scholarly journals Role of a novel fimbrial adhesin in acid-induced host adhesion of escherichia coli 0157:H7

2021 ◽  
Author(s):  
Frances B Chingcuanco
Keyword(s):  
Escherichia Coli ◽  
Cell Adhesion ◽  
Acid Stress ◽  
Whole Genome ◽  
Deficient Mutant ◽  
Wild Type ◽  
Array Analysis ◽  
Genome Array ◽  
Fimbrial Adhesin

Enterohemorrhagic Eschericia coli (EHEC) 0157:H7 must be able to survive the acid stress of gastric passage. Previous studies show that adhesion of EHEC 0157:H7 to human epithelial cells is increased if EHEC was pre-exposed to acid. Whole genome array analysis of EHEC reveals that the putative fimbrial gene yadK is significantly upregulated after EHEC exposure to acid treatments. In this study, a YadK deficient mutant (△yadK) in EHEC 0157:H7 wild-type (WT) strain 85-170 was constructed and phenotyped. The results of this study demonstrate that △yadK exposed to acid stress showed no acid-induced increase in bacteria-host cell adhesion observed in similarly acid-induced wild type cells. This study concludes that YadK plays a role in acid-induced host adhesion of EHEC 0157:H7. It also indicates that acid stress, which is a part of the host’s natural assault to resist invasion, may regulate factors responsible for enhanced bacteria-host attachment, resulting in increased EHEC virulence.

scholarly journals Role of a novel fimbrial adhesin in acid-induced host adhesion of escherichia coli 0157:H7

2021 ◽  
Author(s):  
Frances B Chingcuanco
Keyword(s):  
Escherichia Coli ◽  
Cell Adhesion ◽  
Acid Stress ◽  
Whole Genome ◽  
Deficient Mutant ◽  
Wild Type ◽  
Array Analysis ◽  
Genome Array ◽  
Fimbrial Adhesin

Enterohemorrhagic Eschericia coli (EHEC) 0157:H7 must be able to survive the acid stress of gastric passage. Previous studies show that adhesion of EHEC 0157:H7 to human epithelial cells is increased if EHEC was pre-exposed to acid. Whole genome array analysis of EHEC reveals that the putative fimbrial gene yadK is significantly upregulated after EHEC exposure to acid treatments. In this study, a YadK deficient mutant (△yadK) in EHEC 0157:H7 wild-type (WT) strain 85-170 was constructed and phenotyped. The results of this study demonstrate that △yadK exposed to acid stress showed no acid-induced increase in bacteria-host cell adhesion observed in similarly acid-induced wild type cells. This study concludes that YadK plays a role in acid-induced host adhesion of EHEC 0157:H7. It also indicates that acid stress, which is a part of the host’s natural assault to resist invasion, may regulate factors responsible for enhanced bacteria-host attachment, resulting in increased EHEC virulence.

scholarly journals Dps Protects Enterohemorrhagic Escherichia coli against Acid-Induced Antimicrobial Peptide Killing

2020 ◽  
Vol 202 (11) ◽  
Author(s):  
Tracy Lackraj ◽  
Sarah Birstonas ◽  
Michele Kacori ◽  
Debora Barnett Foster
Keyword(s):  
Escherichia Coli ◽  
Dna Damage ◽  
Antimicrobial Peptide ◽  
Hydroxyl Radicals ◽  
Acid Stress ◽  
Dna Binding Protein ◽  
Wild Type ◽  
Content Type ◽  
Flow Cytometric

