New approach for detection of Escherichia coli invasion to HeLa cells.

To establish a successful infection, microorganisms have developed strategies to invade host cells. One of the most important human pathogens and the greatest cause of urinary tract infections, Escherichia coli, still do not have its invasion mechanisms fully understood. This work aims to present a new approach for detecting bacterial invasion of lineage cells, based on an enzymatic-fluorogenic method. The focus of this technique is the detection of E. coli invasion of HeLa cells, exploring ß-glucuronidase, a specific constitutive enzyme of this bacterium. This enzyme hydrolyses the key substrate of this work, 4-methylumbelliferyl-ß-d-glucuronide (MUG), resulting in a fluorogenic molecule, 4-methylumbelliferone. The fluorescence curve created by this method, analyzed by Tukey statistical test, demonstrated that this detection can be efficiently performed after 5 h incubation with MUG. When testing uropathogenic E. coli and E. coli isolated from human gastrointestinal microbiota, the proposed method presented similar results to those exhibited by plate counting invasion detection. Data examination by Duncan statistical test allowed the creation of an intensity range of bacterial invasion, which is part of the process of results interpretation. Detection by this enzymatic-fluorogenic method, compared to other existing bacterial invasion detection techniques, is less burdensome, more sensitive and allows fast achievement of reliable results.

Bacteria arise at the border of mycoplasma-infected HeLa cells, containing cytoplasm with either malformed cytosol, mitochondria and endoplasmic reticulum or tightly adjoined smooth vacuoles.

A study with transmission electron microscopy of mycoplasma-contaminated HeLa cells using five cell donors referred to as donors A, B, C, D and E, observations are herein presented. Experiments performed with cells from donors B, C and D, revealed the presence of Mycoplasma hyorhinis after PCR and sequencing experiments. Bacteria probably originated from a cytoplasm with compacted tiny granular particles replacing the normal cytosol territories, or from the contact with the cytoplasm through a clear semi-solid material. The compact granularity (CG) of the cytoplasm was crossed by stripes of smooth and rough endoplasmic reticulum cisternae. Among apparently normal mitochondria, it was noted, in variable proportions, mitochondria with crista-delimited lucent central regions that expand to and occupied the interior of a crista-less organelle, which can undergo fission. Other components of the scenarios of mycoplasma-induced cell demolition are villus-like structures with associated 80-200 nm vesicles and a clear, flexible semi-solid, process-sensitive substance that we named jam-like material. This material coated the cytoplasmic surface, its recesses, irregular protrusions and detached cytoplasmic fragments. It also cushioned forming bacteria. Cyst-like structures were often present in the cytoplasm. Cells, mainly apoptotic, exhibiting ample cytoplasmic sectors with characteristic net-like profile due to adjoined vacuoles, as well as ovoid or elongated profiles, consistently appeared in all cells from the last four cell donors. These cells were named "modified host cells" because bacteria arose in the vacuoles. The possibility that, in some samples, there was infection and/or coinfection of the host cell by another organism(s) cannot be ruled out.

Apoptosis in HEp-2 cells infected with Ureaplasma diversum.

BACKGROUND: Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location of Ureaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversum invasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit. RESULTS: The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed. CONCLUSIONS: The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.

