Phenotypic Expression Of Type Research Articles

Biofilm formation in uropathogenic Escherichia coli: association with adhesion factor genes

Background/aim: Biofilm formation is a major determinant factor in the development of urinary tract infections (UTIs) by uropathogenic Escherichia coli (UPEC). Several adhesion factors are involved in attachment of bacterial cells to the urinary tract and biofilm development. Materials and methods: The possible relationship between different adhesion factor genes (AFGs) and biofilm formation among UPEC isolates was investigated. Prevalence of different AFGs including fimA, fimH, papAH, papC, papEF, sfa-S, foc/G, and bmaE and their association to biofilm formation were investigated. Phenotypic expression of type 1 and P fimbriae was also investigated. Results: Our results showed that 84% of UPEC were moderate to strong biofilm producers. Prevalence of the type 1 and P fimbriae associated genes (fimA, fimH, papEF, papC) as well as the F1C fimbriae gene (foc/G) did not show a difference among different biofilm-producing groups, while higher prevalence of papAH, bmaE (M fimbriae), and sfaS (S-fimbriae) was observed for the strains producing moderate to strong biofilms. Phenotypic expression of type 1 fimbriae was associated with biofilm formation, while no association was observed for P fimbriae. Conclusion: Different AFGs (especially M and S fimbria) and their expression levels affect the biofilm formation ability of UPEC.

Variable age of onset and clinical severity in transferrin receptor 2 related haemochromatosis: novel observations

Variable age of onset and clinical severity in transferrin receptor 2 related haemochromatosis: novel observations

A previously uncharacterized gene stm0551 plays a repressive role in the regulation of type 1 fimbriae in Salmonella enterica serotype Typhimurium

BackgroundSalmonella enterica serotype Typhimurium produces surface-associated fimbriae that facilitate adherence of the bacteria to a variety of cells and tissues. Type 1 fimbriae with binding specificity to mannose residues are the most commonly found fimbrial type. In vitro, static-broth culture favors the growth of S. Typhimurium with type 1 fimbriae, whereas non-type 1 fimbriate bacteria are obtained by culture on solid-agar media. Previous studies demonstrated that the phenotypic expression of type 1 fimbriae is the result of the interaction and cooperation of the regulatory genes fimZ, fimY, fimW, and fimU within the fim gene cluster. Genome sequencing revealed a novel gene, stm0551, located between fimY and fimW that encodes an 11.4-kDa putative phosphodiesterase specific for the bacterial second messenger cyclic-diguanylate monophosphate (c-di-GMP). The role of stm0551 in the regulation of type 1 fimbriae in S. Typhimurium remains unclear.ResultsA stm0551-deleted stain constructed by allelic exchange constitutively produced type 1 fimbriae in both static-broth and solid-agar medium conditions. Quantative RT-PCR revealed that expression of the fimbrial major subunit gene, fimA, and one of the regulatory genes, fimZ, were comparably increased in the stm0551-deleted strain compared with those of the parental strain when grown on the solid-agar medium, a condition that normally inhibits expression of type 1 fimbriae. Following transformation with a plasmid possessing the coding sequence of stm0551, expression of fimA and fimZ decreased in the stm0551 mutant strain in both culture conditions, whereas transformation with the control vector pACYC184 relieved this repression. A purified STM0551 protein exhibited a phosphodiesterase activity in vitro while a point mutation in the putative EAL domain, substituting glutamic acid (E) with alanine (A), of STM0551 or a FimY protein abolished this activity.ConclusionsThe finding that the stm0551 gene plays a negative regulatory role in the regulation of type 1 fimbriae in S. Typhimurium has not been reported previously. The possibility that degradation of c-di-GMP is a key step in the regulation of type 1 fimbriae warrants further investigation.