ABSTRACT Dps, a DNA-binding protein from starved cells in Escherichia coli, is part of the bacterial defense system that protects DNA against various cellular stresses. Our lab previously demonstrated that a novel antimicrobial peptide, WRWYCR, enhances acid-induced killing of enterohemorrhagic Escherichia coli (EHEC) and ameliorates infection in a Citrobacter rodentium mouse model of EHEC infection. WRWYCR has previously been shown to compromise DNA damage repair and to increase chelatable iron within the cell. These findings, combined with the effects of peptide and acid stress on DNA damage, suggest a key defense role for Dps in peptide-induced killing of EHEC. The goal of this study is to evaluate the role of Dps in peptide-induced killing of EHEC through survival assays and flow cytometric analyses of DNA damage and hydroxyl radical formation. Our results demonstrate that disruption of the dps gene in stationary-phase EHEC O157:H7 cells, but not in exponential-phase cells, enhances acid-, peptide-, and peptide-acid-induced killing relative to that of wild-type (WT) EHEC. Using flow cytometric analysis, we have also demonstrated increased levels of hydroxyl radicals in peptide-treated wild-type EHEC relative to those in the untreated control. Disruption of the dps gene further increases this. These findings indicate that peptide treatment of EHEC enhances the formation of hydroxyl radicals, likely through the Fenton reaction, thereby contributing to the killing action of the peptide, and that dps protects against peptide killing of EHEC. This study provides important insights into peptide WRWYCR-mediated killing of EHEC, which could be exploited in the development of more effective antimicrobials. IMPORTANCE The research presented in this paper explores the role of the DNA-binding protein Dps as a key defense mechanism of enterohemorrhagic Escherichia coli (EHEC) strains in protecting against killing by the novel antimicrobial peptide WRWYCR. Our results demonstrate that Dps protects against peptide-induced killing of EHEC through direct protection against acid stress and hydroxyl radical formation, both of which are mechanisms targeted by the antimicrobial peptide. This study provides important insights into peptide WRWYCR-mediated killing of EHEC, which could be exploited in the development of more effective antimicrobials through specific targeting of Dps in order to allow a more potent response to the antimicrobial WRWYCR.

scholarly journals Role Of Selected Fimbrial Adheins In Pathogenesis Of Acid-Induced Eneterohemorrhagic Escherichia Coli 0157:H7

2021 ◽  
Author(s):  
Shahnaz Haque
Keyword(s):  
Fatty Acid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Short Chain Fatty Acid ◽  
Acid Stress ◽  
Chain Fatty Acid ◽  
Short Chain ◽  
Wild Type ◽  
Food Borne

Enterohemorrhagic Escherichia coli (EHEC) 0157:H7 is a food-borne pathogen that causes hemolytic uremic syndrome and hemorrhagic colitis. The mechanisms underlying the adhesion of EHEC 0157:H7 to intestinal epithelial cells are not well understood. Like other food-borne pathogens, ECEC 0157:H7 must survive the acid stress of the gastric juice in the stomach and short chain fatty acid in the intestine in order to colonize the large intestine. We have found that acid stress and short chain fatty acid stress significantly enhance host-adhesion of EHEC 0157:H7 and also upregulates expression of EHEC fimbrial genes, lpfA1, lpfA2 and yagZ, as demonstrated by our DNA microarray. We now report that disruption of the yagZ (also known as the E. coli common pilus A) gene results in loss of the acid-induced and short chain fatty acid-induced adhesion increase seen for the wild type strain. When the yagZ mutant is complemented with yagZ, the sress-induced and short chain fatty acid-induced adhesion increase seen for the wild type strain. When the yagZ mutant is complemented with yagZ, the stress-induced adhesion pehnotype is restored, confirming the role of yagZ in the acid as well as short chain fatty acid induced adhesion to HEp-2 cells. On the other hand, neither disruption in the long polar fimbria genes lpfA1 or lpfA2 in the wild type showed any effect in adherence to HEp-2 cells; rather displaying a hyperadherant phenotype to HEp-2 cells after acid-induced or short chain fatty acid-induced stress. The results also indicate that acid or short chain fatty acid stress, which is a part of the host's natural defense mechanism against pathogens, may regulate virulence factors resulting in enhanced bacteria-host attachment during colonization in the human or bovine host.

Abstract 110: Thrombospondin Type-1 Domain-Containing Protein 1 (THSD1), an Intracranial Aneurysm Susceptibility Gene, Mediates Cell Adhesion in Human Umbilical Vein Endothelial Cells

Stroke ◽  
2015 ◽  
Vol 46 (suppl_1) ◽  
Author(s):  
Xiaoqian Fang ◽  
Dong H Kim ◽  
Teresa Santiago-Sim
Keyword(s):  
Endothelial Cells ◽  
Cell Adhesion ◽  
Focal Adhesion ◽  
Umbilical Vein ◽  
Adhesion Formation ◽  
Wild Type ◽  
Focal Adhesion Formation ◽  
Umbilical Vein Endothelial Cells

