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In depth analysis of the Helicobacter pylori cag pathogenicity island transcriptional responses.

Vannini A, Roncarati D, Spinsanti M, Scarlato V, Danielli A - PLoS ONE (2014)

Bottom Line: Most cag promoters are transcriptionally responsive to growth-phase, pH and other stress-factors, although in many cases in a pleiotropic fashion.Finally, we demonstrate that the highly transcribed cagA oncogene is repressed by iron limitation through a direct apo-Fur regulation mechanism.Together the results shed light on regulatory aspects of the cag-PAI, which may be involved in relevant molecular and etiological aspects of H. pylori pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.

ABSTRACT
The severity of symptoms elicited by the widespread human pathogen Helicobacter pylori is strongly influenced by the genetic diversity of the infecting strain. Among the most important pathogen factors that carry an increased risk for gastric cancer are specific genotypes of the cag pathogenicity island (cag-PAI), encoding a type IV secretion system (T4SS) responsible for the translocation of the CagA effector oncoprotein. To date, little is known about the regulatory events important for the expression of a functional cag-T4SS. Here we demonstrate that the cag-PAI cistrons are subjected to a complex network of direct and indirect transcriptional regulations. We show that promoters of cag operons encoding structural T4SS components display homogeneous transcript levels, while promoters of cag operons encoding accessory factors vary considerably in their basal transcription levels and responses. Most cag promoters are transcriptionally responsive to growth-phase, pH and other stress-factors, although in many cases in a pleiotropic fashion. Interestingly, transcription from the Pcagζ promoter controlling the expression of transglycolase and T4SS stabilizing factors, is triggered by co-culture with a gastric cell line, providing an explanation for the increased formation of the secretion system observed upon bacterial contact with host cells. Finally, we demonstrate that the highly transcribed cagA oncogene is repressed by iron limitation through a direct apo-Fur regulation mechanism. Together the results shed light on regulatory aspects of the cag-PAI, which may be involved in relevant molecular and etiological aspects of H. pylori pathogenesis.

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Acid-dependent response of cag promoters in Δfur, ΔnikR and ΔarsS mutant strains.Cultures were grown to exponential phase and exposed to acid-shock (pH = 5.2) for 30 min. Transcript levels at the Pcagζ, PcagU, PcagF, PcagA, PcagS and PcagB promoters were assayed by quantitative primer extensions. Asterisks mark the significant differences of n-fold variations deriving from the average band intensity of three independent primer extension experiments. Error bars indicate the standard deviation.
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pone-0098416-g005: Acid-dependent response of cag promoters in Δfur, ΔnikR and ΔarsS mutant strains.Cultures were grown to exponential phase and exposed to acid-shock (pH = 5.2) for 30 min. Transcript levels at the Pcagζ, PcagU, PcagF, PcagA, PcagS and PcagB promoters were assayed by quantitative primer extensions. Asterisks mark the significant differences of n-fold variations deriving from the average band intensity of three independent primer extension experiments. Error bars indicate the standard deviation.

Mentions: As the acidic-response in H. pylori is primarily controlled by the ArsRS two-component system, together with the metal responsive transcriptional regulators NikR and Fur [2], [24], [44], [46], we cultured wild type, Δfur, ΔnikR and ΔarsS strains to mid-log phase, exposed to acidic shock for 30 min and evaluated the mRNA levels at the acid-responsive Pcagζ, PcagU, PcagF, PcagA, PcagS and PcagB promoters (Fig. 4 A and B) by quantitative primer extension assays with results reported in Fig. 5. Intriguingly, PcagF and PcagS promoters showed a loss of the pH-inducible response in the ΔnikR mutant, displaying unchanged transcript levels after acidic treatment with respect to the untreated sample, while in the Δfur and ΔarsS mutants an acid response similar to the wild type strain was observed. Likely, the acidic response at these promoters is directly or indirectly mediated by NikR. Similarly, transcript levels at the PcagB promoter were unchanged after acidic treatment in the Δfur mutant, while the wild type strain and the other mutants showed a pH-induced reduction in the mRNA levels. These results suggest that Fur is involved in the acid-dependent repression of PcagB. On the other hand, Pcagζ appears to loose the pH-inducible response both in fur and nikR knockout strains, suggesting a role for both regulators on its acidic regulation. Finally, variations of transcript levels in the mutant strains similar to that in the wild type strain were observed at the PcagA and PcagU promoters, hence acid response of these promoters is mediated by still unknown factors.


In depth analysis of the Helicobacter pylori cag pathogenicity island transcriptional responses.