Apoptosis in HEp-2 cells infected with Ureaplasma diversum

BACKGROUND: Bacterial pathogens have many strategies for infecting and persisting in host cells. Adhesion, invasion and intracellular life are important features in the biology of mollicutes. The intracellular location ofUreaplasma diversum may trigger disturbances in the host cell. This includes activation or inhibition of pro and anti-apoptotic factors, which facilitate the development of host damage. The aim of the present study was to associate U. diversum infection in HEp-2 cells and apoptosis induction. Cells were infected for 72hs with four U. diversum clinical isolates and an ATCC strain. The U. diversuminvasion was analyzed by Confocal Laser Scanning Microscopy and gentamicin invasion assay. The apoptosis was evaluated using pro-apoptotic and anti-apoptotic gene expression, and FITC Annexin V/Dead Cell Apoptosis Kit. RESULTS: The number of internalized ureaplasma in HEp-2 cells increased significantly throughout the infection. The flow cytometry analysis with fluorochromes to detect membrane depolarization and gene expression for caspase 2, 3 and 9 increased in infected cells after 24 hours. However, after 72 hours a considerable decrease of apoptotic cells was observed. CONCLUSIONS: The data suggests that apoptosis may be initially induced by some isolates in association with HEp-2 cells, but over time, there was no evidence of apoptosis in the presence of ureaplasma and HEp-2 cells. The initial increase and then decrease in apoptosis could be related to bacterial pathogen-associated molecular pattern (PAMPS). Moreover, the isolates of U. diversum presented differences in the studied parameters for apoptosis. It was also observed that the amount of microorganisms was not proportional to the induction of apoptosis in HEp-2 cells.

[Two multidrug-resistant Salmonella infantis isolates behave like hypo-invasive strains but have high intracellular proliferation]. / Dos aislamientos de Salmonella infantis multirresistentes se comportan como hipoinvasivos pero con elevada proliferación intracelular.

In this work, plasmid-encoded virulence factors in two Salmonella Infantis isolates carrying multiresistance plasmids were investigated. In addition, their invasion and proliferative ability in non-phagocytic cells was studied. None of them showed the typical determinants of virulence plasmids (spy operon). The invasion assays of S. Infantis isolates on eukaryotic cells showed a decreased ability to invade but they remained and proliferated in the cytoplasm regardless of having used a permissive (HeLa) or non-permissive (NRK) cell line. Finally, there was no microscopic evidence suggesting a bactericidal effect of these eukaryotic cell lines on the isolates tested.

Dos aislamientos de Salmonella Infantis multirresistentes se comportan como hipoinvasivos pero con elevada proliferación intracelular

En este trabajo se investigó la presencia de determinantes característicos de plásmidos de virulencia en dos aislamientos clínicos de Salmonella Infantis portadores de plásmidos de multirresistencia. Además, se estudió la capacidad de invasión y proliferación en células eucariotas no fagocíticas. Ninguno de los aislamientos de S. Infantis mostró los determinantes genéticos que caracterizan a los plásmidos de virulencia para este género (operón spv). Los ensayos de invasión sobre líneas celulares eucariotas mostraron que los aislamientos de S. Infantis presentan una capacidad de invasión disminuida pero persisten y proliferan en el citoplasma, independientemente de utilizar una línea celular permisiva (HeLa) o no permisiva (NRK) para tal fin. Finalmente, no se observaron indicios microscópicos que podrían hacer sospechar un efecto bactericida de estas líneas celulares sobre los aislamientos estudiados.

Two multidrug-resistant Salmonella Infantis isolates behave like hypo-invasive strains but have high intracellular proliferation. In this work, plasmid-encoded virulence factors in two Salmonella Infantis isolates carrying multiresistance plasmids were investigated. In addition, their invasion and proliferative ability in non-phagocytic cells was studied. None of them showed the typical determinants of virulence plasmids (spv operon). The invasion assays of S. Infantis isolates on eukaryotic cells showed a decreased ability to Invade but they remained and proliferated In the cytoplasm regardless of having used a permissive (HeLa) or non-permissive (NRK) cell line. Finally, there was no microscopic evidence suggesting a bactericidal effect of these eukaryotic cell lines on the Isolates tested.

