Ler Gene Research Articles

Regulation of gene transcription in Escherichia coli O157:H7 in response to a natural derivate peptide of esculentin-1a used in combination with essential oils from plants of the Cymbopogon genus.

We analyzed the expression of several genes implicated in the pathogenicity of Escherichia coli O157:H7, treating bacteria with Esc(1-21), a derivative of peptide esculentin-1 in combination with three essential oils obtained from plants from the Cympopogon genus. We used the checkerboard assay to determine the antimicrobial activity of the combinations. We analyzed the expression of some genes implicated in the pathogenicity and quorum sensing system of E. coli O157:H7 by real-time RT-PCR technique. Treatment of the bacteria with the peptide combined with oils had an efficacious antimicrobial activity. The analysis of gene expression showed that all used combinations regulate positively the espAD and ler genes, located in the pathogenicity island, named the locus of enterocyte effacement. None of the combinations affects the quorum sensing genes: lsrABCFKR and qseBC. This study demonstrates that the use of essential oil/peptide combinations can be effective in fighting microbial infections.

Molecular Study of Regulatory Gene (Ler) in Enteropathogenic Escherichia Coli (EPEC) of Diarrheagenic Patients

The locus of enterocyte effacement LEE-encoded regulator (Ler( is a global regulator of multiple virulence genes expression in the Enteropathogenic Escherichia coli (EPEC), including those encoding the type III secretion pathway and adhesion proteins such as intimin. Ler is central to the process of the formation of the attaching and effacing (AE) lesions. This study aimed to perform the molecular detection of Ler gene in EPEC, since there is no related previous study in Iraq. Two hundred and fifty stool specimens from children under two years of age for both sexes were collected from some Iraqi hospitals. All isolates were diagnosed according to morphological characteristics and biochemical tests. The results showed that 140 (56%) samples were identified as E.coli, while 8 (5.7%) isolates were identified as EPEC as confirmed by using VITEK 2 system. Susceptibility test was determined for all EPEC isolates to 16 different antibiotics. The results showed that 100%of these isolates were resistant to Ampicillin, Cefazolin, Ceftriaxone, Cefepime, Trimethoprim and Ceftazidime, whereas resistance values to Nitrofurantoin, Cefoxitin and Gentamicin were 66%, 40%, and 15% respectively. However 100% of the isolates were sensitive to Piperacillin, Ertapenem, Imipenem, Amikacin, Ciprofloxacin, Levofloxacin and Tigecycline. Monoplex pattern of PCR was used for detecting 16SrRNA, eae, stx1, lifA and Ler genes in EPEC. The results showed that the isolates of E.coli were positive for 16SrRNA, eae, lifA and Ler, while no bands of stx1 appeared.

The impact of oregano (Origanum heracleoticum) essential oil and carvacrol on virulence gene transcription by Escherichia coli O157:H7.

The aim of the current study was to determine, via reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis, the effect of oregano essential oil (Origanum heracleoticum) and carvacrol, its major component, on the expression of virulence-associated genes in enterohaemorrhagic Escherichia coli (EHEC) O157:H7 ATCC strain 35150. Both oregano oil and carvacrol demonstrated their efficacy firstly, by inhibiting the transcription of the ler gene involved in upregulation of the LEE2, LEE3 and LEE4 promoters and of attaching and effacing lesions and secondly by decreasing both Shiga toxin and fliC genes expression. In addition, a decrease in luxS gene transcription involved in quorum sensing was observed. These results were dose dependent and showed a specific effect of O. heracleoticum and carvacrol in downregulating the expression of virulence genes in EHEC O157:H7. These findings suggest that oregano oil and carvacrol have the potential to mitigate the adverse health effects caused by virulence gene expression in EHEC O157:H7, through the use of these substances as natural antibacterial additives in foods or as an alternative to antibiotics.

Analysis of a plant complex resistance gene locus underlying immune-related hybrid incompatibility and its occurrence in nature.

