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Ulcerogenic Helicobacter pylori strains isolated from children: a contribution to get insight into the virulence of the bacteria.

Vitoriano I, Saraiva-Pava KD, Rocha-Gonçalves A, Santos A, Lopes AI, Oleastro M, Roxo-Rosa M - PLoS ONE (2011)

Bottom Line: Corroborating this, our in vitro infection assays comparing a pool of five H. pylori strains isolated from children with PUD to a pool of five other pediatric clinical isolates associated with non-ulcer dyspepsia (NUD) showed the greater ability of PUD strains to induce a marked decrease in the viability of gastric cells and to cause severe damage in the cells cytoskeleton as well as an impairment in the production/secretion of mucins.To uncover virulence features, we compared the proteome of these two groups of H. pylori strains.In conclusion, the enhanced virulence of the pediatric ulcerogenic H. pylori strains may result from a synergy between their natural ability to better adapt to the hostile human stomach and the expression of the established virulence factors.

View Article: PubMed Central - PubMed

Affiliation: Faculdade de Engenharia, Universidade Católica Portuguesa, Rio de Mouro, Portugal.

ABSTRACT
Infection with Helicobacter pylori is the major cause for the development of peptic ulcer disease (PUD). In children, with no other etiology for the disease, this rare event occurs shortly after infection. In these young patients, habits of smoking, diet, consumption of alcohol and non-steroid anti-inflammatory drugs and stress, in addition to the genetic susceptibility of the patient, represent a minor influence. Accordingly, the virulence of the implicated H. pylori strain should play a crucial role in the development of PUD. Corroborating this, our in vitro infection assays comparing a pool of five H. pylori strains isolated from children with PUD to a pool of five other pediatric clinical isolates associated with non-ulcer dyspepsia (NUD) showed the greater ability of PUD strains to induce a marked decrease in the viability of gastric cells and to cause severe damage in the cells cytoskeleton as well as an impairment in the production/secretion of mucins. To uncover virulence features, we compared the proteome of these two groups of H. pylori strains. Two-dimensional gel electrophoresis followed by mass-spectrometry allowed us to detect 27 differentially expressed proteins between them. In addition to the presence of genes encoding well established virulence factors, namely cagA, vacAs1, oipA "on" status, homB and jhp562 genes, the pediatric ulcerogenic strains shared a proteome profile characterized by changes in the abundance of: motility-associated proteins, accounting for higher motility; antioxidant proteins, which may confer increased resistance to inflammation; and enzymes involved in key steps in the metabolism of glucose, amino acids and urea, which may be advantageous to face fluctuations of nutrients. In conclusion, the enhanced virulence of the pediatric ulcerogenic H. pylori strains may result from a synergy between their natural ability to better adapt to the hostile human stomach and the expression of the established virulence factors.

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Related in: MedlinePlus

Impact of the two isogenic groups of pediatric H. pylori strains on the NCI-N87 cells morphology.NCI-N87 cells grown to 80–90% confluence were co-cultured for 24 h with the pool of five isogenic NUD-associated H. pylori strains (NUD) and in parallel with the pool of five isogenic PUD strains (PUD). The control corresponds to non-infected NCI-N87 cells. A), B), and C) light microscopy observations. D), E), and F) immunodetection of the microtubule network (green) (1∶1000 α-tubulin antibody plus a FITC conjugated secondary antibody). G), H), and I) PAS staining alone, and in J), L), and M) in conjugation with hematoxylin. Dark arrow indicates extracellular vesicles that reacted with PAS.
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pone-0026265-g002: Impact of the two isogenic groups of pediatric H. pylori strains on the NCI-N87 cells morphology.NCI-N87 cells grown to 80–90% confluence were co-cultured for 24 h with the pool of five isogenic NUD-associated H. pylori strains (NUD) and in parallel with the pool of five isogenic PUD strains (PUD). The control corresponds to non-infected NCI-N87 cells. A), B), and C) light microscopy observations. D), E), and F) immunodetection of the microtubule network (green) (1∶1000 α-tubulin antibody plus a FITC conjugated secondary antibody). G), H), and I) PAS staining alone, and in J), L), and M) in conjugation with hematoxylin. Dark arrow indicates extracellular vesicles that reacted with PAS.

