EnteropathogenicEscherichia coliinhibits butyrate uptake in Caco-2 cells by altering the apical membrane MCT1 level

2006 ◽  
Vol 290 (1) ◽  
pp. G30-G35 ◽  
Author(s):  
Alip Borthakur ◽  
Ravinder K. Gill ◽  
Kim Hodges ◽  
Krishnamurthy Ramaswamy ◽  
Gail Hecht ◽  
...  
Keyword(s):  
Surface Expression ◽  
Monocarboxylate Transporter ◽  
Wild Type ◽  
Infantile Diarrhea ◽  
Monocarboxylate Transporter 1 ◽  
E Coli ◽  
Food Borne ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Fatty Acid Absorption

Enteropathogenic Escherichia coli (EPEC), a food-borne human pathogen, is responsible for infantile diarrhea, especially in developing countries. The pathophysiology of EPEC-induced diarrhea, however, is not completely understood. Our recent studies showed modulation of Na+/H+and Cl−/HCO3−exchange activities in Caco-2 cells in response to EPEC infection. We hypothesized that intestinal short-chain fatty acid absorption mediated by monocarboxylate transporter 1 (MCT1) might also be altered by EPEC infection. The aim of the current studies was to examine the effect of EPEC infection on butyrate uptake. Caco-2 cells were infected with wild-type EPEC, various mutant strains, or nonpathogenic E. coli HS4, and [14C]butyrate uptake was determined. EPEC, but not nonpathogenic E. coli, significantly decreased butyrate uptake. Infection of cells with strains harboring mutations in escN, which encodes a putative ATPase for the EPEC type III secretion system (TTSS), or in the espA, espB, or espD genes encoding structural components of the TTSS, had no effect on butyrate uptake, indicating the TTSS dependence. On the other hand, strains with mutations in the effector protein genes espF, espG, espH, and map inhibited butyrate uptake, similar to the wild-type EPEC. Surface expression of MCT1 decreased considerably after EPEC but not after nonpathogenic E. coli infection. In conclusion, our studies demonstrate inhibition of MCT1-mediated butyrate uptake in Caco-2 cells in response to EPEC infection. This inhibition was dependent on a functional TTSS and the structural proteins EspA, -B, and -D of the translocation apparatus.

scholarly journals Mechanisms of DRA recycling in intestinal epithelial cells: effect of enteropathogenic E. coli

2015 ◽  
Vol 309 (12) ◽  
pp. C835-C846 ◽  
Author(s):  
Tarunmeet Gujral ◽  
Anoop Kumar ◽  
Shubha Priyamvada ◽  
Seema Saksena ◽  
Ravinder K. Gill ◽  
...  
Keyword(s):  
Virulence Genes ◽  
Surface Expression ◽  
Intestinal Epithelial ◽  
Basal Surface ◽  
Infantile Diarrhea ◽  
E Coli ◽  
Food Borne ◽  
Cell Surface Biotinylation ◽  
Exchange Activity

Enteropathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl−/HCO3− exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl−/HCO3− exchange activity was measured as DIDS-sensitive 125I− uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 μM) increased apical Cl−/HCO3− exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis.

scholarly journals act Operon Control of Developmental Gene Expression in Myxococcus xanthus

2002 ◽  
Vol 184 (4) ◽  
pp. 1172-1179 ◽  
Author(s):  
Thomas M. A. Gronewold ◽  
Dale Kaiser
Keyword(s):  
Fruiting Body ◽  
Myxococcus Xanthus ◽  
The Other ◽  
Loss Of Function ◽  
Wild Type ◽  
Developmental Gene ◽  
Developmental Gene Expression ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Signal Production

ABSTRACT Cell-bound C-signal guides the building of a fruiting body and triggers the differentiation of myxospores. Earlier work has shown that transcription of the csgA gene, which encodes the C-signal, is directed by four genes of the act operon. To see how expression of the genes encoding components of the aggregation and sporulation processes depends on C-signaling, mutants with loss-of-function mutations in each of the act genes were investigated. These mutations were found to have no effect on genes that are normally expressed up to 3 h into development and are C-signal independent. Neither the time of first expression nor the rate of expression increase was changed in actA, actB, actC, or actD mutant strains. Also, there was no effect on A-signal production, which normally starts before 3 h. By contrast, the null act mutants have striking defects in C-signal production. These mutations changed the expression of four gene reporters that are related to aggregation and sporulation and are expressed at 6 h or later in development. The actA and actB null mutations substantially decreased the expression of all these reporters. The other act null mutations caused either premature expression to wild-type levels (actC) or delayed expression (actD), which ultimately rose to wild-type levels. The pattern of effects on these reporters shows how the C-signal differentially regulates the steps that together build a fruiting body and differentiate spores within it.