Mechanisms underlying speciation in plants include detrimental (incompatible) genetic interactions between parental alleles that incur a fitness cost in hybrids. We reported on recessive hybrid incompatibility between an Arabidopsis thaliana strain from Poland, Landsberg erecta (Ler), and many Central Asian A. thaliana strains. The incompatible interaction is determined by a polymorphic cluster of Toll/interleukin-1 receptor-nucleotide binding-leucine rich repeat (TNL) RPP1 (Recognition of Peronospora parasitica1)-like genes in Ler and alleles of the receptor-like kinase Strubbelig Receptor Family 3 (SRF3) in Central Asian strains Kas-2 or Kond, causing temperature-dependent autoimmunity and loss of growth and reproductive fitness. Here, we genetically dissected the RPP1-like Ler locus to determine contributions of individual RPP1-like Ler (R1–R8) genes to the incompatibility. In a neutral background, expression of most RPP1-like Ler genes, except R3, has no effect on growth or pathogen resistance. Incompatibility involves increased R3 expression and engineered R3 overexpression in a neutral background induces dwarfism and sterility. However, no individual RPP1-like Ler gene is sufficient for incompatibility between Ler and Kas-2 or Kond, suggesting that co-action of at least two RPP1-like members underlies this epistatic interaction. We find that the RPP1-like Ler haplotype is frequent and occurs with other Ler RPP1-like alleles in a local population in Gorzów Wielkopolski (Poland). Only Gorzów individuals carrying the RPP1-like Ler haplotype are incompatible with Kas-2 and Kond, whereas other RPP1-like alleles in the population are compatible. Therefore, the RPP1-like Ler haplotype has been maintained in genetically different individuals at a single site, allowing exploration of forces shaping the evolution of RPP1-like genes at local and regional population scales.

LeuO enhances butyrate-induced virulence expression through a positive regulatory loop in enterohaemorrhagic Escherichia coli.

Enterohaemorrhagic Escherichia coli (EHEC) causes bloody diarrhoea and other severe symptoms such as haemorrhagic uraemic syndrome. The expression of virulence genes on the locus for enterocyte effacement (LEE) and associated genes is regulated by a variety of factors, including transcriptional regulators and environmental signals. Butyrate, one of the major short-chain fatty acids present in the intestine, enhances expression of LEE genes and flagella biosynthesis genes in EHEC O157:H7, resulting in increased bacterial adherence and motility. Here, we show that expression of the leuO gene, which encodes a LysR-type transcriptional regulator, is enhanced by butyrate via Lrp, which is also necessary for butyrate-induced responses of LEE genes. LeuO expression induces prolonged activation of the promoter of LEE1 operon, including the ler gene, as well as virulence mechanisms such as microcolony formation. Activation of the LEE1 promoter by LeuO depends on another regulator, called Pch. The response of the leuO promoter to butyrate requires two virulence regulators, Pch and Ler, in addition to Lrp. Pch, Ler and Lrp bind the upstream region of the leuO promoter. Thus, leuO is involved in butyrate-enhanced expression of LEE genes through a positive feedback mechanism, but its expression and action on the LEE1 promoter are dependent on the virulence regulators Pch and Ler.

Antisense transcription regulates the expression of the enterohemorrhagic Escherichia coli virulence regulatory gene ler in response to the intracellular iron concentration.

Enteric pathogens, such as enterohemorrhagic E. coli (EHEC) O157:H7, encounter varying concentrations of iron during their life cycle. In the gastrointestinal tract, the amount of available free iron is limited because of absorption by host factors. EHEC and other enteric pathogens have developed sophisticated iron-responsive systems to utilize limited iron resources, and these systems are primarily regulated by the Fur repressor protein. The iron concentration could be a signal that controls gene expression in the intestines. In this study, we explored the role of iron in LEE (locus for enterocyte effacement) virulence gene expression in EHEC. In contrast to the expression of Fur-regulated genes, the expression of LEE genes was greatly reduced in fur mutants irrespective of the iron concentration. The expression of the ler gene, the LEE-encoded master regulator, was affected at a post-transcription step by fur mutation. Further analysis showed that the loss of Fur affected the translation of the ler gene by increasing the intracellular concentration of free iron, and the transcription of the antisense strand was necessary for regulation. The results indicate that LEE gene expression is closely linked to the control of intracellular free iron homeostasis.