Mentions: To clarify whether the virulence of the strains was the determining factor for the development of PUD in children from whom they were collected, we compared their impact on human gastric epithelial cells with that of NUD strains, by in vitro infection experiments. These experiments were carried out using the NCI-N87 cell line, known for its unique differentiation status as it is able to form coherent monolayers, expressing the correct cellular distribution of both E-cadherin and ZO-1 junction proteins [13]. Confluent NCI-N87 cells monolayers were thus incubated in parallel with two different pools of H. pylori strains, one including the five PUD associated strains, and another including the five NUD strains, both at a multiplicity of infection (MOI) of 100. Under our experimental co-culture conditions, the viability of NCI-N87 cells was clearly reduced over time with both pools of H. pylori strains (Figure 1). However, this effect was more pronounced in the first hours following infection with the pool of PUD-associated strains. Indeed, cell viability was significantly lower (p<0.05) in NCI-N87 cells infected with PUD-H. pylori strains at 1 h (89.90%±6.23) and 12 h post-infection (73.14%±8.96), compared to cells infected with NUD strains (99.80%±3.52 and 89.94%±6.70, respectively) (Figure 1). At 24 h post-infection, the viability of the epithelial cells was very low in both cases, 5.40%±4.51 and 10.78%±11.91, for cells infected with NUD and PUD strains, respectively (Figure 1). However, the morphological analysis of the remaining cells (Figure 2) showed dramatic differences in the impact of each pool of H. pylori strains. Indeed, light microscopy observations revealed much more pronounced cell damage in NCI-N87 cells infected with the pool of PUD-H. pylori strains (Figures 2A, 2B and 2C). Moreover, the pool of PUD strains caused the total destabilization of the NCI-N87 cells' microtubule network, as demonstrated by the immunocytochemical assays (Figures 2F), which seemed almost unchanged in cells infected with NUD-H. pylori strains (Figure 2E). In addition, epithelial cells infected with the PUD-H. pylori strains showed a drastic reduction in the cytoplasmic Periodic Acid Schiff (PAS) reactivity (Figure 2G, 2H and 2I), which was supported by the destruction of the cell membrane observed with hematoxylin and PAS double staining (Figure 2J, 2L and 2M). We also observed that the nuclear envelope of the NCI-N87 cells infected with the H. pylori strains associated with PUD was undisrupted, despite having a very irregular appearance, suggesting cell death by necrosis (Figure 2I and 2M). In contrast, NCI-N87 cells co-cultured with the NUD-associated H. pylori strains showed an accumulation of extracellular vesicles positive for PAS (dark arrow in Figure 2L) suggesting the expected exocytosis of intracellular mucus-containing vesicles, also observed in the non-infected control (data not shown). After 24 h of co-culture, NUD strains maintained their bacillary shape and were adherent to the epithelial cell surface (Figure 2H and 2L), however, PUD strains acquired a coccoidal shape (Figure 2I and 2M) which was already observed at 12 h post infection (data not shown).


Ulcerogenic Helicobacter pylori strains isolated from children: a contribution to get insight into the virulence of the bacteria.

Vitoriano I, Saraiva-Pava KD, Rocha-Gonçalves A, Santos A, Lopes AI, Oleastro M, Roxo-Rosa M - PLoS ONE (2011)

Impact of the two isogenic groups of pediatric H. pylori strains on the NCI-N87 cells morphology.NCI-N87 cells grown to 80–90% confluence were co-cultured for 24 h with the pool of five isogenic NUD-associated H. pylori strains (NUD) and in parallel with the pool of five isogenic PUD strains (PUD). The control corresponds to non-infected NCI-N87 cells. A), B), and C) light microscopy observations. D), E), and F) immunodetection of the microtubule network (green) (1∶1000 α-tubulin antibody plus a FITC conjugated secondary antibody). G), H), and I) PAS staining alone, and in J), L), and M) in conjugation with hematoxylin. Dark arrow indicates extracellular vesicles that reacted with PAS.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198394&req=5

