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Marina Baquerizo Martinez - One of the best experts on this subject based on the ideXlab platform.
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Recognition of Enteroinvasive Escherichia Coli and Shigella flexneri by dendritic cells: distinct dendritic cell activation states
Memorias do Instituto Oswaldo Cruz, 2012Co-Authors: Ana Carolina Ramos Moreno, Karen Spadari Ferreira, Lucas Gonçalves Ferreira, Sandro Rogério De Almeida, Marina Baquerizo MartinezAbstract:The innate and adaptive immune responses of dendritic cells (DCs) to Enteroinvasive Escherichia Coli (EIEC) infection were compared with DC responses to Shigella flexneri infection. EIEC triggered DCs to produce interleukin (IL)-10, IL-12 and tumour necrosis factor (TNF)-α, whereas S. flexneri induced only the production of TNF-α. Unlike S. flexneri, EIEC strongly increased the expression of toll like receptor (TLR)-4 and TLR-5 in DCs and diminished the expression of co-stimulatory molecules that may cooperate to inhibit CD4+ T-lymphocyte proliferation. The inflammation elicited by EIEC seems to be related to innate immunity both because of the aforementioned results and because only EIEC were able to stimulate DC transmigration across polarised Caco-2 cell monolayers, a mechanism likely to be associated with the secretion of CC chemokine ligands (CCL)20 and TNF-α. Understanding intestinal DC biology is critical to unravelling the infection strategies of EIEC and may aid in the design of treatments for infectious diseases.
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expression of bacterial virulence factors and cytokines during in vitro macrophage infection by Enteroinvasive Escherichia Coli and shigella flexneri a comparative study
Memorias Do Instituto Oswaldo Cruz, 2010Co-Authors: Silvia Yumi Bando, Ana Carolina Ramos Moreno, Juliana Mota Khalil Amhaz, Carlos Alberto Moreirafilho, Jose Antonio Tavares De Albuquerque, Marina Baquerizo MartinezAbstract:Enteroinvasive Escherichia Coli (EIEC) and Shigella spp cause bacillary dysentery in humans by invading and multiplying within epithelial cells of the colonic mucosa. Although EIEC and Shigella share many genetic and biochemical similarities, the illness caused by Shigella is more severe. Thus, genomic and structure-function molecular studies on the biological interactions of these invasive enterobacteria with eukaryotic cells have focused on Shigella rather than EIEC. Here we comparatively studied the interactions of EIEC and of Shigella flexneri with cultured J774 macrophage-like cells. We evaluated several phenotypes: (i) bacterial escape from macrophages after phagocytosis, (ii) macrophage death induced by EIEC and S. flexneri, (iii) macrophage cytokine expression in response to infection and (iv) expression of plasmidial (pINV) virulence genes. The results showed that S. flexneri caused macrophage killing earlier and more intensely than EIEC. Both pathogens induced significant macrophage production of TNF, IL-1 and IL-10 after 7 h of infection. Transcription levels of the gene invasion plasmid antigen-C were lower in EIEC than in S. flexneri throughout the course of the infection; this could explain the diminished virulence of EIEC compared to S. flexneri.
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Enteroinvasive Escherichia Coli vs shigella flexneri how different patterns of gene expression affect virulence
Fems Microbiology Letters, 2009Co-Authors: Ana Carolina Ramos Moreno, Lucas Gona Alves Ferreira, Marina Baquerizo MartinezAbstract:Important features of the Enteroinvasive Escherichia Coli (EIEC) phenotype and gene expression likely to confer EIEC with a lower ability to cause disease than Shigella flexneri were described here for the first time. To confirm the lower pathogenicity of EIEC, we have analyzed the keratoconjunctivitis developed in guinea-pigs with EIEC or S. flexneri. Shigella flexneri induced a more pronounced proinflammatory response, whereas EIEC induced a mild form of the disease. EIEC showed a significantly less efficient cell-to-cell Caco-2 dissemination when compared with S. flexneri. Plaques formed by EIEC during intercellular spreading were four times smaller than those formed by S. flexneri. At the molecular level, the lower expression of virulence genes by EIEC during infection of Caco-2 cells highlighted the importance of effective gene transcription for bacterial pathogenicity.
