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Co-Transcriptomes of Initial Interactions In Vitro between Streptococcus Pneumoniae and Human Pleural Mesothelial Cells.

Heath CJ, del Mar Cendra M, Watson A, Auger JP, Pandey A, Tighe P, Christodoulides M - PLoS ONE (2015)

Bottom Line: After Spn initial contact, 870 human genes were differentially regulated and the largest numbers of significant gene expression changes were found in canonical pathways for eukaryotic initiation factor 2 signaling (60 genes out of 171), oxidative phosphorylation (32/103), mitochondrial dysfunction (37/164), eIF4 and p70S6K signaling (28/142), mTOR signaling (27/182), NRF2-mediated oxidative stress response (20/177), epithelial adherens junction remodeling (11/66) and ubiquitination (22/254).Spn did not activate NF-kB or phosphorylate p38 MAPK or induce cytokine production from PMC.Moreover, Spn infection of TNF-α pre-stimulated PMC inhibited production of IL-6 and IL-8 secretion by >50% (p<0.01).

View Article: PubMed Central - PubMed

Affiliation: Neisseria Research Group, Molecular Microbiology, Clinical and Experimental Sciences, Sir Henry Wellcome laboratories, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, United Kingdom.

ABSTRACT
Streptococcus pneumoniae (Spn) is a major causative organism of empyema, an inflammatory condition occurring in the pleural sac. In this study, we used human and Spn cDNA microarrays to characterize the transcriptional responses occurring during initial contact between Spn and a human pleural mesothelial cell line (PMC) in vitro. Using stringent filtering criteria, 42 and 23 Spn genes were up-and down-regulated respectively. In particular, genes encoding factors potentially involved in metabolic processes and Spn adherence to eukaryotic cells were up-regulated e.g. glnQ, glnA, aliA, psaB, lytB and nox. After Spn initial contact, 870 human genes were differentially regulated and the largest numbers of significant gene expression changes were found in canonical pathways for eukaryotic initiation factor 2 signaling (60 genes out of 171), oxidative phosphorylation (32/103), mitochondrial dysfunction (37/164), eIF4 and p70S6K signaling (28/142), mTOR signaling (27/182), NRF2-mediated oxidative stress response (20/177), epithelial adherens junction remodeling (11/66) and ubiquitination (22/254). The cellular response appeared to be directed towards host cell survival and defense. Spn did not activate NF-kB or phosphorylate p38 MAPK or induce cytokine production from PMC. Moreover, Spn infection of TNF-α pre-stimulated PMC inhibited production of IL-6 and IL-8 secretion by >50% (p<0.01). In summary, this descriptive study provides datasets and a platform for examining further the molecular mechanisms underlying the pathogenesis of empyema.

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Related in: MedlinePlus

Interactions of Streptococcus pneumoniae with pleural mesothelial cells in vitro.A) Association: monolayers of Met-5A cells were infected with various MOI of Streptococcus pneumoniae strain D39. Total associated (defining adherent and internalised bacteria combined) bacteria were counted following saponin lysis of infected monolayers over time. The data are representative of >3 independent experiments; the symbols represent mean adherence and the error bars the standard deviations of wells infected in triplicate. B) Invasion: monolayers of Met5A cells were infected for 9h with MOI 0.025 of D39 and intracellular bacteria were recovered after gentamicin treatment and saponin lysis. Monolayers were also pre-treated with cytochalasin D prior to bacterial infection. The bars represent the mean bacterial cfu of three independent experiments and the error bars the standard error of the means.
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pone.0142773.g001: Interactions of Streptococcus pneumoniae with pleural mesothelial cells in vitro.A) Association: monolayers of Met-5A cells were infected with various MOI of Streptococcus pneumoniae strain D39. Total associated (defining adherent and internalised bacteria combined) bacteria were counted following saponin lysis of infected monolayers over time. The data are representative of >3 independent experiments; the symbols represent mean adherence and the error bars the standard deviations of wells infected in triplicate. B) Invasion: monolayers of Met5A cells were infected for 9h with MOI 0.025 of D39 and intracellular bacteria were recovered after gentamicin treatment and saponin lysis. Monolayers were also pre-treated with cytochalasin D prior to bacterial infection. The bars represent the mean bacterial cfu of three independent experiments and the error bars the standard error of the means.

Mentions: Initially, the dynamics of D39 association with Met-5A cells and any subsequent invasion were quantified over time (Fig 1). High levels of saturating association of D39 were observed with initial infective MOI>2 by 3h, whereas with lower initial MOI ≤0.025, bacteria adhered in a dose and time-dependent manner. An initial MOI of 250 of D39 induced significant cytotoxicity by 6h, whereas the MOI of 2.5 only destroyed the monolayers by 24h. Bacteria could be recovered at 24h from intact monolayers infected with the lowest initial MOI tested (Fig 1A). Cell death following infection with D39 was measured by LDH release. Background levels (spontaneous) of LDH release by uninfected Met-5A cells were between 3–11%. Cell death induced by D39 that was visualized with high MOI (2–250) was not associated with significant LDH release; only 22% LDH release was detected following infection with the highest MOI tested (250) and the levels quantified after infection with MOI of <25 were not significantly different to background levels (p>0.05).