Dos aislamientos de Salmonella Infantis multirresistentes se comportan como hipoinvasivos pero con elevada proliferación intracelular

En este trabajo se investigó la presencia de determinantes característicos de plásmidos de virulencia en dos aislamientos clínicos de Salmonella Infantis portadores de plásmidos de multirresistencia. Además, se estudió la capacidad de invasión y proliferación en células eucariotas no fagocíticas. Ninguno de los aislamientos de S. Infantis mostró los determinantes genéticos que caracterizan a los plásmidos de virulencia para este género (operón spv). Los ensayos de invasión sobre líneas celulares eucariotas mostraron que los aislamientos de S. Infantis presentan una capacidad de invasión disminuida pero persisten y proliferan en el citoplasma, independientemente de utilizar una línea celular permisiva (HeLa) o no permisiva (NRK) para tal fin. Finalmente, no se observaron indicios microscópicos que podrían hacer sospechar un efecto bactericida de estas líneas celulares sobre los aislamientos estudiados.(AU)

Two multidrug-resistant Salmonella Infantis isolates behave like hypo-invasive strains but have high intracellular proliferation. In this work, plasmid-encoded virulence factors in two Salmonella Infantis isolates carrying multiresistance plasmids were investigated. In addition, their invasion and proliferative ability in non-phagocytic cells was studied. None of them showed the typical determinants of virulence plasmids (spv operon). The invasion assays of S. Infantis isolates on eukaryotic cells showed a decreased ability to Invade but they remained and proliferated In the cytoplasm regardless of having used a permissive (HeLa) or non-permissive (NRK) cell line. Finally, there was no microscopic evidence suggesting a bactericidal effect of these eukaryotic cell lines on the Isolates tested.(AU)

The type VI secretion system plays a role in type 1 fimbria expression and pathogenesis of an avian pathogenic Escherichia coli strain.

Avian pathogenic Escherichia coli (APEC) strains frequently cause extraintestinal infections and are responsible for significant economic losses in the poultry industry worldwide. APEC isolates are closely related to human extraintestinal pathogenic E. coli (ExPEC) strains and may also act as pathogens for humans. Known APEC virulence factors include adhesins such as type 1 fimbriae and curli, iron acquisition systems, and cytotoxins. Here we show that APEC strain SEPT362, isolated from a septicemic hen, expresses a type VI secretion system (T6SS); causes cytoskeleton rearrangements; and invades epithelial cells, replicates within macrophages, and causes lethal disease in chicks. To assess the contribution of the T6SS to SEPT362 pathogenesis, we generated two mutants, hcp (which encodes a protein suggested to be both secreted and a structural component of the T6SS) and clpV (encoding the T6SS ATPase). Both mutants showed decreased adherence and actin rearrangement on epithelial cells. However, only the hcp mutant presented a mild decrease in its ability to invade epithelial cells, and none of these mutants were defective for intramacrophage replication. Transcriptome studies showed that the level of expression of type 1 fimbriae was decreased in these mutants, which may account for the diminished adhesion and invasion of epithelial cells. The T6SS seems to be important for the disease process, given that both mutants were attenuated for infection in chicks. These results suggest that the T6SS influences the expression of type 1 fimbriae and contributes to APEC pathogenesis.

Apoptotic effect on HeLa cells produced by Chlamydia trachomatis-LPS

La interacción entre el lipopolisacárido (LPS) de Chlamydia trachomatis y las células de mamíferos permanece sin ser dilucidado. Chlamydia trachomatis es una bacteria intracelular responsable de diversas enfermedades en los humanos y animales. En este trabajo mediante el aislamiento del lipopolisacárido de dos serovares de Chlamydia trachomatis (LGV1-LGV2) y usando una coloración Supravital fluorescente (Hoechst 33258) fue posible investigar la respuesta de las células HeLa. El efecto apoptótico que sufren este tipo de células fue visible cuando fueron expuestas a dicho LPS en concentraciones iguales o mayores que 0,5 µg/mL por un periodo de 48 horas, sin embargo se observó la falta de repuesta celular en su ausencia o en presencia de LPS de otras bacterias. Adicionalmente, el uso en iguales condiciones de polimyxina B conocido como un neutralizador de la acción del LPS demostró una disminución del efecto apoptótico en dichas células, indicando que la respuesta celular observada fue producida por C. trachomatis-LPS. Los resultados de este trabajo le dan fuerza a la teoría de que el LPS de C. trachomatis pudiera ser el responsable del efecto tóxico que se observa sobre las células cervicales infectadas con esta bacteria intracelular.