Vannini A, Roncarati D, Spinsanti M, Scarlato V, Danielli A - PLoS ONE (2014)

Acid-dependent response of cag promoters in Δfur, ΔnikR and ΔarsS mutant strains.Cultures were grown to exponential phase and exposed to acid-shock (pH = 5.2) for 30 min. Transcript levels at the Pcagζ, PcagU, PcagF, PcagA, PcagS and PcagB promoters were assayed by quantitative primer extensions. Asterisks mark the significant differences of n-fold variations deriving from the average band intensity of three independent primer extension experiments. Error bars indicate the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043881&req=5

pone-0098416-g005: Acid-dependent response of cag promoters in Δfur, ΔnikR and ΔarsS mutant strains.Cultures were grown to exponential phase and exposed to acid-shock (pH = 5.2) for 30 min. Transcript levels at the Pcagζ, PcagU, PcagF, PcagA, PcagS and PcagB promoters were assayed by quantitative primer extensions. Asterisks mark the significant differences of n-fold variations deriving from the average band intensity of three independent primer extension experiments. Error bars indicate the standard deviation.
Mentions: As the acidic-response in H. pylori is primarily controlled by the ArsRS two-component system, together with the metal responsive transcriptional regulators NikR and Fur [2], [24], [44], [46], we cultured wild type, Δfur, ΔnikR and ΔarsS strains to mid-log phase, exposed to acidic shock for 30 min and evaluated the mRNA levels at the acid-responsive Pcagζ, PcagU, PcagF, PcagA, PcagS and PcagB promoters (Fig. 4 A and B) by quantitative primer extension assays with results reported in Fig. 5. Intriguingly, PcagF and PcagS promoters showed a loss of the pH-inducible response in the ΔnikR mutant, displaying unchanged transcript levels after acidic treatment with respect to the untreated sample, while in the Δfur and ΔarsS mutants an acid response similar to the wild type strain was observed. Likely, the acidic response at these promoters is directly or indirectly mediated by NikR. Similarly, transcript levels at the PcagB promoter were unchanged after acidic treatment in the Δfur mutant, while the wild type strain and the other mutants showed a pH-induced reduction in the mRNA levels. These results suggest that Fur is involved in the acid-dependent repression of PcagB. On the other hand, Pcagζ appears to loose the pH-inducible response both in fur and nikR knockout strains, suggesting a role for both regulators on its acidic regulation. Finally, variations of transcript levels in the mutant strains similar to that in the wild type strain were observed at the PcagA and PcagU promoters, hence acid response of these promoters is mediated by still unknown factors.

Bottom Line: Most cag promoters are transcriptionally responsive to growth-phase, pH and other stress-factors, although in many cases in a pleiotropic fashion.Finally, we demonstrate that the highly transcribed cagA oncogene is repressed by iron limitation through a direct apo-Fur regulation mechanism.Together the results shed light on regulatory aspects of the cag-PAI, which may be involved in relevant molecular and etiological aspects of H. pylori pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacy and Biotechnology (FaBiT), University of Bologna, Bologna, Italy.

ABSTRACT
The severity of symptoms elicited by the widespread human pathogen Helicobacter pylori is strongly influenced by the genetic diversity of the infecting strain. Among the most important pathogen factors that carry an increased risk for gastric cancer are specific genotypes of the cag pathogenicity island (cag-PAI), encoding a type IV secretion system (T4SS) responsible for the translocation of the CagA effector oncoprotein. To date, little is known about the regulatory events important for the expression of a functional cag-T4SS. Here we demonstrate that the cag-PAI cistrons are subjected to a complex network of direct and indirect transcriptional regulations. We show that promoters of cag operons encoding structural T4SS components display homogeneous transcript levels, while promoters of cag operons encoding accessory factors vary considerably in their basal transcription levels and responses. Most cag promoters are transcriptionally responsive to growth-phase, pH and other stress-factors, although in many cases in a pleiotropic fashion. Interestingly, transcription from the Pcagζ promoter controlling the expression of transglycolase and T4SS stabilizing factors, is triggered by co-culture with a gastric cell line, providing an explanation for the increased formation of the secretion system observed upon bacterial contact with host cells. Finally, we demonstrate that the highly transcribed cagA oncogene is repressed by iron limitation through a direct apo-Fur regulation mechanism. Together the results shed light on regulatory aspects of the cag-PAI, which may be involved in relevant molecular and etiological aspects of H. pylori pathogenesis.

Show MeSH
Related in: MedlinePlus