Differentiation of typical and atypical enteropathogenic Escherichia coli using colony immunoblot for detection of bundle-forming pilus expression.

AIMS: The aim of study was to develop a colony immunoblot assay to differentiate typical from atypical enteropathogenic Escherichia coli (EPEC) by detection of bundle-forming pilus (BFP) expression. METHODS AND RESULTS: Anti-BFP antiserum was raised in rabbits and its reactivity was confirmed by immunoelectron microscopy and by immunoblotting recognizing bundlin, the major pilus repeating subunit. The bacterial isolates tested in the colony immunoblot assay were grown in different media. Proteins from bacterial isolates were transferred to nitrocellulose membrane after treatment with phosphate buffer containing Triton X-100, EDTA and sodium chloride salts. When 24 typical EPEC and 96 isolates including, 72 atypical EPEC, 13 Gram-negative type IV-expressing strains and 11 enterobacteriaceae were cultivated in Dulbecco's Modified Eagle's Medium agar containing fetal bovine serum or in blood agar in the presence of CaCl(2) , they showed a positivity of 92 and 83%, and specificity of 96 and 97%, respectively. CONCLUSION: The assay enables reliable identification of BFP-expressing isolates and contributes to the differentiation of typical and atypical EPEC. SIGNIFICANCE AND IMPACT OF THE STUDY: The colony immunoblot for BFP detection developed in this study combines the simplicity of an immunoserological assay with the high efficiency of testing a large number of EPEC colonies.

Differentiation of typical and atypical enteropathogenic Escherichia coli using colony immunoblot for detection of bundle-forming pilus expression

The aim of study was to develop a colony immunoblot assay to differentiatetypical from atypical enteropathogenic Escherichia coli (EPEC) by detectionof bundle-forming pilus (BFP) expression. Anti-BFP antiserum was raised in rabbits and itsreactivity was confirmed by immunoelectron microscopy and by immunoblotting recognizing bundlin, the major pilus repeating subunit. The bacterial isolates tested in the colony immunoblot assay were grown in different media. Proteins from bacterial isolates were transferred to nitrocellulose membrane after treatment with phosphate buffer containing Triton X-100, EDTA and sodium chloride salts. When 24 typical EPEC and 96 isolates including, 72 atypical EPEC, 13 Gram-negative type IV-expressing strains and 11 enterobacteriaceae were cultivated in Dulbecco’s Modified Eagle’s Medium agar containing fetal bovine serum or in blood agar in the presence of CaCl2, they showed a positivity of 92 and 83%, and specificity of 96 and 97%, respectively. The assay enables reliable identification of BFP-expressing isolatesand contributes to the differentiation of typical and atypical EPEC.The colony immunoblot for BFP detectiondeveloped in this study combines the simplicity of an immunoserologicalassay with the high efficiency of testing a large number of EPECcolonies.

Comparison of the pulmonary response against lethal and non-lethal intranasal challenges with two different pneumococcal strains

The differences between the immune response elicited during a self-limiting and a life-threatening lung infection with Streptococcus pneumoniae was analyzed in a mouse model of intranasal challenge using two different pneumococcal strains. M10, a serotype 11A strain, induced an early response within the first 12 h after the challenge, which was characterized by the early local secretion of TNF-á and IL-6, followed by a sharp and rapid neutrophil influx. Bacterial loads in the lungs already started to fall at 12 h after the challenge and no pneumococci could be recovered after 36 h, at the time point when the animals started to show improvement in disease symptoms. ATCC6303, a serotype 3 strain, on the other hand, showed only a late increase in local TNF-á and IL-6 levels, when bacterial growth already seems to be out of control. Although cell influx was also observed, neutrophil rise was not as marked as with M10 (type 11A). Pneumococcal loads increased constantly and bacteria started to be recovered from the blood at 30 h after the challenge. After this time point, animals showed worsening of symptoms and became lethargic. The resolution of the acute infection could be thus correlated with the early induction of proinflammatory cytokines, which could be due to the presence of a thinner polysaccharide capsule in M10 (type 11A), rendering bacterial components capable of activating the innate immune response more accessible.

Leptospiral TlyC is an extracellular matrix-binding protein and does not present hemolysin activity

The role of TlyA, TlyB and TlyC proteins in the biology of Leptospira is still uncertain. Although these proteins have been considered as putative hemolysins, we demonstrate that leptospiral recombinant TlyB and TlyC do not possess hemolytic activity. However, further experiments showed that TlyC is a surface-exposed protein that seems to bind to laminin, collagen IV and fibronectin. The expression of both proteins was detected both in vitro and in vivo. Our findings suggest that TlyB and TlyC are not directly involved in hemolysis, and that TlyC may contribute to Leptospira binding to extracellular matrix (ECM) during host infection.

Adherence and invasion of Bacteroidales isolated from the human intestinal tract.