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molecular evolutionary relationships of Enteroinvasive Escherichia Coli and shigella spp
Infection and Immunity, 2004Co-Authors: Ruiting Lan, Marina Baquerizo Martinez, Chehani M Alles, Kathy Donohoe, Peter R ReevesAbstract:Enteroinvasive Escherichia Coli (EIEC), a distinctive pathogenic form of E. Coli causing dysentery, is similar in many properties to bacteria placed in the four species of Shigella. Shigella has been separated as a genus but in fact comprises several clones of E. Coli. The evolutionary relationships of 32 EIEC strains of 12 serotypes have been determined by sequencing of four housekeeping genes and two plasmid genes which were used previously to determine the relationships of Shigella strains. The EIEC strains were grouped in four clusters with one outlier strain, indicating independent derivation of EIEC several times. Three of the four clusters contain more than one O antigen type. One EIEC strain (an O112ac:H− strain) was found in Shigella cluster 3 but is not identical to the Shigella cluster 3 D2 and B15 strains with the same O antigen. Two forms of the virulence plasmid pINV have been identified in Shigella strains by using the sequences of ipgD and mxiA genes, and all but two of our EIEC strains have pINV A. The EIEC strains were grouped in two subclusters with a very low level of variation, generally not intermingled with Shigella pINV A strains. The EIEC clusters based on housekeeping genes were reflected in the plasmid gene sequences, with some exceptions. Two strains were found in the pINV B form by using the ipgD sequence, with one strain having an mxiA sequence similar to the divergent sequence of D1. Clearly, EIEC and Shigella spp. form a pathovar of E. Coli.
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molecular typing and phylogenetic analysis of Enteroinvasive Escherichia Coli using the flic gene sequence
Fems Microbiology Letters, 2004Co-Authors: Juliana Mota Khalil Amhaz, Agda Andrade, Silvia Yumi Bando, Carlos Alberto Moreirafilho, Ta Nia L Tanaka, Marina Baquerizo MartinezAbstract:Non-motile Enteroinvasive Escherichia Coli (EIEC) is serotyped based only on O antigen polymorphism, since H antigen epitopes, present on the flagellins, cannot be characterised in these bacteria. In this study, we demonstrate the presence of the flagellin-coding fliC gene in non-motile EIEC strains. Moreover, we were able to group the 11 most common non-motile EIEC serotypes into six different RFLP patterns of the fliC gene. Amplicons representing each RFLP pattern were sequenced. Sequencing data were used to construct a phylogenetic tree which showed two main clusters: one sharing similarity with Shigella dysenteriae and pathogenic E. Coli, and the other being closer to non-pathogenic E. Coli.
Peter Echeverria - One of the best experts on this subject based on the ideXlab platform.
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detection of pcr products of the ipah gene from shigella and Enteroinvasive Escherichia Coli by enzyme linked immunosorbent assay
Diagnostic Microbiology and Infectious Disease, 2000Co-Authors: Orntipa Sethabutr, Peter Echeverria, David N Taylor, Malabi M Venkatesan, Sylvia Yam, Lorrin W Pang, Bonnie L Smoak, Willie Sang, Daniel W IsenbargerAbstract:PCR techniques applied to diarrheal stools reliably diagnose Shigella and Enteroinvasive Escherichia Coli (EIEC) infections. Identification of PCR products using agarose gel electrophoresis (AGE) and hybridization with DNA probes has several shortcomings. Automated methods of identifying PCR products that process larger numbers of specimens can facilitate epidemiologic studies and standardize results. In this study, we used ELISA following PCR to detect ipaH gene sequences of Shigella and EIEC from 89 diarrheal stools. Results of ELISA were compared with AGE with and without DNA probe, and with culture. Two specimen preparation methods were compared as well: boiling/centrifugation, and purification with silicon dioxide (SiO(2)). Both PCR product-detection methods identified significantly more infections than did culture. PCR-ELISA detected significantly more infections than PCR-AGE when processed using SiO2 (P = 0.014). PCR-ELISA allows screening of larger numbers of specimens, automates test results, and avoids use of mutagenic reagents. PCR-ELISA is faster than PCR-AGE when testing large numbers of specimens, although not when testing small numbers of specimens.