Introduction: An intracranial aneurysm (IA) is a weak spot in cerebral blood vessel wall that can lead to its abnormal bulging. Previously, we reported that mutations in THSD1 , encoding thrombospondin type-1 domain-containing protein 1, are associated with IA in a subset of patients. THSD1 is a transmembrane molecule with a thrombospondin type-1 repeat (TSR). Proteins with TSR domain have been implicated in a variety of processes including regulation of matrix organization, cell adhesion and migration. We have shown that in mouse brain Thsd1 is expressed in endothelial cells. Hypothesis: THSD1 plays an important role in maintaining the integrity of the endothelium by promoting adhesion of endothelial cells to the underlying basement membrane. Methods: Human umbilical vein endothelial cells are used to investigate the role of THSD1 in vitro . THSD1 expression was knocked-down by RNA interference. Cell adhesion assay was done on collagen I-coated plates and focal adhesion formation was visualized using immunofluorescence by paxillin and phosphorylated focal adhesion kinase (pFAK) staining. THSD1 re-expression is accomplished by transfection with a pCR3.1-THSD1-encoding plasmid. Results: Knockdown of THSD1 caused striking change in cell morphology and size. Compared to control siRNA-treated cells that exhibited typical cobblestone morphology, THSD1 knockdown cells were narrow and elongated, and were significantly smaller ( p <0.01). Cell adherence to collagen I-coated plates was also attenuated in THSD1 knockdown cells ( p <0.01). Consistent with this finding is the observation that the number and size of focal adhesions, based on paxillin and pFAK staining, were significantly reduced after THSD1 knockdown ( p <0.01). These defects in cell adhesion and focal adhesion formation were rescued by re-expression of wild type THSD1 ( p <0.05). In contrast, initial studies indicate that expression of mutated versions of THSD1 as seen in human patients (L5F, R450*, E466G, P639L) could not restore cell adhesion and focal adhesion formation to wild type levels. Conclusions: Our studies provide evidence for a role of THSD1 and THSD1 mutations in endothelial cell adhesion and suggest a possible mechanism underlying THSD1 -mediated aneurysm disease.

Abstract 361: Creg1 Interacts With Sec8 to Promote Cell-cell Adhesion During Cardiomyogenesis

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Jie Liu ◽  
Yanmei Qi ◽  
Shu-Chan Hsu ◽  
Siavash Saadat ◽  
Saum Rahimi ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Es Cells ◽  
Embryonic Stem ◽  
Intercellular Junctions ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Wild Type ◽  
Adhesion Sites ◽  
Cell Cell

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a 24 kD glycoprotein essential for early embryonic development. Our immunofluorescence studies revealed that CREG1 is highly expressed at myocyte junctions in both embryonic and adult hearts. To explore it role in cardiomyogenesis, we employed gain- and loss-of-function analyses demonstrating that CREG1 is required for the differentiation of mouse embryonic stem (ES) cell into cohesive myocardium-like structures. Chimeric cultures of wild-type and CREG1 knockout ES cells expressing cardiac-specific reporters showed that the cardiomyogenic effect of CREG1 is cell autonomous. Furthermore, we identified a novel interaction between CREG1 and Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Mutations of the amino acid residues D141 and P142 to alanine in CREG1 abolished its binding to Sec8. To address the role of the CREG1-Sec8 interaction in cardiomyogenesis, we rescued CREG1 knockout ES cells with wild-type and Sec8-binding mutant CREG1 and showed that CREG1 binding to Sec8 promotes cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8 and N-cadherin all localize at cell-cell adhesion sites. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. Finally, shRNA-mediated knockdown of Sec8 leads to cardiomyogenic defects similar to CREG1 knockout. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis.

scholarly journals Fimbria-Encoding GeneyadCHas a Pleiotropic Effect on Several Biological Characteristics and Plays a Role in Avian Pathogenic Escherichia coli Pathogenicity

2015 ◽  
Vol 84 (1) ◽  
pp. 187-193 ◽  
Author(s):  
Renu Verma ◽  
Thaís Cabrera Galvão Rojas ◽  
Renato Pariz Maluta ◽  
Janaína Luisa Leite ◽  
Livia Pilatti Mendes da Silva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Soft Agar ◽  
Pleiotropic Effect ◽  
Biological Characteristics ◽  
Wild Type ◽  
Content Type ◽  
Pathogenic Escherichia Coli