pone-0026265-g002: Impact of the two isogenic groups of pediatric H. pylori strains on the NCI-N87 cells morphology.NCI-N87 cells grown to 80–90% confluence were co-cultured for 24 h with the pool of five isogenic NUD-associated H. pylori strains (NUD) and in parallel with the pool of five isogenic PUD strains (PUD). The control corresponds to non-infected NCI-N87 cells. A), B), and C) light microscopy observations. D), E), and F) immunodetection of the microtubule network (green) (1∶1000 α-tubulin antibody plus a FITC conjugated secondary antibody). G), H), and I) PAS staining alone, and in J), L), and M) in conjugation with hematoxylin. Dark arrow indicates extracellular vesicles that reacted with PAS.
Mentions: To clarify whether the virulence of the strains was the determining factor for the development of PUD in children from whom they were collected, we compared their impact on human gastric epithelial cells with that of NUD strains, by in vitro infection experiments. These experiments were carried out using the NCI-N87 cell line, known for its unique differentiation status as it is able to form coherent monolayers, expressing the correct cellular distribution of both E-cadherin and ZO-1 junction proteins [13]. Confluent NCI-N87 cells monolayers were thus incubated in parallel with two different pools of H. pylori strains, one including the five PUD associated strains, and another including the five NUD strains, both at a multiplicity of infection (MOI) of 100. Under our experimental co-culture conditions, the viability of NCI-N87 cells was clearly reduced over time with both pools of H. pylori strains (Figure 1). However, this effect was more pronounced in the first hours following infection with the pool of PUD-associated strains. Indeed, cell viability was significantly lower (p<0.05) in NCI-N87 cells infected with PUD-H. pylori strains at 1 h (89.90%±6.23) and 12 h post-infection (73.14%±8.96), compared to cells infected with NUD strains (99.80%±3.52 and 89.94%±6.70, respectively) (Figure 1). At 24 h post-infection, the viability of the epithelial cells was very low in both cases, 5.40%±4.51 and 10.78%±11.91, for cells infected with NUD and PUD strains, respectively (Figure 1). However, the morphological analysis of the remaining cells (Figure 2) showed dramatic differences in the impact of each pool of H. pylori strains. Indeed, light microscopy observations revealed much more pronounced cell damage in NCI-N87 cells infected with the pool of PUD-H. pylori strains (Figures 2A, 2B and 2C). Moreover, the pool of PUD strains caused the total destabilization of the NCI-N87 cells' microtubule network, as demonstrated by the immunocytochemical assays (Figures 2F), which seemed almost unchanged in cells infected with NUD-H. pylori strains (Figure 2E). In addition, epithelial cells infected with the PUD-H. pylori strains showed a drastic reduction in the cytoplasmic Periodic Acid Schiff (PAS) reactivity (Figure 2G, 2H and 2I), which was supported by the destruction of the cell membrane observed with hematoxylin and PAS double staining (Figure 2J, 2L and 2M). We also observed that the nuclear envelope of the NCI-N87 cells infected with the H. pylori strains associated with PUD was undisrupted, despite having a very irregular appearance, suggesting cell death by necrosis (Figure 2I and 2M). In contrast, NCI-N87 cells co-cultured with the NUD-associated H. pylori strains showed an accumulation of extracellular vesicles positive for PAS (dark arrow in Figure 2L) suggesting the expected exocytosis of intracellular mucus-containing vesicles, also observed in the non-infected control (data not shown). After 24 h of co-culture, NUD strains maintained their bacillary shape and were adherent to the epithelial cell surface (Figure 2H and 2L), however, PUD strains acquired a coccoidal shape (Figure 2I and 2M) which was already observed at 12 h post infection (data not shown).

Bottom Line: Corroborating this, our in vitro infection assays comparing a pool of five H. pylori strains isolated from children with PUD to a pool of five other pediatric clinical isolates associated with non-ulcer dyspepsia (NUD) showed the greater ability of PUD strains to induce a marked decrease in the viability of gastric cells and to cause severe damage in the cells cytoskeleton as well as an impairment in the production/secretion of mucins.To uncover virulence features, we compared the proteome of these two groups of H. pylori strains.In conclusion, the enhanced virulence of the pediatric ulcerogenic H. pylori strains may result from a synergy between their natural ability to better adapt to the hostile human stomach and the expression of the established virulence factors.

View Article: PubMed Central - PubMed

Affiliation: Faculdade de Engenharia, Universidade Católica Portuguesa, Rio de Mouro, Portugal.

ABSTRACT
Infection with Helicobacter pylori is the major cause for the development of peptic ulcer disease (PUD). In children, with no other etiology for the disease, this rare event occurs shortly after infection. In these young patients, habits of smoking, diet, consumption of alcohol and non-steroid anti-inflammatory drugs and stress, in addition to the genetic susceptibility of the patient, represent a minor influence. Accordingly, the virulence of the implicated H. pylori strain should play a crucial role in the development of PUD. Corroborating this, our in vitro infection assays comparing a pool of five H. pylori strains isolated from children with PUD to a pool of five other pediatric clinical isolates associated with non-ulcer dyspepsia (NUD) showed the greater ability of PUD strains to induce a marked decrease in the viability of gastric cells and to cause severe damage in the cells cytoskeleton as well as an impairment in the production/secretion of mucins. To uncover virulence features, we compared the proteome of these two groups of H. pylori strains. Two-dimensional gel electrophoresis followed by mass-spectrometry allowed us to detect 27 differentially expressed proteins between them. In addition to the presence of genes encoding well established virulence factors, namely cagA, vacAs1, oipA "on" status, homB and jhp562 genes, the pediatric ulcerogenic strains shared a proteome profile characterized by changes in the abundance of: motility-associated proteins, accounting for higher motility; antioxidant proteins, which may confer increased resistance to inflammation; and enzymes involved in key steps in the metabolism of glucose, amino acids and urea, which may be advantageous to face fluctuations of nutrients. In conclusion, the enhanced virulence of the pediatric ulcerogenic H. pylori strains may result from a synergy between their natural ability to better adapt to the hostile human stomach and the expression of the established virulence factors.

Show MeSH
Related in: MedlinePlus