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a simple polymerase chain reaction technique to detect and differentiate shigella and Enteroinvasive Escherichia Coli in human feces
Diagnostic Microbiology and Infectious Disease, 1997Co-Authors: Huoshu H Houng, Orntipa Sethabutr, Peter EcheverriaAbstract:Abstract A simple polymerase chain reaction (PCR) procedure using IS630-specific primers was developed as a general diagnostic probe to detect Shigella and Enteroinvasive Escherichia Coli (EIEC). However, IS630 and the other two previously reported molecular probes, ipa H and ial , cannot be used to differentiate among Shigella serotypes and EIEC strains that cause dysentery. The sensitivity of PCR protocol was determined to be 100–200 shigellae for each PCR reaction. An enrichment incubation would allow the detection of shigellae in stool samples with low bacterial concentration; i.e., 4 CFU/gram. Serotype-specific primers derived from the rfc genes of different Shigella strains were used in PCR reactions to differentiate among Shigella serotypes in the laboratory, such as S. sonnei , S. flexneri , and S. dysenteriae 1. It was demonstrated further that the multiplex PCR system containing rfc -specific primers can efficiently identify the most prominent Shigella serotypes in raw stool samples of acute diarrheal patients.
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detection of shigellae and Enteroinvasive Escherichia Coli by amplification of the invasion plasmid antigen h dna sequence in patients with dysentery
The Journal of Infectious Diseases, 1993Co-Authors: Orntipa Sethabutr, Malabi M Venkatesan, Gerald S Murphy, Boonchuey Eampokalap, Charles W Hoge, Peter EcheverriaAbstract:Detection of Shigella organisms and Enteroinvasive Escherichia Coli (EIEC) by polymerase chain reaction (PCR) was evaluated in 20 patients with dysentery before and in 17 of the 20 after treatment with ciprofloxacin. DNA sequences coding for IpaH antigen, a multiple copy sequence found on the chromosome, and the invasion plasmid locus (ial) was detected after DNA amplification in 13 stools from patients from whom shigellae or EIEC were isolated but not in 21 nondysenteric stools containing other enteric bacteria. Although shigellae or EIEC were not isolated from any patient with dysentery after ciprofloxacin treatment, IpaH and ial sequences were found after PCR amplification in 7 patients after treatment with ciprofloxacin. IpaH sequences alone were detected in 4 patients; DNA augmentation of IpaH in stools in a specific way to identify Shigella or EIEC infection in persons from whom cultures cannot be obtained promptly after the onset of diarrhea or who have received antibiotics.
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microbiology and diagnosis of infections with shigella and Enteroinvasive Escherichia Coli
Clinical Infectious Diseases, 1991Co-Authors: Peter Echeverria, Orntipa Sethabutr, Chittima PitarangsiAbstract:The etiology of dysentery in Thailand and the existing methods of diagnosing infections with Shigella and Enteroinvasive Escherichia Coli (EIEC) are reviewed. The four Shigella species (S. dysenteriae, S. flexneri, S. boydii, and S. sonnei) are classically identified by culture of fecal specimens on selective media and testing of isolates for agglutination in species-specific antisera. DNA probes have been used to identify both lactose-fermenting and non-lactose-fermenting EIEC as well as Shigella isolates that do not agglutinate in antisera. These DNA probes are not necessary for the identification of Shigella if a competent bacteriology laboratory with shigella antisera is available. In Thailand Shigella and EIEC are isolated more often from children greater than 2 years of age than from younger children. The clinical illness associated with EIEC infections is similar to shigellosis. Fewer children with EIEC infections than with shigellosis, however, have occult blood in stool (36% vs. 82%) and more than 10 fecal leukocytes per high-power field (36% vs. 67%). Standard bacteriologic methods and testing of E. Coli isolates for hybridization with the shigella/EIEC probe are currently the most sensitive means of diagnosing infections caused by these enteric pathogens. A more rapid method of identifying Shigella and EIEC infections in a situation where a bacteriology laboratory is not available will probably involve immunologic assays.
Orntipa Sethabutr - One of the best experts on this subject based on the ideXlab platform.