LisRK is required for optimal fitness of Listeria monocytogenes in soil

2020 ◽  
Vol 367 (22) ◽  
Author(s):  
Maja Z Brunhede ◽  
Patrícia T Dos Santos ◽  
Laurent Gal ◽  
Dominique Garmyn ◽  
Birgitte H Kallipolitis ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Soil Environment ◽  
Sterile Soil ◽  
Natural Habitats ◽  
Growth And Survival ◽  
Food Borne ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Two Component Systems ◽  
Or Genes

ABSTRACT Listeria monocytogenes is a food-borne pathogen responsible for the disease listeriosis. It is ubiquitously found in the environment and soil is one of its natural habitats. Listeria monocytogenes is highly capable of coping with various stressful conditions. We hypothesized that stress-responsive two-component systems such as LisRK might contribute to the adaptation of L. monocytogenes to the soil environment. Indeed, investigations of the population dynamics of wild-type and mutant strains suggest an important role of LisRK for optimal fitness of L. monocytogenes in sterile soil. Results from non-sterile soil showed that the parental strain was capable of surviving longer than mutant strains lacking lisRK or genes encoding the LisRK-regulated LhrC small RNAs (sRNAs), suggesting that LisRK as well as the LhrC sRNAs were important for survival. Transcription of five LisRK-regulated genes was assessed after 1 h incubation in sterile soil. We observed that LisRK and the LhrC sRNAs contribute to the upregulation of lmo2522 in the soil environment. Notably, lmo2522 encodes an equivalent of the resuscitation promoting factors, Rpfs, in actinobacteria. Collectively, our study demonstrates that LisRK is important for growth and survival in sterile and non-sterile soil and suggests a role for LisRK-regulation of Lmo2522 in resuscitation from dormancy in the soil environment.

scholarly journals Electrical Phenotypes of Calcium Transport Mutant Strains of a Filamentous Fungus, Neurospora crassa

2012 ◽  
Vol 11 (5) ◽  
pp. 694-702 ◽  
Author(s):  
Ahmed Hamam ◽  
Roger R. Lew
Keyword(s):  
Atpase Activity ◽  
Calcium Transport ◽  
Tip Growth ◽  
Electrical Characterization ◽  
Mechanosensitive Channel ◽  
Wild Type ◽  
Current Voltage ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Hyphal Tip

ABSTRACT We characterized the electrical phenotypes of mutants with mutations in genes encoding calcium transporters—a mechanosensitive channel homolog ( MscS ), a Ca 2+ /H + exchange protein ( cax ), and Ca 2+ -ATPases ( nca-1 , nca-2 , nca-3 )—as well as those of double mutants (the nca-2 cax , nca-2 nca-3 , and nca-3 cax mutants). The electrical characterization used dual impalements to obtain cable-corrected current-voltage measurements. Only two types of mutants (the MscS mutant; the nca-2 mutant and nca-2 -containing double mutants) exhibited lower resting potentials. For the nca-2 mutant, on the basis of unchanged conductance and cyanide-induced depolarization of the potential, the cause is attenuated H + -ATPase activity. The growth of the nca-2 mutant-containing strains was inhibited by elevated extracellular Ca 2+ levels, indicative of lesions in Ca 2+ homeostasis. However, the net Ca 2+ effluxes of the nca-2 mutant, measured noninvasively with a self-referencing Ca 2+ -selective microelectrode, were similar to those of the wild type. All of the mutants exhibited osmosensitivity similar to that of the wild type (the turgor of the nca-2 mutant was also similar to that of the wild type), suggesting that Ca 2+ signaling does not play a role in osmoregulation. The hyphal tip morphology and tip-localized mitochondria of the nca-2 mutant were similar to those of the wild type, even when the external [Ca 2+ ] was elevated. Thus, although Ca 2+ homeostasis is perturbed in the nca-2 mutant (B. J. Bowman et al., Eukaryot. Cell 10:654–661, 2011), the phenotype does not extend to tip growth or to osmoregulation but is revealed by lower H + -ATPase activity.

