RESUMO
This study analysed the invasiveness of Listeria monocytogenes into enterocyte-like Caco-2 cells in which iron depletion was achieved by picolinic acid treatment. Both entry and intracellular multiplication varied depending on the endogenous iron content of bacterial and eukaryotic cells. The behaviour within enterocytes was correlated with a 10-fold increased transcription of the actA gene observed in bacterial cells grown under conditions of iron stress.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Ferro/fisiologia , Listeria monocytogenes/genética , Proteínas de Membrana/genética , Células CACO-2 , Humanos , Listeria monocytogenes/patogenicidade , VirulênciaRESUMO
Thiobacillus ferrooxidans is a Gram-negative chemolithotrophic bacterium able to oxidize ferrous iron, elemental sulfur and inorganic sulfur compounds. The oxidation of sulfur by T. ferrooxidans resulted in an expression of some outer membrane proteins (OMPs) at a level higher than that observed during ferrous iron oxidation. Among these OMPs, a protein with a molecular mass of 54 kDa was purified and 18 amino acids of the N-terminal sequence determined. Using a 54 bp PCR generated DNA product as a probe for the protein, we isolated a 4.5 kb Pst I DNA chromosomal fragment containing the corresponding gene. Sequencing 2169 bp of this fragment revealed the open reading frame codifying for the protein, consisting of 467 amino acids and a molecular mass of 49,674 Da. The mature protein was produced by the removal of a 32 amino acid signal peptide-like sequence from the N-terminus of a 499 amino acid peptide. Although no significant homology with any known protein has been found and its physiological role remains unclear, its high expression on sulfur substrates suggests a role in sulfide mineral oxidation.
Assuntos
Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Enxofre/metabolismo , Thiobacillus/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , DNA Bacteriano , Dados de Sequência MolecularRESUMO
We developed a method of identification of Listeria monocytogenes based on colony hybridization with nonradioactively labeled DNA probes, represented by the hly and inlA virulence-associated genes. The procedure described in this paper results simple, rapid, specific and reproducible. Since it can be performed in a short time, the above technique can be applied to detect L. monocytogenes from different source and constitutes a noteworthy and alternative tool to identify this gram-positive pathogenic bacterium.
Assuntos
Técnicas de Tipagem Bacteriana , Sondas de DNA/genética , Genes Bacterianos/genética , Listeria monocytogenes/classificação , Microbiologia de Alimentos , Listeria monocytogenes/genética , Listeria monocytogenes/patogenicidade , Virulência/genéticaRESUMO
The influence of iron on the entry of Listeria monocytogenes into Caco-2 cells was studied. Iron availability was found to modify the surface hydrophobicity and protein profile of L. monocytogenes, with the result that cell invasion strongly increased upon bacterial growth in iron-rich medium. The enhanced invasive capability of iron-overloaded L. monocytogenes cells correlates to the higher-level expression of the inlAB virulence genes, which were positively iron regulated at the transcriptional level.
Assuntos
Aderência Bacteriana , Proteínas de Bactérias/genética , Toxinas Bacterianas , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Ferro/metabolismo , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Proteínas de Bactérias/química , Células CACO-2 , Proteínas Hemolisinas , Hemólise , Humanos , Listeria monocytogenes/química , Listeria monocytogenes/genética , RNA Bacteriano/genética , RNA Mensageiro/genética , Solubilidade , Propriedades de Superfície , Transcrição GênicaRESUMO
The aim of the present study was to evaluate the role of temperature in the virulence of Listeria monocytogenes, a Gram-positive facultative intracellular food-borne pathogen. The capacity of bacteria grown at 37, 25 and 4 degrees C to develop haemolytic activity, to enter the Caco-2 enterocyte-like cell line and to multiply intracellularly was investigated. We demonstrated that L. monocytogenes penetration was not significantly influenced by the growth temperature of cultures and that bacteria grown at low temperature were capable of synthesizing internalin and, during the infection process, of restoring the haemolytic phenotype which is normally lacking in the extracellular environment at 4 and 25 degrees C. It can be concluded that L. monocytogenes, frequently present in numerous environmental sources and also in refrigerated food products, produces at low temperature, the virulence factors necessary to invade intestinal cells.
Assuntos
Neoplasias do Colo/microbiologia , Listeria monocytogenes/patogenicidade , Listeriose/microbiologia , Rifampina/farmacologia , Humanos , Técnicas In Vitro , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/crescimento & desenvolvimento , Listeriose/tratamento farmacológico , Rifampina/uso terapêutico , Temperatura , Células Tumorais Cultivadas , VirulênciaRESUMO
The effect of growth in iron-excess or iron-limitation conditions on the invasiveness for HeLa cells of Escherichia coli HB101 carrying plasmid pRI203 which bears the invasion gene of Yersinia pseudotuberculosis was examined. Iron-limitation reduced adhesion and the number of organisms internalised by HeLa cells by about 100-fold. The reduced adhesion of iron-starved bacteria correlated with reduced hydrophobicity and the reduced invasiveness appeared to depend on the plasmid copy number, which was 3.5-fold less than in bacteria grown in iron excess.
Assuntos
Escherichia coli/efeitos dos fármacos , Ferro/farmacologia , Yersinia pseudotuberculosis/genética , Aderência Bacteriana/efeitos dos fármacos , Proteínas da Membrana Bacteriana Externa/análise , DNA Bacteriano/análise , Escherichia coli/genética , Escherichia coli/patogenicidade , Células HeLa , Humanos , Plasmídeos , Inoculações Seriadas , Propriedades de SuperfícieRESUMO
Lactoferrin (Lf) is an iron-binding protein which plays an important role in the host defense systems of different mucosal surfaces including the intestinal mucosa. In the present research the role of apo-Lf and iron-saturated Lf in the invasion process of enteroinvasive bacteria, grown in iron stress or excess, was investigated. As enteroinvasive bacterium, Escherichia coli HB101 strain harboring a plasmid which contains the chromosomal inv gene from Yersinia pseudotuberculosis was utilized. The product of this gene (invasin) enables this microorganism to invade human epithelial cultured cells (HeLa). The results obtained showed that apo-Lf and iron-saturated Lf added at physiological concentration during the infection exerted a significant inhibition of adhesion (3.2 x 10(5) instead 3.4 x 10(6) adherent bacteria grown in iron excess; 1.6 x 10(3) instead of 2.3 x 10(4) adherent bacteria grown in iron-limited medium) and internalization (4.0 x 10(5) instead of 3.7 x 10(6) internalized bacteria grown in iron excess; 2.1 x 10(3) instead 2.8 x 10(4) internalized bacteria grown in iron-limited medium). It has also been demonstrated that in these experimental conditions Lf binds to HeLa cell membrane as well as to bacterial outer membrane. It is likely that this binding interfere with the early events of interaction between bacteria and eukaryotic cells. This inhibiting effect of Lf on the invasion efficiency of E. coli HB101 (pRI203) could be related to the cationic nature of the molecule, although other mechanisms cannot be ruled out.