Co-Transcriptomes of Initial Interactions In Vitro between Streptococcus Pneumoniae and Human Pleural Mesothelial Cells.

Heath CJ, del Mar Cendra M, Watson A, Auger JP, Pandey A, Tighe P, Christodoulides M - PLoS ONE (2015)

Interactions of Streptococcus pneumoniae with pleural mesothelial cells in vitro.A) Association: monolayers of Met-5A cells were infected with various MOI of Streptococcus pneumoniae strain D39. Total associated (defining adherent and internalised bacteria combined) bacteria were counted following saponin lysis of infected monolayers over time. The data are representative of >3 independent experiments; the symbols represent mean adherence and the error bars the standard deviations of wells infected in triplicate. B) Invasion: monolayers of Met5A cells were infected for 9h with MOI 0.025 of D39 and intracellular bacteria were recovered after gentamicin treatment and saponin lysis. Monolayers were also pre-treated with cytochalasin D prior to bacterial infection. The bars represent the mean bacterial cfu of three independent experiments and the error bars the standard error of the means.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4643877&req=5

pone.0142773.g001: Interactions of Streptococcus pneumoniae with pleural mesothelial cells in vitro.A) Association: monolayers of Met-5A cells were infected with various MOI of Streptococcus pneumoniae strain D39. Total associated (defining adherent and internalised bacteria combined) bacteria were counted following saponin lysis of infected monolayers over time. The data are representative of >3 independent experiments; the symbols represent mean adherence and the error bars the standard deviations of wells infected in triplicate. B) Invasion: monolayers of Met5A cells were infected for 9h with MOI 0.025 of D39 and intracellular bacteria were recovered after gentamicin treatment and saponin lysis. Monolayers were also pre-treated with cytochalasin D prior to bacterial infection. The bars represent the mean bacterial cfu of three independent experiments and the error bars the standard error of the means.
Mentions: Initially, the dynamics of D39 association with Met-5A cells and any subsequent invasion were quantified over time (Fig 1). High levels of saturating association of D39 were observed with initial infective MOI>2 by 3h, whereas with lower initial MOI ≤0.025, bacteria adhered in a dose and time-dependent manner. An initial MOI of 250 of D39 induced significant cytotoxicity by 6h, whereas the MOI of 2.5 only destroyed the monolayers by 24h. Bacteria could be recovered at 24h from intact monolayers infected with the lowest initial MOI tested (Fig 1A). Cell death following infection with D39 was measured by LDH release. Background levels (spontaneous) of LDH release by uninfected Met-5A cells were between 3–11%. Cell death induced by D39 that was visualized with high MOI (2–250) was not associated with significant LDH release; only 22% LDH release was detected following infection with the highest MOI tested (250) and the levels quantified after infection with MOI of <25 were not significantly different to background levels (p>0.05).

Bottom Line: After Spn initial contact, 870 human genes were differentially regulated and the largest numbers of significant gene expression changes were found in canonical pathways for eukaryotic initiation factor 2 signaling (60 genes out of 171), oxidative phosphorylation (32/103), mitochondrial dysfunction (37/164), eIF4 and p70S6K signaling (28/142), mTOR signaling (27/182), NRF2-mediated oxidative stress response (20/177), epithelial adherens junction remodeling (11/66) and ubiquitination (22/254).Spn did not activate NF-kB or phosphorylate p38 MAPK or induce cytokine production from PMC.Moreover, Spn infection of TNF-α pre-stimulated PMC inhibited production of IL-6 and IL-8 secretion by >50% (p<0.01).

View Article: PubMed Central - PubMed

Affiliation: Neisseria Research Group, Molecular Microbiology, Clinical and Experimental Sciences, Sir Henry Wellcome laboratories, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, United Kingdom.

ABSTRACT
Streptococcus pneumoniae (Spn) is a major causative organism of empyema, an inflammatory condition occurring in the pleural sac. In this study, we used human and Spn cDNA microarrays to characterize the transcriptional responses occurring during initial contact between Spn and a human pleural mesothelial cell line (PMC) in vitro. Using stringent filtering criteria, 42 and 23 Spn genes were up-and down-regulated respectively. In particular, genes encoding factors potentially involved in metabolic processes and Spn adherence to eukaryotic cells were up-regulated e.g. glnQ, glnA, aliA, psaB, lytB and nox. After Spn initial contact, 870 human genes were differentially regulated and the largest numbers of significant gene expression changes were found in canonical pathways for eukaryotic initiation factor 2 signaling (60 genes out of 171), oxidative phosphorylation (32/103), mitochondrial dysfunction (37/164), eIF4 and p70S6K signaling (28/142), mTOR signaling (27/182), NRF2-mediated oxidative stress response (20/177), epithelial adherens junction remodeling (11/66) and ubiquitination (22/254). The cellular response appeared to be directed towards host cell survival and defense. Spn did not activate NF-kB or phosphorylate p38 MAPK or induce cytokine production from PMC. Moreover, Spn infection of TNF-α pre-stimulated PMC inhibited production of IL-6 and IL-8 secretion by >50% (p<0.01). In summary, this descriptive study provides datasets and a platform for examining further the molecular mechanisms underlying the pathogenesis of empyema.

Show MeSH
Related in: MedlinePlus