Identification of the genetic determinants of Salmonella enterica serotype Typhimurium that may regulate the expression of the type 1 fimbriae in response to solid agar and static broth culture conditions

BackgroundType 1 fimbriae are the most commonly found fimbrial appendages on the outer membrane of Salmonella enterica serotype Typhimurium. Previous investigations indicate that static broth culture favours S. Typhimurium to produce type 1 fimbriae, while non-fimbriate bacteria are obtained by growth on solid agar media. The phenotypic expression of type 1 fimbriae in S. Typhimurium is the result of the interaction and cooperation of several genes in the fim gene cluster. Other gene products that may also participate in the regulation of type 1 fimbrial expression remain uncharacterized.ResultsIn the present study, transposon insertion mutagenesis was performed on S. Typhimurium to generate a library to screen for those mutants that would exhibit different type 1 fimbrial phenotypes than the parental strain. Eight-two mutants were obtained from 7,239 clones screened using the yeast agglutination test. Forty-four mutants produced type 1 fimbriae on both solid agar and static broth media, while none of the other 38 mutants formed type 1 fimbriae in either culture condition. The flanking sequences of the transposons from 54 mutants were cloned and sequenced. These mutants can be classified according to the functions or putative functions of the open reading frames disrupted by the transposon. Our current results indicate that the genetic determinants such as those involved in the fimbrial biogenesis and regulation, global regulators, transporter proteins, prophage-derived proteins, and enzymes of different functions, to name a few, may play a role in the regulation of type 1 fimbrial expression in response to solid agar and static broth culture conditions. A complementation test revealed that transforming a recombinant plasmid possessing the coding sequence of a NAD(P)H-flavin reductase gene ubiB restored an ubiB mutant to exhibit the type 1 fimbrial phenotype as its parental strain.ConclusionGenetic determinants other than the fim genes may involve in the regulation of type 1 fimbrial expression in S. Typhimurium. How each gene product may influence type 1 fimbrial expression is an interesting research topic which warrants further investigation.

Behavior and biology: the prevention of type 2 diabetes.

Type 2 diabetes mellitus (DM), characterized by insulin resistance and a beta-cell secretory defect, appears to result from a number of gene and environmental interactions. There are marked differences in the phenotypic expression of type 2 DM with individuals exhibiting varying levels of insulin resistance and impairments in insulin secretion. Study results indicate that a number of healthy lifestyle behaviors, such as increased physical activity and reduced intake of dietary fat, are associated with decreased development of type 2 DM. This article explores the genetic and environmental factors associated with the development of type 2 DM along with the role of lifestyle modifications in the prevention of this disease.

Type 2 diabetes mellitus in children and adolescents.

1. J. Darrell Nesmith, MD, MPH* 1. 2. *Assistant Professor of Pediatrics, University of Arkansas for Medical Sciences, Little Rock, AR. Objectives After completing this article, readers should be able to: 1. Delineate the population in which type 2 diabetes mellitus is reaching epidemic proportions. 2. List the characteristics of type 2 diabetes mellitus. 3. Explain the increasing prevalence of type 2 diabetes mellitus among children. 4. Characterize the clinical and laboratory features as well as the course of the disease that will assist in classification of the patient who has diabetes mellitus. 5. Describe the treatments for type 2 diabetes mellitus. Type 2 diabetes mellitus (DM) is a common disease affecting more than 15 million Americans. Its prevalence among adults is increasing, but more worrisome is the recent recognition of type 2 DM among children and adolescents. Little is known about the etiology, management, and changing epidemiology of this disease in pediatric populations. Nonetheless, practitioners who care for children and adolescents must consider the diagnosis of type 2 DM in the child presenting with hyperglycemia. The diagnostic criteria and etiologic classification of DM are listed in Tables 1⇓ and 2⇓ . The diagnosis of DM is based on blood glucose measurements that may be obtained randomly, in the fasting state, or during an oral glucose tolerance test. The fasting blood glucose test is easy and inexpensive to perform and is recommended for screening by the American Diabetes Association (ADA). The criteria for making the diagnosis of DM were changed in 1997 by the Expert Committee on the Diagnosis and Classification of Diabetes Mellitus to propose simple and sensitive screening tests for carbohydrate metabolism derangements. View this table: Table 1. Criteria for the Diagnosis of Diabetes Mellitus (DM)1 View this table: Table 2. Etiologic Classification of Diabetes Mellitus (DM)1 DM represents a group of diseases characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. Type 1 DM is due to insulin deficiency usually caused by autoimmune destruction of …

