : H7

Microbiology ◽  
2010 ◽  
Vol 156 (2) ◽  
pp. 408-419 ◽  
Author(s):  
Galeb S. Abu-Ali ◽  
Lindsey M. Ouellette ◽  
Scott T. Henderson ◽  
Thomas S. Whittam ◽  
Shannon D. Manning
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Severe Disease ◽  
Expression Patterns ◽  
Disease Process ◽  
Bacterial Attachment ◽  
Significant Differential Expression ◽  
Enterohaemorrhagic Escherichia Coli

The Escherichia coli O157 : H7 TW14359 strain was implicated in a multi-state outbreak in North America in 2006, which resulted in high rates of severe disease. Similarly, the O157 : H7 RIMD0509952 (Sakai) strain caused the largest O157 : H7 outbreak to date. Both strains were shown to represent divergent phylogenetic lineages. Here we compared global gene expression patterns before and after epithelial cell exposure, as well as the ability to adhere to and invade epithelial cells, between the two outbreak strains. Epithelial cell assays demonstrated a 2.5-fold greater adherence of the TW14359 strain relative to Sakai, while whole-genome microarrays detected significant differential expression of 914 genes, 206 of which had a fold change ≥1.5. Interestingly, most locus of enterocyte effacement (LEE) genes were upregulated in TW14359, whereas flagellar and chemotaxis genes were primarily upregulated in Sakai, suggesting discordant expression of these genes between the two strains. The Shiga toxin 2 genes were also upregulated in the TW14359 strain, as were several pO157-encoded genes that promote adherence, including type II secretion genes and their effectors stcE and adfO. Quantitative RT-PCR confirmed the expression differences detected in the microarray analysis, and expression levels were lower for a subset of LEE genes before versus after exposure to epithelial cells. In all, this study demonstrated the upregulation of major and ancillary virulence genes in TW14359 and of flagellar and chemotaxis genes in Sakai, under conditions that precede intimate bacterial attachment to epithelial cells. Differences in the level of adherence to epithelial cells were also observed, implying that these two phylogenetically divergent O157 : H7 outbreak strains vary in their ability to colonize, or initiate the disease process.

scholarly journals Influence of Cranberry Extract on Tamm-Horsfall Protein in Human Urine and its Antiadhesive Activity Against Uropathogenic Escherichia coli

Planta Medica ◽  
2018 ◽  
Vol 85 (02) ◽  
pp. 126-138 ◽  
Author(s):  
Birte Scharf ◽  
Jandirk Sendker ◽  
Ulrich Dobrindt ◽  
Andreas Hensel
Keyword(s):  
Escherichia Coli ◽  
Bacterial Adhesion ◽  
Ex Vivo ◽  
Transmembrane Protein ◽  
Bacterial Attachment ◽  
Urine Samples ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Upec Strain ◽  
Bladder Cells

AbstractLC-MS characterized cranberry extract from the fruits of Vaccinium macrocarpon inhibited under in vitro conditions the bacterial adhesion of Escherichia coli strain 2980 uropathogenic E. coli (UPEC strains UTI89, NU14) to T24 bladder cells and adhesion of UPEC strain CFT073 to A498 kidney cells in a concentration-dependent manner. Within a biomedical study, urine samples from 16 volunteers (8 male, 8 female) consuming cranberry extract for 7 d (900 mg/d) were analyzed for potential antiadhesive activity against UPEC by ex vivo experiments. Results indicated inhibition of adhesion of UPEC strain UTI89 to human T24 bladder cells. Subgroup analysis proved significant inhibition of bacterial adhesion in case of urine samples obtained from male volunteers while female urine did not influence the bacterial attachment. Differences between antiadhesive capacity of urine samples from male/female volunteers were significant. Protein analysis of the urine samples indicated increased amounts of Tamm-Horsfall protein (THP, syn. uromodulin) in the active samples. Inhibition of bacterial adhesion by the urine samples was correlated to the respective amount of THP. As it is known that THP, a highly mannosylated glycoprotein, strongly interacts with FimH of UPEC, this will lead to a decreased interaction with uroplakin, a FimH-binding transmembrane protein of urothelial lining cells. From these data it can be concluded that the antiadhesive effect of cranberry after oral intake is not only related to the direct inhibition of bacterial adhesins by extract compounds but is additionally due to an induction of antiadhesive THP in the kidney.

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