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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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pH-dependent response of cag promoters.Primer extension analyses were performed on total RNA extracted from bacterial cultures of H. pylori wild type strain, grown to exponential phase and treated for 30 or 90 min with 22 mM HCl to adjust the pH of the medium to 5.2, or maintained at neutral pH (pH 7.0). The intensity of the bands of four independent experiments were quantified and reported in the graphs as n-fold variation of the transcript levels in the acidic-treated samples with respect to the untreated sample. A. Pcag promoters with transcript levels increased after an acidic treatment for 30 min; B. Promoters with reduced mRNA levels after the 30 min acidic treatment; C. Promoters with unchanged transcript levels after the treatment. Error bars indicate the standard deviation and significant variations between treated and untreated samples are marked with asterisks (P<0.05). D. Response of PcagV, PcagQ, PcagP, PcagM and PcagC promoters after an acidic treatment of 90 min.
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pone-0098416-g004: pH-dependent response of cag promoters.Primer extension analyses were performed on total RNA extracted from bacterial cultures of H. pylori wild type strain, grown to exponential phase and treated for 30 or 90 min with 22 mM HCl to adjust the pH of the medium to 5.2, or maintained at neutral pH (pH 7.0). The intensity of the bands of four independent experiments were quantified and reported in the graphs as n-fold variation of the transcript levels in the acidic-treated samples with respect to the untreated sample. A. Pcag promoters with transcript levels increased after an acidic treatment for 30 min; B. Promoters with reduced mRNA levels after the 30 min acidic treatment; C. Promoters with unchanged transcript levels after the treatment. Error bars indicate the standard deviation and significant variations between treated and untreated samples are marked with asterisks (P<0.05). D. Response of PcagV, PcagQ, PcagP, PcagM and PcagC promoters after an acidic treatment of 90 min.

Mentions: To investigate the transcriptional responses to acidic pH, liquid cultures of H. pylori grown to mid-log phase were divided in two subcultures and treated for 30 min or 90 min either with HCl to adjust the pH of the medium to a value of 5.2 (acid shock) or with the same volume of sterile water (untreated sample). The RNAs extracted from three independent cultures were assayed by primer extension experiments and bands were quantified with results reported in Fig. 4.


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)

pH-dependent response of cag promoters.Primer extension analyses were performed on total RNA extracted from bacterial cultures of H. pylori wild type strain, grown to exponential phase and treated for 30 or 90 min with 22 mM HCl to adjust the pH of the medium to 5.2, or maintained at neutral pH (pH 7.0). The intensity of the bands of four independent experiments were quantified and reported in the graphs as n-fold variation of the transcript levels in the acidic-treated samples with respect to the untreated sample. A. Pcag promoters with transcript levels increased after an acidic treatment for 30 min; B. Promoters with reduced mRNA levels after the 30 min acidic treatment; C. Promoters with unchanged transcript levels after the treatment. Error bars indicate the standard deviation and significant variations between treated and untreated samples are marked with asterisks (P<0.05). D. Response of PcagV, PcagQ, PcagP, PcagM and PcagC promoters after an acidic treatment of 90 min.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098416-g004: pH-dependent response of cag promoters.Primer extension analyses were performed on total RNA extracted from bacterial cultures of H. pylori wild type strain, grown to exponential phase and treated for 30 or 90 min with 22 mM HCl to adjust the pH of the medium to 5.2, or maintained at neutral pH (pH 7.0). The intensity of the bands of four independent experiments were quantified and reported in the graphs as n-fold variation of the transcript levels in the acidic-treated samples with respect to the untreated sample. A. Pcag promoters with transcript levels increased after an acidic treatment for 30 min; B. Promoters with reduced mRNA levels after the 30 min acidic treatment; C. Promoters with unchanged transcript levels after the treatment. Error bars indicate the standard deviation and significant variations between treated and untreated samples are marked with asterisks (P<0.05). D. Response of PcagV, PcagQ, PcagP, PcagM and PcagC promoters after an acidic treatment of 90 min.
Mentions: To investigate the transcriptional responses to acidic pH, liquid cultures of H. pylori grown to mid-log phase were divided in two subcultures and treated for 30 min or 90 min either with HCl to adjust the pH of the medium to a value of 5.2 (acid shock) or with the same volume of sterile water (untreated sample). The RNAs extracted from three independent cultures were assayed by primer extension experiments and bands were quantified with results reported in Fig. 4.

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