Adherence to HeLa cells, typing by killer toxins and susceptibility to antifungal agents of Candida dubliniensis strains.

The aim of this study was to evaluate the adherence capability to HeLa cells, the susceptibility to killer toxins and the in vitro susceptibility to antifungal agents (eTest method--AB Biodisk, Solna, Sweden) of 9 Candida dubliniensis isolates recovered from HIV+ and AIDS patients. The adherence test was strongly positive for strain ATCC 777 and positive for all other strains. Typing by killer toxins revealed two different biotypes among the 9 isolates studied: 888 and 688. Only biotype 688 (ATCC 777) was susceptible to the K2 toxin. There was a significant inverse correlation between adherence and killer toxin susceptibility (r = -0.8525 - p = 0.0035). No strains presented resistance to fluconazole, itraconazole, ketoconazole, voriconazole, flucytosine or amphotericin-B. With the exception of ATCC 777, all the other isolates presented similar behavior.

Adherence to HeLa cells, typing by killer toxins and susceptibility to antifungal agents of Candida dubliniensis strains

The aim of this study was to evaluate the adherence capability to HeLa cells, the susceptibility to killer toxins and the in vitro susceptibility to antifungal agents (eTest? method - AB Biodisk, Solna, Sweden) of 9 Candida dubliniensis isolates recovered from HIV+ and AIDS patients. The adherence test was strongly positive for strain ATCC 777 and positive for all other strains. Typing by killer toxins revealed two different biotypes among the 9 isolates studied: 888 and 688. Only biotype 688 (ATCC 777) was susceptible to the K2 toxin. There was a significant inverse correlation between adherence and killer toxin susceptibility (r = -0.8525 - p = 0.0035). No strains presented resistance to fluconazole, itraconazole, ketoconazole, voriconazole, flucytosine or amphotericin-B. With the exception of ATCC 777, all the other isolates presented similar behavior.

O objetivo do presente trabalho foi avaliar o comportamento de cepas de Candida dubliniensis recuperadas de pacientes HIV+ e com AIDS por meio da pesquisa de capacidade de adesão a células HeLa, susceptibilidade a toxinas "Killer" e resistência in vitro a antifúngicos (eTest® AB Biodisk, Solna, Suécia). O ensaio de adesão foi fortemente aderente para a amostra padrão ATCC 777, e aderente para os demais isolados. Os testes de tipagem das amostras frente às cepas-padrão produtoras de toxinas "Killer" mostraram dois biótipos diferentes dos 9 isolados estudados: 888 e 688. Somente o biótipo 688 (ATCC 777) de C. dubliniensis foi sensível à toxina K2. Houve correlação inversa significativa entre adesão e sensibilidade a toxinas "killer" (r = -0,8525 - p = 0,0035). Em relação à pesquisa de resistência a antifúngicos, as amostras de C. dubliniensis foram sensíveis ao fluconazol, itraconazol, cetoconazol, voriconazol, à flucitosina e anfotericina B. Com exceção da amostra ATCC 777, todas as demais mostraram comportamento similar.

Lactoferrin and free secretory component of human milk inhibit the adhesion of enteropathogenic Escherichia coli to HeLa cells.