Members of the genera Bacteroides and Parabacteroides are important constituents of both human and animal intestinal microbiota, and are significant facultative pathogens. In this study, the ability of Bacteroides spp. and Parabacteroides distasonis isolated from both diarrhoeal and normal stools (n = 114) to adhere to and invade HEp-2 cells was evaluated. The presence of putative virulence factors such as capsule and fimbriae was also investigated. Adherence to HEp-2 cells was observed in 75.4% of the strains, which displayed non-localized clusters. Invasion was observed in 37.5% and 26% of the strains isolated from diarrhoeal and non-diarrhoeal stools, respectively. All strains displayed a capsule, whereas none of them showed fimbriae-like structures. This is the first report of the ability of Bacteroides spp. and P. distasonis to adhere to and invade cultured HEp-2 epithelial cells.

Haemoglobin biosynthesis site in rabbit embryo erythroid cells

Properly metabolized globin synthesis and iron uptake are indispensable for erythroid cell differentiation and maturation. Mitochondrial participation is crucial in the process of haeme synthesis for cytochromes and haemoglobin. We studied the final biosynthesis site of haemoglobin using an ultrastructural approach, with erythroid cells obtained from rabbit embryos, in order to compare these results with those of animals treated with saponine or phenylhydrazine. Our results are similar to those obtained in assays with adult mammals, birds, amphibians, reptiles and fish, after induction of haemolytic anaemia. Therefore, the treatment did not interfere with the process studied, confirming our previous findings. Immunoelectron microscopy showed no labelling of mitochondria or other cellular organelles supposedly involved in the final biosynthesis of haemoglobin molecules, suggesting instead that it occurs free in the cytoplasm immediately after the liberation of haeme from the mitochondria, by electrostatic attraction between haeme and globin chains.

Ultrastructural chemical reaction to detect saturated phospholipids of a natural surfactant from pig lungs

The ultrastructure of the natural surfactant from pig lungs fixed by the tricomplex method with Pb(NO 3) 2 and K 3Fe(CN) 6 reveals its lamellar phase (ordered configuration). The lamellar forms are usually associated with being surface active

Mitochondrial kinetics during amphibian erythropoiesis related to haeme synthesis.

Flow cytometry, light and epifluorescence microscopies and transmission electron microscopy were used to follow the mitochondrial kinetics during amphibian erythropoiesis. A similar behaviour in response to the induction of anaemia was observed in the diploid Bufo ictericus and the tetraploid Odontophrynus americanus. A high cellular activity was observed ten days after haemolytic anaemia induced by phenylhydrazine, based on the higher Rhodamine 123 uptake by the erythroid cells. In addition, the more intense expression of the mitochondrial enzyme cytochrome oxidase, isocitrate and succinic dehydrogenases were cytochemically detected at this stage. This suggests that erythroid cell mitochondria, at this time, could be in a more active functional state than at other stages. In both species, mitochondrial plasticity was observed during cell maturation. A progressive loss of oxidation-reduction enzyme expression seemed to follow changes at the mitochondrial cristae morphology, from transverse to longitudinal form, mainly at the 20th day of recovery from anaemia, possibly related to a natural loss of function. The presence of these mitochondrial enzymes in mitochondrion-like organelles also favours their participation in the haeme synthesis, although with a reduced expression, since this suggests the presence of a complete and active enzymatic complex in these modified organelles. This also supports the idea that all these organelles are mitochondria in distinct metabolic stages, and not mitochondrion-like organelles or haemosomes, as proposed by some authors.

Immunocytochemical mapping of the hemoglobin biosynthesis site in amphibian erythroid cells.

During the past 25 years, several studies have attempted to determine the site of integration of the heme and the four globin chains in vertebrate erythroid cells that is important in the formation of the hemoglobin molecule. Mitochondrion-like organelles or hemosomes were pointed out as responsible for this task. We performed several experiments to investigate this hypothesis. The intracellular distribution of hemoglobin in amphibian erythroid cells was detected by post-embedding immuno-electron microscopy, using a polyclonal anti-human hemoglobin-proteinA-gold complex. Hemoglobin mapping showed an intense labeling in the cell cytoplasm, but none in cytoplasmic structures such as endoplasmic reticulum, mitochondria, mitochondrion-like organelles, Golgi complex, ribosomes or ferruginous inclusions. The mitochondrial fraction obtained according to the protocol described for some authors, showed by ultrastructural examination that this fraction has a heterogeneous content, also composed by microvesicles rich in cytoplasmic hemoglobin, an artifact generated by mechanical action during cell fractionation. Thus, when this fraction is lysed and its content submitted to electrophoresis, hemoglobin bands would be found inevitably, causing false-positive results, erroneously attributed to hemoglobin content of mitochondrion-like organelles. Our data do not confirm the hypothesis that the final hemoglobin biosynthesis occurs inside mitochondrion-like organelles. They suggest that the hemoglobin molecule be assembled in the erythrocyte cytoplasm outside of mitochondria or hemosomes.