Molecular Cloning of the gyrA andgyrB Genes of Bacteroides fragilis Encoding DNA Gyrase

1999 ◽  
Vol 43 (10) ◽  
pp. 2423-2429 ◽  
Author(s):  
Yoshikuni Onodera ◽  
Kenichi Sato
Keyword(s):  
Hot Spot ◽  
Bacteroides Fragilis ◽  
Dna Gyrase ◽  
E Coli ◽  
Important Target ◽  
Mutant Strains ◽  
Genes Encoding ◽  
Gyrase A ◽  
Selection For

ABSTRACT The genes encoding the DNA gyrase A and B subunits ofBacteroides fragilis were cloned and sequenced. ThegyrA and gyrB genes code for proteins of 845 and 653 amino acids, respectively. These proteins were expressed inEscherichia coli, and the combination of GyrA and GyrB exhibited ATP-dependent supercoiling activity. To analyze the role of DNA gyrase in quinolone resistance of B. fragilis, we isolated mutant strains by stepwise selection for resistance to increasing concentrations of levofloxacin. We analyzed the resistant mutants and showed that Ser-82 of GyrA, equivalent to resistance hot spot Ser-83 of GyrA in E. coli, was in each case replaced with Phe. These results suggest that DNA gyrase is an important target for quinolones in B. fragilis.

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 Sensitivity of Escherichia albertii, a Potential Food-Borne Pathogen, to Food Preservation Treatments

2007 ◽  
Vol 73 (13) ◽  
pp. 4351-4353 ◽  
Author(s):  
Manan Sharma ◽  
Kalmia E. Kniel ◽  
Alexandra Derevianko ◽  
Jason Ling ◽  
Arvind A. Bhagwat
Keyword(s):  
Diarrheal Disease ◽  
Food Preservation ◽  
Wild Type ◽  
E Coli ◽  
Acid Ph ◽  
Food Borne ◽  
Potential Food

ABSTRACT Escherichia albertii is a potential food-borne pathogen because of its documented ability to cause diarrheal disease by producing attachment and effacement lesions. Its tolerances to heat (56°C), acid (pH 3.0), and pressure (500 MPa [5 min]) were evaluated and found to be significantly less than those of wild-type E. coli O157:H7.

scholarly journals Mutant analysis of glyceraldehyde 3-phosphate dehydrogenase in Escherichia coli

1979 ◽  
Vol 179 (1) ◽  
pp. 99-107 ◽  
Author(s):  
Jeffrey D. Hillman
Keyword(s):  
Escherichia Coli ◽  
Simple Procedure ◽  
Rabbit Antiserum ◽  
Double Diffusion ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Glycolytic Intermediate ◽  
E Coli ◽  
Catalytic Function ◽  
Mutant Strains

NAD+-specific glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12) from Escherichia coli was purified to homogeneity by a relatively simple procedure involving affinity chromatography on agarose–hexane–NAD+ and repeated crystallization. Rabbit antiserum directed against this protein produced one precipitin line in double-diffusion studies against the pure enzyme, and two lines against crude extracts of wild-type E. coli strains. Both precipitin lines represent the interaction of antibody with determinants specific for glyceraldehyde 3-phosphate dehydrogenase. Nine independent mutants of E. coli lacking glyceraldehyde 3-phosphate dehydrogenase activity all possessed some antigenic cross-reacting material to the wild-type enzyme. The mutants could be divided into three groups on the basis of the types and amounts of precipitin lines observed in double-diffusion experiments; one group formed little cross-reacting material. The cross-reacting material in crude cell-free extracts of several of the mutant strains were also tested for alterations in their affinity for NAD+ and their phosphorylative activity. The cumulative data indicate that the protein in several of the mutant strains is severely altered, and thus that glyceraldehyde 3-phosphate dehydrogenase is unlikely to have an essential, non-catalytic function such as buffering nicotinamide nucleotide or glycolytic-intermediate concentrations. Others of the mutants tested have cross-reacting material which behaved like the wild-type enzyme for the several parameters studied; the proteins from these strains, once purified, might serve as useful analogues of the wild-type enzyme.