BACKGROUND: Diarrhoea caused by Escherichia coli is an important cause of infant morbidity and mortality in developing countries. Enteropathogenic Escherichia coli (EPEC) is considered one of the major causes of diarrhoea in children living in developing countries. The ability of diarrhoeagenic strains of E. coli to adhere to and colonize the intestine is the first step towards developing the disease. EPEC strains adhere to enterocytes and HeLa cells in a characteristic pattern known as localized adherence. Many epidemiological studies of diarrhoea have shown that breast-feeding protects infants from intestinal infections. Both immunoglobulin and non-immunoglobulin elements of human milk are thought to contribute to the protection from diarrhoeal agents. RESULTS: The effects of human milk and its protein components on the localized adherence of EPEC were investigated. Non-immunoglobulin components of human milk responsible for the inhibition of EPEC adhesion to HeLa cells were isolated by chromatographic fractionation of human whey proteins. Besides secretory immunoglobulin A, which has been previously reported to affect the adhesion of EPEC, free secretory component (fSC) and lactoferrin (Lf) were isolated. Even in concentrations lower than those usually found in whole milk, fSC and Lf were able to inhibit the adhesion of EPEC. alpha-lactalbumin was also isolated, but showed no activity on EPEC adhesion. CONCLUSIONS: This study demonstrated that the immunoglobulin fraction, the free secretory component and lactoferrin of human milk inhibit EPEC adhesion to HeLa cells. These results indicate that fSC and Lf may be important non-specific defence factors against EPEC infections.

Biological and genetic characteristics of uropathogenic Escherichia coli strains.

The aim of the present study was to determine biological characteristics such as expression of fimbriae, Congo red binding, production of hemolysin and aerobactin, adhesion to HeLa and uroepithelial cells and invasion of HeLa cells by Escherichia coli isolates obtained from patients showing clinical signs of urinary tract infection (UTI). Also, the presence of genes (apa, afa, spa) for fimbria expression and cytotoxic necrotizing factors (CNF1, CNF2) was assayed using specific primers in PCR. The data obtained were compared with the clonal relationships obtained by analysis of multilocus enzyme electrophoresis (MLEE), restriction fragment length polymorphism (RFLP) of the rDNA (ribotyping) and enterobacterial repetitive intergenic consensus-PCR (ERIC-PCR). All isolates but one presented a combination of at least two of the characteristics studied, a fact suggesting the presence of pathogenicity islands (PAIs). Diffuse adherence type to HeLa cells was observed to occur in most of the strains, but adhesion to uroepithelial cells seems to be a more reliable test to verify pathogenicity. Although four strains seemed to be able to invade HeLa cells when assayed by light microscopy, electron microscopy studies demonstrated that these strains were not invasive. MLEE, RFLP and ERIC-PCR were able to group the isolates differently into main clusters that were not correlated with the presence of pathogenic traits.

Prevalence of invasive ability and other virulence-associated characteristics in Providencia alcalifaciens strains isolated in São Paulo, Brazil.

Providencia alcalifaciens is an invasive enteric pathogen. The present study determined the prevalence of invasive ability in P. alcalifaciens strains isolated in São Paulo, Brazil, mainly from patients with diarrhoea. Invasion of HeLa cells was found in 17 (42%) of 41 strains studied. Most (88%) of the invasive strains were isolated from diarrhoeal stools. The invasive property was identified in 50% of P. alcalifaciens strains isolated as pure cultures or from stool samples where no other enteropathogen was identified. All the invasive strains caused actin condensation in infected cells. Plasmid profile analysis showed the presence of plasmids of 35.8-180 kb in 70% of the strains regardless of their invasive ability, suggesting that invasiveness in P. alcalifaciens is not plasmid related. No homology with a probe for gene sequences for invasion of enteroinvasive Escherichia coli and Shigella strains was identified in colony hybridisation assays. The invasive property of P. alcalifaciens was confirmed in the present study, but this characteristic did not predominate among strains isolated from patients with diarrhoea in São Paulo City. The presence of other virulence mechanisms and the role of non-invasive P. alcalifaciens strains as a cause of diarrhoea remain to be established.