The extraintestinal pathogen termed avian pathogenicEscherichia coli(APEC) is known to cause colibacillosis in chickens. The molecular basis of APEC pathogenesis is not fully elucidated yet. In this work, we deleted a component of the Yad gene cluster (yadC) in order to understand the role of Yad in the pathogenicity of the APEC strain SCI-07.In vitro, the transcription level ofyadCwas upregulated at 41°C and downregulated at 22°C. TheyadCexpressionin vivowas more pronounced in lungs than in spleen, suggesting a role in the early steps of the infection. Chicks infected with the wild-type and mutant strains presented, respectively, 80% and 50% mortality rates. The ΔyadCstrain presented a slightly decreased ability to adhere to HeLa cells with or without thed-mannose analog compared with the wild type. Real-time PCR (RT-PCR) assays showed thatfimHwas downregulated (P< 0.05) andcsgAandecpAwere slightly upregulated in the mutant strain, showing thatyadCmodulates expression of other fimbriae. Bacterial internalization studies showed that the ΔyadCstrain had a lower number of intracellular bacteria recovered from Hep-2 cells and HD11 cells than the wild-type strain (P< 0.05). Motility assays in soft agar demonstrated that the ΔyadCstrain was less motile than the wild type (P< 0.01). Curiously, flagellum-associated genes were not dramatically downregulated in the ΔyadCstrain. Taken together, the results show that the fimbrial adhesin Yad contributes to the pathogenicity and modulates different biological characteristics of the APEC strain SCI-07.

scholarly journals Transposon Insertion in the purL Gene Induces Biofilm Depletion in Escherichia coli ATCC 25922

Pathogens ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 774
Author(s):  
Virginio Cepas ◽  
Victoria Ballén ◽  
Yaiza Gabasa ◽  
Miriam Ramírez ◽  
Yuly López ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
De Novo ◽  
Wild Type ◽  
Planktonic Cells ◽  
E Coli ◽  
Qrt Pcr ◽  
Transposon Insertion ◽  
De Novo Purine Biosynthesis

Current Escherichia coli antibiofilm treatments comprise a combination of antibiotics commonly used against planktonic cells, leading to treatment failure. A better understanding of the genes involved in biofilm formation could facilitate the development of efficient and specific new antibiofilm treatments. A total of 2578 E. coli mutants were generated by transposon insertion, of which 536 were analysed in this study. After sequencing, Tn263 mutant, classified as low biofilm-former (LF) compared to the wild-type (wt) strain (ATCC 25922), showed an interruption in the purL gene, involved in the de novo purine biosynthesis pathway. To elucidate the role of purL in biofilm formation, a knockout was generated showing reduced production of curli fibres, leading to an impaired biofilm formation. These conditions were restored by complementation of the strain or addition of exogenous inosine. Proteomic and transcriptional analyses were performed to characterise the differences caused by purL alterations. Thirteen proteins were altered compared to wt. The corresponding genes were analysed by qRT-PCR not only in the Tn263 and wt, but also in clinical strains with different biofilm activity. Overall, this study suggests that purL is essential for biofilm formation in E. coli and can be considered as a potential antibiofilm target.

scholarly journals The RcsCB His-Asp Phosphorelay System Is Essential To Overcome Chlorpromazine-Induced Stress in Escherichia coli

2002 ◽  
Vol 184 (10) ◽  
pp. 2850-2853 ◽  
Author(s):  
Annie Conter ◽  
Rachel Sturny ◽  
Claude Gutierrez ◽  
Kaymeuang Cam
Keyword(s):  
Escherichia Coli ◽  
Type Strain ◽  
Wild Type Strain ◽  
Cell Envelope ◽  
Wild Type ◽  
E Coli ◽  
Induced Stress ◽  
Mutant Strains ◽  
Molecular Nature