Comparative study of immature erythroid cells of the diploid Bufo ictericus and the tetraploid Odontophrynus americanus (Amphibia, Anura): ultrastructural cytochemical detection of nucleic acids and polysaccharides, and mapping of the element phosphorus.

In the present study, we used ultrastructural cytochemistry to analyze the distribution of nuclear and cytoplasmic nucleic acids and polysaccharides, and electron spectroscopic imaging to map the element phosphorus in immature erythroid cells taken from two amphibians, the diploid Bufo ictericus and the tetraploid Odontophrynus americanus. In the cytoplasm of cells from the tetraploid species, we detected numerous inclusions containing a material that was similar to the dispersed chromatin seen in the nucleus of these cells. The RNase-gold complex labeled both the dispersed nuclear chromatin and the cytoplasmic inclusions. The Thiéry technique showed that glycoconjugates were present in all the membranous complexes of the erythroid cells of both types of amphibians under study, although they were absent within or around the cytoplasmic RNA inclusions. Electron spectroscopic imaging revealed the presence of phosphorus in these inclusions. These data suggest that an increase in RNA synthesis occurs in tetraploid amphibian cells, probably as a result of an alteration in the mechanisms of gene regulation.

Hemosomegenesis and hemoglobin biosynthesis in vertebrates.

1. Ultrastructural observations on maturing rabbit embryo erythroid cells led to the finding of hemoglobinized organelles distinguishable from mitochondria due to their highly dense matrix, two or three longitudinally arranged double lamellae, and smaller diameters. Intraorganellar 50-60 A particles identical to those contained in the hemoglobinized cytoplasm were found. 2. Their hemoglobin (Hb) content was demonstrated by electrophoresis of the concentrated supernatant from the isolated, washed, and osmotically lysed organellar fraction. We have proposed that these organelles are the sites for heme integration into the globin (G) polypeptide chains and subunits assembly. The term hemosome has been suggested for such entities. 3. This hypothesis has been sustained by several analytical and experimental works based on the postulation that hemosomes should be found at higher frequencies where the Hb biosynthesis rate is more intensive, or where the induction of this biosynthesis is always dependent on the formation of hemosomes. 4. Maturing erythroid cells of the circulating embryo blood contain hemosomes in higher frequency than in liver erythroid cells, coinciding with the higher Hb biosynthesis rate in peripheral blood than in the liver. In bleeding anemia, the decay of Hb concentration parallels the reduction of the mean number of hemosomes per reticulocyte, in comparison with normal reticulocytes. 5. In HeLa cells and epithelial cultured cells induced to synthesize Hb, it was shown that this biosynthesis is ever concomitant with the formation of hemosomes and depends on the presence of erythropoietin, as occurs in erythroid cells. 6. Studies on hemosomegenesis and Hb biosynthesis experimentally effected in epithelial cultured cells, allowed the interpretation of the sequence of events leading to hemosome formation in maturing erythroid cells. Simultaneously with iron uptake, mitochondria differentiate to lamellated bodies and, successively, expansions rise for ferruginous compounds and G polypeptides gathering, followed by prehemosome vesicles formation, which condense and change to prohemosomes that afterwards evolve to hemosomes. 7. These dynamics, and organellar Hb have been detected in immature erythrocytes of mammalians, including humans, avians, reptilians, amphibians and representative fish specimens. It appears that these events occur in the erythrocytary maturation of all vertebrate classes.

Comparative ultrastructure of maturing toad (Bufo ictericus) and rabbit (Oryctolagus cuniculus) erythroid cells with regard to hemoglobin biosynthesis.

1. Toad and rabbit maturing erythroid cells were comparatively analysed with regard to their ultrastructural modifications involved in hemoglobin (Hb) biosynthesis. 2. The mitochondrial inner membrane differentiates to a lamellated body that, successively, gives rise to prehemosomal vesicles, prohemosomes, and to hemoglobinized organelles called hemosomes. 3. The prehemosomal vesicle involves ferruginous inclusions, taken as iron sources for heme biosynthesis, as well as the polypeptide globin chains, assembling themselves in the course of volume reduction. 4. From the prohemosomal stage onwards, where possibly heme biosynthesis occurs, hemosomes are formed; these organelles are presumably sites where the final Hb biosynthesis could take place. 5. All development stages leading to hemosome formation are similar in toad and rabbit erythroid cells, except that, in the toad, the structural prohemosome characteristics persist in hemosomes. 6. Through toad erythroid cell fractionation and electrophoresis of the organelle lysate supernatant, a wide and a weak cytoplasmic Hb bands were obtained; the latter coincides with the intraorganellar Hb band.