scholarly journals Genetic Evidence for a Molybdopterin-Containing Tellurate Reductase

2013 ◽  
Vol 79 (10) ◽  
pp. 3171-3175 ◽  
Author(s):  
Joanne Theisen ◽  
Gerben J. Zylstra ◽  
Nathan Yee
Keyword(s):  
Genetic Evidence ◽  
Biosynthesis Pathway ◽  
Wild Type ◽  
Transport Pathway ◽  
Content Type ◽  
E Coli ◽  
Reduction Activity ◽  
Mutant Strains ◽  
Molybdate Transport ◽  
K 12

ABSTRACTThe genetic identity and cofactor composition of the bacterial tellurate reductase are currently unknown. In this study, we examined the requirement of molybdopterin biosynthesis and molybdate transporter genes for tellurate reduction inEscherichia coliK-12. The results show that mutants deleted of themoaA,moaB,moaE, ormoggene in the molybdopterin biosynthesis pathway lost the ability to reduce tellurate. Deletion of themodBormodCgene in the molybdate transport pathway also resulted in complete loss of tellurate reduction activity. Genetic complementation by the wild-type sequences restored tellurate reduction activity in the mutant strains. These findings provide genetic evidence that tellurate reduction inE. coliinvolves a molybdoenzyme.

scholarly journals Use of Inducible Feedback-ResistantN-Acetylglutamate Synthetase (argA) Genes for Enhanced Arginine Biosynthesis by Genetically EngineeredEscherichia coli K-12 Strains

1998 ◽  
Vol 64 (5) ◽  
pp. 1805-1811 ◽  
Author(s):  
B. S. Rajagopal ◽  
Joseph DePonte ◽  
Mendel Tuchman ◽  
Michael H. Malamy
Keyword(s):  
Feedback Inhibition ◽  
Expression System ◽  
Point Mutations ◽  
Normal Growth ◽  
Growth Conditions ◽  
Wild Type ◽  
Arginine Biosynthesis ◽  
E Coli ◽  
Genes Encoding ◽  
K 12

ABSTRACT The goal of this work was to construct Escherichia colistrains capable of enhanced arginine production. The arginine biosynthetic capacity of previously engineered E. colistrains with a derepressed arginine regulon was limited by the availability of endogenous ornithine (M. Tuchman, B. S. Rajagopal, M. T. McCann, and M. H. Malamy, Appl. Environ. Microbiol. 63:33–38, 1997). Ornithine biosynthesis is limited due to feedback inhibition by arginine of N-acetylglutamate synthetase (NAGS), the product of the argA gene and the first enzyme in the pathway of arginine biosynthesis in E. coli. To circumvent this inhibition, the argA genes from E. coli mutants with feedback-resistant (fbr) NAGS were cloned into plasmids that contain “arg boxes,” which titrate the ArgR repressor protein, with or without the E. coli carABgenes encoding carbamyl phosphate synthetase and the argIgene for ornithine transcarbamylase. The free arginine production rates of “arg-derepressed” E. coli cells overexpressing plasmid-encoded carAB, argI, and fbr argA genes were 3- to 15-fold higher than that of an equivalent system overexpressing feedback-sensitive wild-type (wt)argA. The expression system with fbr argAproduced 7- to 35-fold more arginine than a system overexpressingcarAB and argI genes on a plasmid in a strain with a wt argA gene on the chromosome. The arginine biosynthetic capacity of arg-derepressed DH5α strains with plasmids containing only the fbr argA gene was similar to that of cells with plasmids also containing the carABand argI genes. Plasmids containing wt or fbrargA were stably maintained under normal growth conditions for at least 18 generations. DNA sequencing identified different point mutations in each of the fbr argA mutants, specifically H15Y, Y19C, S54N, R58H, G287S, and Q432R.

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