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detection of pcr products of the ipah gene from shigella and Enteroinvasive Escherichia Coli by enzyme linked immunosorbent assay
Diagnostic Microbiology and Infectious Disease, 2000Co-Authors: Orntipa Sethabutr, Peter Echeverria, David N Taylor, Malabi M Venkatesan, Sylvia Yam, Lorrin W Pang, Bonnie L Smoak, Willie Sang, Daniel W IsenbargerAbstract:PCR techniques applied to diarrheal stools reliably diagnose Shigella and Enteroinvasive Escherichia Coli (EIEC) infections. Identification of PCR products using agarose gel electrophoresis (AGE) and hybridization with DNA probes has several shortcomings. Automated methods of identifying PCR products that process larger numbers of specimens can facilitate epidemiologic studies and standardize results. In this study, we used ELISA following PCR to detect ipaH gene sequences of Shigella and EIEC from 89 diarrheal stools. Results of ELISA were compared with AGE with and without DNA probe, and with culture. Two specimen preparation methods were compared as well: boiling/centrifugation, and purification with silicon dioxide (SiO(2)). Both PCR product-detection methods identified significantly more infections than did culture. PCR-ELISA detected significantly more infections than PCR-AGE when processed using SiO2 (P = 0.014). PCR-ELISA allows screening of larger numbers of specimens, automates test results, and avoids use of mutagenic reagents. PCR-ELISA is faster than PCR-AGE when testing large numbers of specimens, although not when testing small numbers of specimens.
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a simple polymerase chain reaction technique to detect and differentiate shigella and Enteroinvasive Escherichia Coli in human feces
Diagnostic Microbiology and Infectious Disease, 1997Co-Authors: Huoshu H Houng, Orntipa Sethabutr, Peter EcheverriaAbstract:Abstract A simple polymerase chain reaction (PCR) procedure using IS630-specific primers was developed as a general diagnostic probe to detect Shigella and Enteroinvasive Escherichia Coli (EIEC). However, IS630 and the other two previously reported molecular probes, ipa H and ial , cannot be used to differentiate among Shigella serotypes and EIEC strains that cause dysentery. The sensitivity of PCR protocol was determined to be 100–200 shigellae for each PCR reaction. An enrichment incubation would allow the detection of shigellae in stool samples with low bacterial concentration; i.e., 4 CFU/gram. Serotype-specific primers derived from the rfc genes of different Shigella strains were used in PCR reactions to differentiate among Shigella serotypes in the laboratory, such as S. sonnei , S. flexneri , and S. dysenteriae 1. It was demonstrated further that the multiplex PCR system containing rfc -specific primers can efficiently identify the most prominent Shigella serotypes in raw stool samples of acute diarrheal patients.
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detection of shigella and Enteroinvasive Escherichia Coli by pcr in the stools of patients with dysentery in thailand
Journal of Diarrhoeal Diseases Research, 1994Co-Authors: Orntipa Sethabutr, P Echeverria, Charles W Hoge, L Bodhidatta, C PitarangsiAbstract:The rate of detection of Shigella and Enteroinvasive Escherichia Coli (EIEC) using a PCR technique was compared with the rate detected by standard microbiological methods (bacteriology plus hybridization of E. Coli colonies with a 17 kb EIEC probe) among patients with dysentery before and after antibiotic therapy. The PCR amplified DNA sequences encoding IpaH, a multiple copy sequence located on the chromosome and the invasion plasmid. Shigella or EIEC were detected using the IpaH PCR system among 72 (61%) of 119 patients with dysentery on the first day they were seen at hospital, compared to 50 (42%) using standard microbiological methods (p = 0.006). After three days of antibiotic therapy, IpaH sequences were detected in stools from 38 percent of patients, compared to 10 percent using standard microbiology (p < 0.001). After seven days of therapy, the rates were 26 percent vs. 8 percent respectively (p < 0.001). The IpaH PCR system appeared to be specific for Shigella or EIEC based on low rates of positive reactions among non-diarrhoea controls, and a strong correlation between persistently positive reactions and antibiotic resistance of bacterial isolates. IpaH sequences were detected in 10 (8%) of 119 drinking water samples from homes of patients with disease; none of these specimens were positive for Shigella or EIEC by standard microbiology. In conclusion, PCR amplification of IpaH sequences and detection of target DNA with a non-radioactive probe increased the rates of identification of Shigella and EIEC by 45% in initial clinical specimens and by nearly 300% in specimens obtained from patients receiving antibiotic therapy.