Sorotipos de Escherichia coli enteropatogênica clássica (EPEC) causadores de alteraçöes da ultra-estrutura do intestino delgado e invasäo de células Hela / Serotypes of classical enteropathogic that cause changes in the small bowel ultrastructure and invasion of Hela cells

Escherichia coli enteropatogênica clássica (EPEC) é a principal causa de diarréia em lactentes menores de um ano de idade nos países subdesenvolvidos. In vitro, cepas de EPEC sao capazes de aderir a células HeLa ou Hep-2, obedecendo um padrao característico denominado adesao localizada. Estudos in vivo demonstram que as cepas de EPEC provocam profundas alteraçoes nas micro-vilosidades do enterócito, causando lesoes em pedestal. Objetivos. Neste trabalho, os autores relatam as alteraçoes da ultra-estrutura do intestino delgado causada pelos sorotipos 0111ab:H2, 0119:H6 e 018ab:H14, em lactentes portadores de diarréia aguda, e a capacidade destas bactérias de penetrar e se multiplicar no interior de células HeLa. Pacientes e Métodos. Estes sorotipos de EPEC foram isolados das fezes de três pacientes portadores de diarréia aguda que apresentaram intolerância alimentar e agravo nutricional. Alteraçoes ultra-estruturais do intestino delagado foram observadas nos espécimes estudados com formaçao de pedestal e penetraçao das bactérias no interior do citoplasma dos enterócitos. Estas bactérias também penetraram e se multiplicaram no interior das células HeLa. Conclusao. As alteraçoes ultra-estruturaisdescritas em pacientes com diarréria aguda devem ser levadas em conta quanto ao seu potencial efeito de provocar prolongamento da diarréia, seja por um fenômeno secretor ou por má absorçao dos nutrientes.

[Serotypes of classical enteropathogenic Escherichia coli that produce changes in the small bowel ultrastructure and invasion of HeLa cells]. / Sorotipos de Escherichia coli enteropatogênica clássica (EPEC) causadores de alterações da ultra-estrutura do intestino delgado e invasão de células HeLa.

UNLABELLED: Enteropathogenic Escherichia coli (EPEC) is the main cause of diarrhea in infants up to one year of age in the majority of the developing countries. In vitro EPEC strains attach to HeLa or HEp-2 cells in a specific pattern called localized adherence (LA), which is correlated with 93% of the EPEC serotypes. In vivo, EPEC strains adhere intimately to cuplike projections of the apical enterocyte membrane causing localized destruction of the microvilli, described as an attaching and effacing lesion. PURPOSE: We showed the attaching-effacing lesions and intracellular penetration in the ultrastructural study of the small intestinal cells as well as in the HeLa cell assay by the following EPEC serotypes: O111ab:H2, O119:H6 and O18ab:H14. PATIENTS AND METHODS: These strains were isolated from the stools of three infants less than 2 years of age with watery diarrhea who were hospitalized for fluid and electrolyte repairment. In the three patients there were severe ultrastructural alterations of the enterocytes mainly shortening and destruction of the microvilli, and formation of cuplike pedestal lesions. There was also penetration of microorganisms into the cytoplasm of the enterocytes in the interior of endocytotic vesicles. The serotypes of EPEC were assayed with HeLa cells showing the formation of pedestals and penetration into the cytoplasm. CONCLUSION: The ultrastructural alterations of the small bowel observed in these patients represent an important indication of the possibility of perpetuation of the diarrhea, owing to either a secretory mechanism or malabsorption of the nutrients.

Vibrio fluvialis attachs to but does not enter Hela cell monolayers

Considering the possibility that invasiveness could be a neglected factor of virulence in Vibrio fluvialis-linked enteritis, since a dysenteric form of the disease was seen in Bangladesh, we studied 12 Brazilian strains of the organism, six clinical and six environmental, to determine whether they might be able to enter into HeLa cell monolayers or would carry plasmids incidentally involved in invasiveness. Four human and two environmental isolates attached to but did not enter into the cells. Though five strains harbored plasmids,no relationship was found between the carriage of these genetic elements and adhesiveness