Translation of a minigene in the 5′ leader sequence of the enterohaemorrhagic Escherichia coli LEE1 transcription unit affects expression of the neighbouring downstream gene

The 5′ end of the major RNA transcript of the LEE1 operon of enterohaemorrhagic Escherichia coli contains ~170 bases before the AUG translation start codon of the first recognized gene, ler. This unusually long leader sequence carries three potential alternative AUG start codons. Using a lac fusion expression vector, we confirmed that the ler gene AUG is functional for translation initiation, and we checked for translation initiation at the three alternative AUG codons. Whereas two of the alternative AUG codons appear incompetent for translation initiation, we detected strong initiation at the third AUG, which is followed by one AAA codon and a UAG stop codon. The location of this very short two-codon open reading frame with respect to the ler translation start appears to be critical. Hence mutations that destroy the UAG stop codon, or short deletions between the UAG stop codon and the ler translation initiation region, result in big effects on ler expression. In the context of the full-length LEE1 operon leader sequence, translation of this very short two-codon open reading frame is necessary for optimal expression of the ler gene and for the subsequent interactions of enterohaemorrhagic Escherichia coli with host target cells.

Inhibitory effects of broccoli extract on Escherichia coli O157:H7 quorum sensing and in vivo virulence

Broccoli extract (BE) has numerous beneficial effects on human health including anticancer activity. Quorum sensing (QS), mediated by self-produced autoinducer (AI) molecules, is a key process for the production of virulence determinants in pathogenic bacteria. BE suppressed AI-2 synthesis and AI-2-mediated bacterial motility in a dose-dependent manner in Escherichia coli O157:H7. In addition, expression of the ler gene that regulates AI-3 QS system was also diminished in response to treatment with BE. Furthermore, in an in vivo efficacy test using Caenorhabditis elegans as a host organism, C. elegans fed on E. coli O157:H7 in the presence of BE survived longer than those fed solely on the pathogenic bacteria. Quantitative real-time PCR analysis indicated that quercetin was the most active among the tested broccoli-derived compounds in downregulating virulence gene expression, while treatment with myricetin significantly suppressed the expression of the eae gene involved in type III secretion system. These data suggest that BE and its flavonoid constituents can inhibit expression of QS-associated genes, thereby downregulating the virulence attributes of E. coli O157:H7 both in vitro and in vivo. This study clearly elucidates BE's QS-inhibitory activity and suggests that BE has the potential to be developed as an anti-infective agent.

Ler of pathogenic Escherichia coli forms toroidal protein–DNA complexes

Enteropathogenic and enterohaemorrhagic Escherichia coli are related pathotypes of bacteria that cause acute watery diarrhoea and haemorrhagic colitis, respectively, and enterohaemorrhagic E. coli can lead to a serious complication known as haemolytic uraemic syndrome. In both bacteria the global regulatory protein Ler controls virulence. The ler gene is found within the locus of enterocyte effacement, or LEE, encoding a type III secretion system necessary for injecting effector proteins into intestinal epithelial cells and causing net secretory diarrhoea. The nucleoid-associated protein H-NS silences, whereas Ler serves as an anti-silencer of, multiple LEE operons. Although Ler has a higher affinity for DNA than does H-NS, the precise molecular mechanism by which Ler increases LEE transcription remains to be determined. In this report we investigate the oligomerization activity of Ler. In solution, Ler forms dimers and soluble aggregates of up to 5000 kDa molecular mass, and appears to oligomerize more readily than the related protein H-NS. An insertional mutation into the Ler linker region diminished oligomerization activity. Despite being proteins of similar mass and having homologous DNA-binding domains, Ler and H-NS complexed to DNA migrated to distinct locations, as determined by an electrophoretic mobility shift assay, implying that the related proteins form different 3D shapes in the presence of DNA. Lastly, we present electron microscopy images of toroidal Ler-DNA structures that are predicted to be involved in stimulating gene expression.