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detection of shigellae and Enteroinvasive Escherichia Coli by amplification of the invasion plasmid antigen h dna sequence in patients with dysentery
The Journal of Infectious Diseases, 1993Co-Authors: Orntipa Sethabutr, Malabi M Venkatesan, Gerald S Murphy, Boonchuey Eampokalap, Charles W Hoge, Peter EcheverriaAbstract:Detection of Shigella organisms and Enteroinvasive Escherichia Coli (EIEC) by polymerase chain reaction (PCR) was evaluated in 20 patients with dysentery before and in 17 of the 20 after treatment with ciprofloxacin. DNA sequences coding for IpaH antigen, a multiple copy sequence found on the chromosome, and the invasion plasmid locus (ial) was detected after DNA amplification in 13 stools from patients from whom shigellae or EIEC were isolated but not in 21 nondysenteric stools containing other enteric bacteria. Although shigellae or EIEC were not isolated from any patient with dysentery after ciprofloxacin treatment, IpaH and ial sequences were found after PCR amplification in 7 patients after treatment with ciprofloxacin. IpaH sequences alone were detected in 4 patients; DNA augmentation of IpaH in stools in a specific way to identify Shigella or EIEC infection in persons from whom cultures cannot be obtained promptly after the onset of diarrhea or who have received antibiotics.
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shigella and Enteroinvasive Escherichia Coli infections in households of children with dysentery in bangkok
The Journal of Infectious Diseases, 1992Co-Authors: P Echeverria, Orntipa Sethabutr, O Serichantalergs, U Lexomboon, K TamuraAbstract:Shigellae and Enteroinvasive Escherichia Coli (EIEC) were identified in children with dysentery and their household contacts in Bangkok. Shigellae were isolated from 49% and EIEC from 6% of 306 children with dysentery seen at the outpatient department of Children's Hospital on weekdays during January through June 1989 and October 1989 through October 1990. The same serotype infecting the index child was isolated from 21 (4%) of 522 household contacts of 151 index children with Shigella infections and from none of 60 household contacts of 19 index children with EIEC infections. Amplification of DNA sequences coding for the invasion-associated locus (ial) by polymerase chain reaction increased the identification of Shigella and EIEC infections from 57% (111/193) to 68% (132/193). ial sequences were identified in 3 of 20 drinking water specimens from which shigellae or EIEC were not isolated. Amplification of ial sequences identified more shigellae and EIEC than did bacteriologic and colony hybridization methods in children with dysentery and in drinking water in Bangkok.
Tibor Pal - One of the best experts on this subject based on the ideXlab platform.
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a colony blot immune assay to identify Enteroinvasive Escherichia Coli and shigella in stool samples
Diagnostic Microbiology and Infectious Disease, 2003Co-Authors: Ora D Szakal, Yorgy G Schneider, Tibor PalAbstract:Using an IpaC protein-specific monoclonal antibody a colony blot immune assay was developed for the identification of Enteroinvasive Escherichia Coli (EIEC) and Shigella in fecal specimens, and was evaluated in a field study. By screening the entire culture plates the colony blot assay was significantly more sensitive than the investigation of 16 randomly selected colonies from artificially contaminated fecal specimens. Among the 165 stool samples from 121 patients with diarrhea the immune assay detected IpaC expressing colonies in 16 out of the 17 specimens positive with a Shigella-, and EIEC-specific polymerase chain reaction targeting the ipaH gene. Guided by the colony blots, Shigella was isolated from 12, while EIEC from four of the samples. The IpaC-specific colony blot immune assay is a simple screening method to detect EIEC in stool samples for laboratories not equipped with molecular techniques.
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a colony blot immunoassay to detect Enteroinvasive Escherichia Coli and shigella in water samples
Journal of Applied Microbiology, 2001Co-Authors: D Szakal, I Gado, Tibor PalAbstract:D. SZAKAL, I. GADO AND T. PAL. 2001. Aims: The aim of the study was to develop a colony blot immunoassay to detect Shigella and Enteroinvasive Escherichia Coli (EIEC) in water. Methods and Results: Spiked samples were filtered through nitrocellulose membranes. Colony prints on the filters were tested with a monoclonal antibody specific to IpaC, an antigen coded by the invasion plasmid of Shigella and EIEC. Invasive pathogens could be successfully detected with the technique, even in the presence of a large number of non-pathogenic bacterial cells. The method was significantly more sensitive in identifying pathogen-containing samples then the traditional culture-based approach. Conclusions: The IpaC-specific colony blot immunoassay is an inexpensive method for identifying the aetiological agents of bacillary dysentery in water samples. Significance and Impact of the Study: The technique could be particularly useful in detecting Enteroinvasive E. Coli which often remains undetected by bio- and serotyping.