ABSTRACT The RcsCB His-Asp phosphorelay system regulates the expression of several genes of Escherichia coli, but the molecular nature of the inducing signal is still unknown. We show here that treatment of an exponentially growing culture of E. coli with the cationic amphipathic compound chlorpromazine (CPZ) stimulates expression of a set of genes positively regulated by the RcsCB system. This induction is abolished in rcsB or rcsC mutant strains. In addition, treatment with CPZ inhibits growth. The wild-type strain is able to recover from this inhibition and resume growth after a period of adaptation. In contrast, strains deficient in the RcsCB His-Asp phosphorelay system are hypersensitive to CPZ. These results suggest that cells must express specific RcsCB-regulated genes in order to cope with the CPZ-induced stress. This is the first report of the essential role of the RcsCB system in a stress situation. These results also strengthen the notion that alterations of the cell envelope induce a signal recognized by the RcsC sensor.

scholarly journals Role of Motility and the flhDC Operon in Escherichia coli MG1655 Colonization of the Mouse Intestine

2007 ◽  
Vol 75 (7) ◽  
pp. 3315-3324 ◽  
Author(s):  
Eric J. Gauger ◽  
Mary P. Leatham ◽  
Regino Mercado-Lubo ◽  
David C. Laux ◽  
Tyrrell Conway ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Region ◽  
Flagellar Motor ◽  
Wild Type ◽  
E Coli ◽  
Mouse Intestine ◽  
Flagellum Biosynthesis ◽  
Colonizing Ability

ABSTRACT Previously, we reported that the mouse intestine selected mutants of Escherichia coli MG1655 that have improved colonizing ability (M. P. Leatham et al., Infect. Immun. 73:8039-8049, 2005). These mutants grew 10 to 20% faster than their parent in mouse cecal mucus in vitro and 15 to 30% faster on several sugars found in the mouse intestine. The mutants were nonmotile and had deletions of various lengths beginning immediately downstream of an IS1 element located within the regulatory region of the flhDC operon, which encodes the master regulator of flagellum biosynthesis, FlhD4C2. Here we show that during intestinal colonization by wild-type E. coli strain MG1655, 45 to 50% of the cells became nonmotile by day 3 after feeding of the strain to mice and between 80 and 90% of the cells were nonmotile by day 15 after feeding. Ten nonmotile mutants isolated from mice were sequenced, and all were found to have flhDC deletions of various lengths. Despite this strong selection, 10 to 20% of the E. coli MG1655 cells remained motile over a 15-day period, suggesting that there is an as-yet-undefined intestinal niche in which motility is an advantage. The deletions appear to be selected in the intestine for two reasons. First, genes unrelated to motility that are normally either directly or indirectly repressed by FlhD4C2 but can contribute to maximum colonizing ability are released from repression. Second, energy normally used to synthesize flagella and turn the flagellar motor is redirected to growth.

scholarly journals In Vivo Effect of NusB and NusG on rRNA Transcription Antitermination

2004 ◽  
Vol 186 (5) ◽  
pp. 1304-1310 ◽  
Author(s):  
Martha Torres ◽  
Joan-Miquel Balada ◽  
Malcolm Zellars ◽  
Craig Squires ◽  
Catherine L. Squires
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Protein Complex ◽  
Test System ◽  
Wild Type ◽  
Rrna Transcription ◽  
Transcription Antitermination ◽  
Gene Mrna

ABSTRACT Similarities between lambda and rRNA transcription antitermination have led to suggestions that they involve the same Nus factors. However, direct in vivo confirmation that rRNA antitermination requires all of the lambda Nus factors is lacking. We have therefore analyzed the in vivo role of NusB and NusG in rRNA transcription antitermination and have established that both are essential for it. We used a plasmid test system in which reporter gene mRNA was measured to monitor rRNA antiterminator-dependent bypass of a Rho-dependent terminator. A comparison of terminator read-through in a wild-type Escherichia coli strain and that in a nusB::IS10 mutant strain determined the requirement for NusB. In the absence of NusB, antiterminator-dependent terminator read-through was not detected, showing that NusB is necessary for rRNA transcription antitermination. The requirement for NusG was determined by comparing rRNA antiterminator-dependent terminator read-through in a strain overexpressing NusG with that in a strain depleted of NusG. In NusG-depleted cells, termination levels were unchanged in the presence or absence of the antiterminator, demonstrating that NusG, like NusB, is necessary for rRNA transcription antitermination. These results imply that NusB and NusG are likely to be part of an RNA-protein complex formed with RNA polymerase during transcription of the rRNA antiterminator sequences that is required for rRNA antiterminator-dependent terminator read-through.

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