Towards an attenuated enterohemorrhagic Escherichia coli O157:H7 vaccine characterized by a deleted ler gene and containing apathogenic Shiga toxins

The aim of this study was to develop a candidate vaccine against enterohemorrhagic Escherichia coli O157:H7. A ler deletion mutant derived from wild-type EHEC O157:H7 86-24 was constructed by use of suicide vector pCVD442. The bacteriophage encoding Shiga toxin (Stx) was excised by serial passage to produce a ler/ stx deletion mutant, F25. Stx1 and Stx2 mutants were constructed by site-directed mutagenesis within the active center and membrane-spanning region of the toxin A subunit. Mutants stx1 and stx2 were then introduced into F25 to construct live attenuated candidate vaccine F105. The cytotoxicity of F25 was inactivated and that of F105 was significantly reduced in comparison with wild-type E. coli strain EDL933. Mice injected with candidate vaccine strains F25 and F105 gained weight and showed no clinical signs of disease. F25 and F105 reduced the colonization of wild-type O157:H7 in mouse intestine. Immunized pregnant mice were able to protect their suckling newborns from intragastric challenge with wild-type O157:H7. Immunized mice were protected against infection with wild-type O157:H7 and exhibited normal weight gain. Such attenuated vaccine strains may therefore have potential use as oral vaccines against O157:H7.

Cell attachment properties and infectivity of host-adapted and environmentally adapted Citrobacter rodentium

Citrobacter rodentium belongs to a family of extracellular enteric pathogens that include enterohaemorrhagic and enteropathogenic Escherichia coli, which colonises the gastrointestinal mucosa by the attaching and effacing (A/E) mechanism. We previously described the appearance of a ‘hyper-infectious’ state after passage of C. rodentium through the murine gastrointestinal tract. Here we report that host-adapted C. rodentium is able to efficiently adhere and trigger actin polymerisation on cultured epithelial cells. Consistent with these observations we recorded higher levels of expression of genes carried on the LEE pathogenicity island and type III secretion system effector genes carried on prophages compared with in vitro-grown bacteria; importantly, the level of ler gene expression was unchanged. These phenotypes were lost after shed C. rodentium was adapted to the external environment. Upon exposure of C57Bl/6 mice, environmentally adapted C. rodentium was no longer infectious at the low doses associated with host-adapted bacteria and the bacteria were found to be localised in the caecal patch in a similar way to C. rodentium cultured in laboratory media. Thus, the ‘hyper-infectious’ host-adapted state, allowing efficient transmission and colonisation of naive hosts, is transient in nature and gradually lost after shedding into the environment.

Towards a vaccine for attaching/effacing Escherichia coli: a LEE encoded regulator ( ler) mutant of rabbit enteropathogenic Escherichia coli is attenuated, immunogenic, and protects rabbits from lethal challenge with the wild-type virulent strain

The ler (LEE encoded regulator) gene product is a central regulator for the genes encoded on the locus of enterocyte effacement (LEE) pathogenicity island of attaching/effacing (A/E) pathogens, including human enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) as well as animal isolates. Although an in vivo role for Ler in bacterial virulence has not been documented, we hypothesized that a Ler deletion mutant should be attenuated for virulence but might retain immunogenicity. The goals of this study were to genetically characterize ler of a rabbit EPEC (rEPEC) strain (O103:H2), to examine the effect of ler on in vivo virulence, and to determine if intragastric inoculation of an attenuated rEPEC ler mutant was immunogenic and could protect rabbits against subsequent challenge with the wild-type virulent parent strain. The predicted ler gene product of rEPEC strain O103:H2 shares high homology (over 95% amino acid identity) with the Lers of another rEPEC strain RDEC-1 (O15:H-) and human EPEC and EHEC. A defined internal ler deletion mutant of rEPEC O103:H2 showed reduced production of secreted proteins. Although orogastric inoculation of rabbits with the virulent parent O103:H2 strain induced severe diarrhea, significant weight loss and early mortality with adherent mucosal bacteria found at sacrifice, the isogeneic ler mutant strain was well tolerated. Animals gained weight and showed no clinical signs of disease. Examination of histological sections of intestinal segments revealed the absence of mucosal bacterial adherence. This result demonstrates an essential role for Ler in in vivo pathogenicity of A/E E. coli. Single dose orogastric immunization with the rEPEC ler mutant induced serum IgG antibody to whole bacteria (but not to intimin). Immunized animals were protected against enteric infection with the WT virulent parent strain exhibiting normal weight gain, absence of diarrhea and absence of mucosally adherent bacteria at sacrifice. Such attenuated ler mutant strains may have potential for use as oral vaccines, or as vaccine vectors for delivery of foreign antigens. It remains to be determined whether such regulatory mutants can protect against infection with A/E bacteria of differing serotypes affecting different hosts.