Performance of undergraduate dental students on ICDAS clinical caries detection after different learning strategies.

AIM: This study aimed to evaluate the effect of a digital learning tool on undergraduate dental students' performance in detecting dental caries using ICDAS. METHODS: An experimental digital learning tool (DLT) was created using digital photographs of sound and carious teeth. Thirty-nine students were divided into three groups (n = 13) and each assessed 12 randomly allocated patients before and after learning strategies: G1, ICDAS e-learning program; G2, ICDAS e-learning program plus DLT; G3, no learning strategy. Students (n = 32) reassessed patients 2 weeks after training. RESULTS: Comparing before and after the learning strategies, any difference in the values of specificity and area under the ROC curve for all groups was found. Sensitivity was statistically significantly higher for G1 and G2. Comparing the groups, G2 showed a significant increase in sensitivity at the D2 and D3 thresholds. Spearman's correlations with the gold standard before and after the learning strategy were 0.60 and 0.61 for G1, 0.57 and 0.63 for G2, and 0.54 and 0.54 for G3, respectively. The Wilcoxon test showed a statistically significant difference between the values obtained before and after learning strategies for G1 and G2. CONCLUSIONS: Use of the DLT after the ICDAS e-learning program tended to increase the sensitivity of ICDAS used by undergraduate dental students. The DLT appeared to improve dental students' ability to use ICDAS.

Purification of the autotransporter protein Hbp of Escherichia coli.

The enzyme Hbp (hemoglobin protease) of the pathogenic Escherichia coli strain EB1 has been purified to homogeneity by gel filtration chromatography. The purified protein is capable of binding heme and shows hemoglobin protease activity. Our method of purification is applicable not only to Hbp but also to other autotransporter proteins and will contribute to a better understanding of the function-structure relationship of this family of proteins.

Characterization of a hemoglobin protease secreted by the pathogenic Escherichia coli strain EB1.

Many pathogenic bacteria can use heme compounds as a source of iron. Pathogenic Escherichia coli strains are capable of using hemoglobin as an iron source. However, the mechanism of heme acquisition from hemoglobin is not understood for this microorganism. We present the first molecular characterization of a hemoglobin protease (Hbp) from a human pathogenic E. coli strain. The enzyme also appeared to be a heme-binding protein. Affinity purification of this bifunctional protein enabled us to identify the extracellular gene product, and to clone and analyze its gene. A purification procedure developed for Hbp allowed us to perform functional studies. The protein interacted with hemoglobin, degraded it and subsequently bound the released heme. These results suggest that the protein is involved in heme acquisition by this human pathogen. Hbp belongs to the so-called IgA1 protease-like proteins, as indicated by the kinetics of its membrane transfer and DNA sequence similarity. The gene of this protein appears to be located on the large pColV-K30 episome, that only has been isolated from human and animal pathogens. All these characteristics indicate that Hbp may be an important virulence factor that may play a significant role in the pathogenesis of E. coli infections.

Molecular characterization of a heme-binding protein of Bacteroides fragilis BE1.

An iron-repressible 44-kDa outer membrane protein plays a crucial role in the acquisition of heme by the anaerobic bacterium Bacteroides fragilis. The DNA sequence of the gene encoding the 44-kDa protein (hupA) was determined. The hupA gene encodes a protein of 431 amino acid residues with a calculated molecular mass of 48,189 Da. The hupA gene is preceded by an open reading frame of 480 bp that probably encodes a protein with a calculated molecular mass of 18,073 Da. hupA and this open reading frame are likely organized in an operon, and a sequence homologous to the Escherichia coli consensus Fur box was present in the putative promoter region of the operon. Heme-binding studies showed that HupA binds heme. Analysis of the deduced amino acid sequence revealed signature heme-binding consensus motifs, characteristic of heme lyases. Subcellular localization studies in E. coli revealed that HupA was mainly found in the cytoplasmic membrane but not in the outer membrane of E. coli. This suggested that B. fragilis uses another strategy for the translocation of this outer membrane protein across its cell envelope than E. coli does. HupA did not have significant homology with other putative bacterial heme receptors.

Iron-repressible outer membrane proteins of Helicobacter pylori involved in heme uptake.

Helicobacter pylori is known to be a causative agent of gastritis and peptic ulcer disease in humans. The acquisition of iron from the human host may contribute greatly to the virulence of this organism. To study this, H. pylori was cultured under iron-restrictive conditions to induce synthesis of possible iron-regulated outer membrane proteins. This was achieved by the addition of 20% (vol/vol) heat-inactivated newborn calf serum, which contains iron-binding proteins like transferrin and albumin, and no free iron. The newborn calf serum was able to bind free ionic iron in brucella broth culture medium. Electrophoretic analysis of outer membrane preparations from H. pylori cultured under conditions of iron restriction showed several proteins to be present at elevated levels. These appeared to be iron-repressible outer membrane proteins (IROMPs). In addition, IROMPs with molecular sizes of 77, 50, and 48 kDa were isolated by use of hemin-agarose affinity chromatography. These three heme-binding IROMPs might be involved in the uptake of heme from the host and might therefore be important virulence factors of H. pylori.

Utilization of haem from the haptoglobin-haemoglobin complex by Bacteroides fragilis.