Tracheomalacia in a Neonate with Kniest Dysplasia: Histopathologic and Ultrastructural Features

Kniest dysplasia is an autosomal-dominant chondrodysplastic condition characterized by disproportionate dwarfism, short trunk, small pelvis, kyphoscoliosis, short limbs, prominent joints, premature osteoarthritis, and craniofacial manifestations. The craniofacial abnormalities include tracheomalacia, midface hypoplasia, cleft palate, early onset myopia, retinal detachment, prominent eyes, and sensorineural hearing loss. Radiologic features include dumbbell-shaped femora, platyspondylia with anterior wedging of vertebral bodies, coronal clefts of thoracolumbar vertebral bodies, low broad ilia, and short tubular bones with broad metaphyses and deformed large epiphyses. This form of chondrodysplasia is associated with mutations in type II collagen splicing sequences. Mutations have been identified in the COL2A1 (type II collagen) gene between exons 12 and 24. Type II collagen is the predominant structural protein in cartilage, and mutations in this collagen account for the Kniest dysplasia phenotype. Histopathologic and ultrastructural features of epiphyseal plate cartilage have been described, but tracheal cartilage in an affected neonate has not been examined. The authors report the histopathologic and ultrastructural findings of anterior tracheal cartilage from a 35-day-old female with suspected chondrodysplasia who had tracheomalacia with airway obstruction. The tracheal cartilage was moderately cellular, but lacked cystic and myxoid changes in its matrix. The chondrocytes had abundant cytoplasmic PAS-positive inclusions. Some of these inclusions were diastase-resistant and were also highlighted on Alcian blue staining. Ultrastructural examination revealed chondrocytes with greatly dilated rough endoplasmic reticulum containing granular proteinaceous material. There were also frequent aggregates of typical glycogen. The defect in the COL2A1 gene is secondary to mutations, especially at splice junctions, and this markedly disrupts triple helix formation. The mutated type II procollagen results in intracellular retention within the chondrocytes, as abundant granular proteinaceous material within the dilated RER. A relationship is known to exist between the proportion of mutated to normal type II collagen in the matrix and the severity of the phenotype. With low levels of normal type II collagen, the phenotypic manifestations become more severe, such as in achondrogenesis type II. Both the quantity and quality of type II collagen modulates the phenotypic expression of type II collagenopathies.

Identification of ancillary fim genes affecting fimA expression in Salmonella typhimurium.

Regulation of the gene, fimA, encoding the major fimbrial subunit of S. typhimurium S6704 was examined by using a lambda fimA-lacZ lysogen. Transformation of the lambda fimA-lacZ lysogen with various derivatives of the recombinant plasmid that encodes type 1 fimbrial expression, pISF101, indicated that two regions of this plasmid alter beta-galactosidase production. One plasmid is a deletion resulting in the loss of a 28-kDa polypeptide downstream of fimA, while the other plasmid encodes a 24- and a 27-kDa polypeptide. Northern (RNA) blot analyses indicated that the steady-state fimA mRNA levels of these transformants were high. In addition, phenotypic expression of type 1 fimbriae by agar-grown cultures is observed only in those transformants bearing plasmids which show increased beta-galactosidase and fimA mRNA levels.