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identification of Enteroinvasive Escherichia Coli and shigella strains in pediatric patients by an ipac specific enzyme linked immunosorbent assay
Journal of Clinical Microbiology, 1997Co-Authors: Tibor Pal, Noura Alsweih, Maria Herpay, T D ChughAbstract:A new method, a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) recognizing a secreted, invasion plasmid-coded protein antigen (IpaC), was used to identify Enteroinvasive Escherichia Coli and Shigella strains among colonies from 859 cultures of fecal samples from children in Kuwait. A total of 33.8% of the samples were diarrheal. By the immunoassay, Enteroinvasive E. Coli strains were identified from two diarrheal samples but from none of the samples from children without diarrhea. These strains were fully virulent and belonged to serogroup O28ac. In addition, 26 Shigella strains were also recognized by the ELISA, while only 23 were isolated by routine biotyping and serotyping. For two diarrheal patients, Shigella was identified by culture only. The study showed that the IpaC-specific immunoassay is a simple and useful tool for identifying Enteroinvasive strains. Furthermore, by reporting the first Enteroinvasive E. Coli isolates from Kuwait, the study indicates the presence of this group of pathogens as a potential source of diarrhea in the region.
M Nicoletti - One of the best experts on this subject based on the ideXlab platform.
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ala160 and his116 residues are involved in activity and specificity of apyrase an atp hydrolysing enzyme produced by Enteroinvasive Escherichia Coli
Microbiology, 2005Co-Authors: S Sarli, M Nicoletti, Serena Schippa, Federica Del Chierico, Daniela Santapaola, Piera Valenti, Francesca BerluttiAbstract:The virulence plasmid-carried apy (phoN2) gene of Shigella and related Enteroinvasive Escherichia Coli (EIEC) encodes apyrase, an ATP-diphosphohydrolase belonging to class A of the non-specific acid phosphatases (A-NSAPs). Apyrase and A-NSAPs share three domains of conserved amino acids (domains D1–D3) containing residues forming the putative active site of apyrase. In spite of their similarity, apyrase and A-NSAPs show different substrate specificity, apyrase being able to hydrolyse nucleotide tri- and diphosphates, but not monophosphates, as well as p-nitrophenyl phosphate (pNPP), while A-NSAPs are also active towards monophosphates and pNPP. In this paper, to get further insights into the structure–function relationship of apyrase, a random and site-directed mutagenesis of the apy gene of EIEC strain HN280 was conducted. Results indicate that amino acids located within the D2 and D3 conserved domains (Ser157 and Arg192, respectively) as well as residues located in the N-terminal (Ser97) and C-terminal (Glu233) domains are required for enzyme activity. Surprisingly, Ala160, located near the D2 domain and considered to be important for enzyme specificity, is required for enzyme activity, as its substitution with Thr led to the inactivation of enzyme activity. Furthermore, residue His116 is involved in apyrase specificity, since the H116L apyrase mutant shows substrate specificity resembling that of A-NSAPs.
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Enteroinvasive Escherichia Coli virulence plasmid carried apyrase apy and ospb genes are organized as a bicistronic operon and are subject to differential expression
Microbiology, 2002Co-Authors: Daniela Santapaola, Francesca Berlutti, Mariassunta Casalino, Carlo Zagaglia, Bianca Colonna, Andrea Petrucca, Carlo Presutti, M NicolettiAbstract:In Shigella flexneri and Enteroinvasive Escherichia Coli (EIEC) the expression of the virulence-plasmid(pINV)-carried potential pathogenesis-associated apy gene, which encodes apyrase (ATP diphosphohydrolase), is regulated by the same regulators that govern the expression of virulence genes. To understand the transcriptional organization of the apy gene, the authors sequenced an 8023 bp PstI fragment of the pINV of EIEC strain HN280, which encompasses apy as well as its adjacent genes. The PstI fragment displays 99% identity with the corresponding fragment of pWR100, the pINV of S. flexneri strain M90T, and contains four genes. One of these genes, ospB, encodes a secreted protein of unknown activity and is located immediately upstream of apy. Analyses of sequence, Northern hybridization, RT-PCR and primer extension data and transcriptional fusions indicated that ospB and apy are co-transcribed as a 2 kb bicistronic, temperature-regulated mRNA from an upstream promoter that precedes ospB. The 2 kb mRNA is post-transcriptionally processed in the intercistronic ospB–apy region, leading to the considerable accumulation of a more stable 1 kb apy-specific mRNA (half-life of 2·2±0·3 min, versus 27±4 s for the 2 kb transcript). Upon temperature induction, peak expression of the ospB–apy operon occurs when bacteria enter into the late phases of bacterial growth, where the apy-specific transcript was found to be much more prevalent if compared to the ospB–apy transcript.