Possession of specialized iron acquisition systems is a prerequisite for the survival of pathogenic bacteria in their host. The purpose of this study was to determine whether Bacteroides fragilis, a clinically important Gram-negative anaerobic bacterium, possesses a specific haem-uptake system. Growth studies indicated that this microorganism can utilize haem from either haemoglobin or haptoglobin-haemoglobin as its sole source of iron. Iron-repressible haem-binding protein complexes (HBP complexes), involved in the uptake of haem from haptoglobin-haemoglobin were detected by means of lithium dodecyl sulfate polyacrylamide gel electrophoresis (LDS-PAGE). Four polypeptides of approximately 60, 58, 49 and 35 kDa, which are part of these HBP complexes, were identified as haem-binding proteins. A 44 kDa iron-repressible outer-membrane protein is needed for a functional HBP complex, but the exact role of this protein in the uptake of haem is still unknown.

Transferrins and heme-compounds as iron sources for pathogenic bacteria.

The low concentration of free iron in body fluids creates bacteriostatic conditions for many microorganisms and is therefore an important defense factor of the body against invading bacteria. Pathogenic bacteria have developed several mechanisms for acquiring iron from the host. Siderophore-mediated iron uptake involves the synthesis of low molecular weight iron chelators called siderophores which compete with the host iron-binding glycoproteins lactoferrin (LF) and transferrin (TF) for iron. Other ways to induce iron uptake, without the mediation of siderophores, are the possession of outer membrane protein receptors that actually recognize the complex of TF or LF with iron, resulting in the internalization of this metal, and the use of heme-compounds released into the circulation after lysis of erythrocytes. In this review, the nonsiderophore-mediated iron-uptake systems used by certain pathogenic bacteria are emphasized. The possible contribution of these iron-uptake systems to the virulence of pathogens is also discussed.

Human immune response to an iron-repressible outer membrane protein of Bacteroides fragilis.

Under conditions of iron starvation, Bacteroides fragilis expresses various iron-repressible outer membrane proteins (IROMPs). A 44-kDa protein appears to be one of the major outer membrane proteins (OMPs) in B. fragilis under iron stress and plays a role in heme uptake by this bacterium. To determine whether the 44-kDa IROMP of B. fragilis is expressed in vivo and whether this protein is immunogenic, we used Western immunoblotting to examine serum samples from patients with an infection caused by Bacteroides species. All the serum samples from patients and from normal controls showed reactivity with several proteins of B. fragilis. Only serum samples from patients infected with B. fragilis showed immunoreactivity with the 44-kDa protein. We also used a rat infection model to study the immune response against this protein during the process of an intra-abdominal infection in these animals. During the first 8 days of infection a gradual increase of antibodies to the 44-kDa protein in the rat was detected. These results suggest that the 44-kDa IROMP is expressed in vivo, since it induces an antibody response in patients and animals. We also analyzed 85 strains of the B. fragilis group for the presence of proteins antigenically related to the B. fragilis 44-kDa protein. The data indicate that this protein was conserved in B. fragilis strains and was absent in the other bacterial strains tested.

Iron-regulated outer membrane protein of Bacteroides fragilis involved in heme uptake.

Under iron starvation, Bacteroides fragilis expresses various iron-regulated outer membrane proteins. In this study, a deferrated minimal medium was used in growth experiments, and the role of one of these iron-regulated outer membrane proteins (a 44-kDa protein) in an iron uptake mechanism which acquires iron from heme compounds was elucidated. When a specific 44-kDa protein antiserum was used in a medium with heme as the only iron source, growth inhibition was observed. These results demonstrate that the 44-kDa outer membrane protein plays an important role in the uptake of heme in B. fragilis.

Purification and partial characterization of an iron regulated outer membrane protein of B. fragilis under non-denaturing conditions.

The CHAPS-PAGE gelsystem we applied gave a good separation of the proteins of Bacteroides fragilis under non-denaturing conditions. We succeeded with preparative CHAPS-PAGE in purifying an iron regulated outer membrane protein (a 44 kDa polypeptide on SDS-PAGE) of B. fragilis. This integral membrane protein proved to be a lipopolysaccharide binding protein with an isoelectric point of approximately pH 5.5. This method of purifying membrane proteins could be an important step in research into the function of membrane proteins.

Outer membrane proteins of Bacteroides fragilis and Bacteroides vulgatus in relation to iron uptake and virulence.

A virulent strain B. fragilis BE1 and an avirulent strain B. vulgatus BE20 were grown in a culture medium with and without the addition of a synthetic chelator (Bipyridyl) to induce iron limitation. Cells grew more slowly under iron stress, although the growth rate of the B. vulgatus strain was more affected under these conditions than the strain of B. fragilis. The outer membrane protein profile of these strains was studied in relation to the iron concentration in the growth medium by means of SDS-polyacrylamide gel electrophoresis. Four proteins, with the apparent molecular weights of 89, 49, 44 and 23.5 kDa, were consistently present in the outer membrane of B. fragilis BE1 grown under iron restricted conditions. In B. vulgatus BE20 cells a 44 and a 23.5 kDa protein were absent and only the expression of an 89 kDa protein was clearly seen under these conditions. The iron regulated proteins, particularly the 44 kDa protein, could be involved to an iron uptake mechanism in B. fragilis. So the presence of these proteins might play an important role in the virulence of this anaerobic bacterium.