Expression of type 1 fimbriae and mannose-sensitive hemagglutinin by recombinant plasmids

Deletions within the cloned genes (fimA) encoding the type 1 major fimbrial subunits of two isolates of Klebsiella pneumoniae resulted in a nonfimbriate but hemagglutinating phenotype after transformation of Escherichia coli HB101 or ORN103. Phenotypic expression of type 1 fimbriae could be restored by transformation with plasmids containing the fimA genes of the fimbrial gene clusters from different strains. The surface fimbriae expressed were serologically identical to those of the polymerized product of the introduced fimA gene. The fimA gene products of Salmonella typhimurium and Serratia marcescens could utilize the accessory fimbrial genes of K. pneumoniae to produce surface-associated, hemagglutinating fimbriae. The relatedness of the type 1 fimbrial gene clusters from multiple isolates of members of the family Enterobacteriaceae was examined by DNA hybridization techniques. These analyses demonstrated little nucleotide sequence agreement among distinct genera of the enteric bacteria.

Cloning and characterization of the gene cluster encoding type 3 (MR/K) fimbriae ofKlebsiella pneumoniae

The gene cluster encoding the type 3 fimbriae of a Klebsiella pneumoniae isolate was cloned using the cosmid-cloning technique. Escherichia coli transformants, expressing type 3 fimbriae, were selected by reactivity with a monoclonal antibody directed against an epitope of the purified type 3 fimbriae. The phenotypic expression of type 3 fimbriae by transformants possessing the parental plasmid was dependent upon the host strain used. However, subcloning of this plasmid resulted in the construction of a chimeric molecule which imparted a stable phenotype regardless of the host strain. In addition, subcloning of the parental recombinant plasmid suggested that the minimal size of DNA necessary for production and expression of fimbriae was approximately 5.5 kb.

Cloning and characterization of the gene cluster encoding type 3 (MR/K) fimbriae of Klebsiella pneumoniae

The gene cluster encoding the type 3 fimbriae of a Klebsiella pneumoniae isolate was cloned using the cosmid-cloning technique. Escherichia coli transformants, expressing type 3 fimbriae, were selected by reactivity with a monoclonal antibody directed against an epitope of the purified type 3 fimbriae. The phenotypic expression of type 3 fimbriae by transformants possessing the parental plasmid was dependent upon the host strain used. However, subcloning of this plasmid resulted in the construction of a chimeric molecule which imparted a stable phenotype regardless of the host strain. In addition, subcloning of the parental recombinant plasmid suggested that the minimal size of DNA necessary for production and expression of fimbriae was approximately 5.5 kb.

Collagen biosynthesis by human skin fibroblasts II. Isolation and further characterization of type I and type III procollagens synthesized in culture

Human skin fibroblasts in culture have previously been shown to synthesize genetically distinct procollagens type I and type III. In the present study, cultured human skin fibroblasts were incubated under conditions optimized for synthesis of these procollagens in medium containing [ 3H]proline. The newly synthesized type I and type III 3H-labeled procollagens in the culture medium were then isolated as native proteins by DEAE-cellulose chromatography, or by gel filtration and SDS-polyacrylamide slab gel electrophoresis under denaturing conditions after limited pepsin proteolysis. The chromatographic procedures were optimized to yield reliable and reproducible results with good recoveries. The isolated procollagens were identified by cyanogen bromide peptide mapping and characterized by cleavage with highly purified collagenase synthesized by human skin fibroblasts. Assay of the relative synthesis of type I/III procollagens by normal human skin fibroblasts using DEAE-cellulose chromatography indicated that 80% of the procollagen in the medium was type I while the remaining 20% consisted of type III. When the ratio of newly-synthesized type I/III collagens was estimated by gel filtration or using SDS-polyacrylamide slab gel electrophoresis after limited pepsin proteolysi, relatively fewer type III collagen α-chains were recovered. This observation suggests that some of the type III collagen molecules are in a conformation which is less resistant to digestion by pepsin than the triple-helix of type I procollagen. The coefficient of variation for the relative synthesis of type I and type III procollagens by control cultures was relatively small (16%), indicating that the phenotypic expression of type I and type III procollagen genes, under optimized culture conditions, is under a relatively tight control. The results further suggest that the optimized methodology developed for assay of the relative synthesis of type I and type III procollagens and collagens by cultured human skin fibroblasts can be utilized in studies on collagen aberrations in acquired and inherited diseases of connective tissue.