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expression of the virulence plasmid carried apyrase gene apy of Enteroinvasive Escherichia Coli and shigella flexneri is under the control of h ns and the virf and virb regulatory cascade
Infection and Immunity, 1998Co-Authors: Francesca Berlutti, Mariassunta Casalino, Carlo Zagaglia, Piera Assunta Fradiani, Paolo Visca, M NicolettiAbstract:The transcription of the virulence plasmid (pINV)-carried invasion genes of Shigella flexneri and Enteroinvasive Escherichia Coli (EIEC) is induced at 37°C and repressed at 30°C. In this work, we report that the O135: K−:H− EIEC strain HN280 and S. flexneri SFZM53, M90T, and 454, of serotypes 4, 5, and 2a, respectively, produce apyrase (ATP-diphosphohydrolase), the product of the apy gene. In addition, the S. flexneri strains, but not the EIEC strain, produce a nonspecific phosphatase encoded by the phoN-Sf gene. Both apy and phoN-Sf are pINV-carried loci whose contribution to the pathogenicity of Enteroinvasive microorganisms has been hypothesized but not yet established. We found that, like that of virulence genes, the expression of both the apy and the phoN-Sf genes was temperature regulated. Strain HN280/32 (a pINV-integrated avirulent derivative of HN280 which has a severe reduction of virB transcription) expressed the apy gene in a temperature-regulated fashion but to a much lower extent than wild-type HN280, while the introduction of the Δhns deletion in HN280 and in HN280/32 induced the wild-type temperature-independent expression of apyrase. These results indicated that a reduction of virB transcription, which is known to occur in the pINV-integrated strain HN280/32, accounts for reduced apyrase expression and that the histone-like protein H-NS is involved in this regulatory network. Independent spontaneously generated mutants of HN280 and of SFZM53 which had lost the capacity to bind Congo red dye (Crb−) were isolated, and the molecular alterations of pINV were evaluated by PCR analysis. Alterations of pINV characterized by the absence of virF or virB and by the presence of the intact apy locus or intact apy and phoN-Sf loci were detected among Crb− mutants of HN280 and SFZM53, respectively. While all Crb− apy+ mutants of HN280 failed to produce apyrase, Crb− apy+ phoN-Sf+ mutants of SFZM53 lacked apyrase activity but produced a nonspecific phosphatase, like parental SFZM53. Moreover, the introduction of recombinant plasmids carrying cloned virF (pMYSH6504) or virB (pBN1) into Crb− mutants of HN280 and SFZM53 lacking virF or virB, respectively, fully restored temperature-dependent apyrase expression to levels resembling those of the parental strains. Taken together, our results demonstrate that, as has already been shown for invasion genes, apy is another locus whose expression is controlled by temperature, H-NS, and the VirF and VirB regulatory cascade. In contrast, the temperature-regulated expression of the nonspecific phosphatase does not appear to be under the control of the same regulatory network. These findings led us to speculate that apyrase may play a role in the pathogenicity of Enteroinvasive bacteria.
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a role for h ns in the regulation of the virf gene of shigella and Enteroinvasive Escherichia Coli
Research in Microbiology, 1998Co-Authors: Gianni Prosseda, M Nicoletti, Mariassunta Casalino, Piera Assunta Fradiani, Maurizio Falconi, M Di Lorenzo, Gioacchino Micheli, Bianca ColonnaAbstract:We have investigated the role of H-NS, one of the major components of the bacterial nucleoid, in the expression of the virF gene present on the large virulence plasmid of Shigella and Enteroinvasive Escherichia Coli in response to different environmental conditions. VirF is an AraC-like protein which activates at least two promoters, virB and virG, both repressed by H-NS. Band shift experiments reveal that the affinity of H-NS for the virF and virB promoters is comparable, while the affinity for the virG promoter is higher. Polyacrylamide gel electrophoresis of three DNA fragments containing the virF, the virB and the VirG promoters demonstrates, in agreement with computer predictions, that they have an intrinsically curved structure, confirming the preference of H-NS for bent DNA. In vivo transcriptional analysis of virF mRNA shows that H-NS negatively controls the expression of virF at 30 degrees C. The expression of a virF-lacZ translational fusion in E.Coli wild type and in an hns-defective derivative grown at 30 degrees or 37 degrees C and at pH 6.0 or 7.0 indicates that, in the absence of H-NS, virF expression becomes insensitive to temperature and to limited pH changes. Our results strongly suggest that H-NS controls virF expression by binding to the virF promoter and by repressing its expression at low temperature and at low pH.
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h ns regulation of virulence gene expression in Enteroinvasive Escherichia Coli harboring the virulence plasmid integrated into the host chromosome
Journal of Bacteriology, 1995Co-Authors: Bianca Colonna, Mariassunta Casalino, Carlo Zagaglia, Piera Assunta Fradiani, Gianni Prosseda, Silvia Naitza, Livia Leoni, Annamaria Coppo, Patrizia Ghelardini, M NicolettiAbstract:We have previously shown that integration of the virulence plasmid pINV into the chromosome of Enteroinvasive Escherichia Coli and of Shigella flexneri makes these strains noninvasive (C. Zagaglia, M. Casalino, B. Colonna, C. Conti, A. Calconi, and M. Nicoletti, Infect. Immun. 59:792-799, 1991). In this work, we have studied the transcription of the virulence regulatory genes virB, virF, and hns (virR) in wild-type Enteroinvasive E. Coli HN280 and in its pINV-integrated derivative HN280/32. While transcription of virF and of hns is not affected by pINV integration, transcription of virB is severely reduced even if integration does not occur within the virB locus. This indicates that VirF cannot activate virB transcription when pINV is integrated, and this lack of expression accounts for the noninvasive phenotype of HN280/32. Virulence gene expression in strains HN280 and HN280/32, as well as in derivatives harboring a mxiC::lacZ operon fusion either on the autonomously replicating pINV or on the integrated pINV, was studied. The effect of the introduction of plasmids carrying virB (pBNI) or virF (pHW745 and pMYSH6504), and of a delta hns deletion, in the different strains was evaluated by measuring beta-galactosidase activity, virB transcription, and virB-regulated virulence phenotypes like synthesis of Ipa proteins, contact-mediated hemolysis, and capacity to invade HeLa cells. The introduction of pBN1 or of the delta hns deletion in pINV-integrated strains induces temperature-regulated expression or temperature-independent expression, respectively, of beta-galactosidase activity and of all virulence phenotypes, while an increase in virF gene dosage does not, in spite of a high-level induction of virB transcription. Moreover, a wild-type hns gene placed in trans fully reversed the induction of beta-galactosidase activity due to the delta hns deletion. These results indicate that virB transcription is negatively regulated by H-NS both at 30 and at 37 degrees C in pINV-integrated strains and that there is also a dose-dependent effect of VirF on virB transcription. The negative effect of H-NS on virB transcription at the permissive temperature of 37 degrees C could be due to changes in the DNA topology occurring upon pINV integration that favor more stable binding of H-NS to the virB promoter DNA region. At 30 degrees C, the introduction of the high-copy-number plasmid pMYSH6504 (but not of the low-copy-number pHW745) or of the deltahns deletion induces, in strains harboring an autonomously replicating pINV, beta-galactosidase activity, virB transcription, and expression of the virulence phenotypes, indicating that, as for HN280/32, the increase in virF gene dosage overcomes the negative regulatory effect of H-NS on virB transcription. Moreover, we have found that virF transcription is finely modulated by temperature and, with E. Coli K-12 strains containing a virF-lacZ gene fusion, by H-NS. This leads us to speculate that, in Enteroinvasive bacteria, the level of Virf inside the cell controls the temperature-regulated expression of invasion genes.