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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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Comparison of the Pcag-lux and Pcag-5′UTR-lux reporter signals.A. Schematic representation of the Pcag-lux and Pcag-5′UTR-lux fusion constructs, obtained transforming the G27lux acceptor strain with the PVCC vector. The promoter sequences with or without the 5′untranslated regions (5′UTRs) carried by the pVCC vector are inserted upstream the luxCDABE operon by double homologous recombination and selected by cat chloramphenicol resistance. B. Luminescence signals from three independent experiments were normalized according to the optical density of the cultures and the means values were reported in the graph, with Pcag-lux signals on the X-axis and Pcag-5′UTR-lux signals on the Y-axis. Error bars indicate the standard deviation. A dashed line was added to the graph, corresponding to the 1∶1 ratio of the two signals. Grey dots: cag promoters with 1∶1 signal ratio; black dots: cag promoters with altered Pcag-lux/Pcag-5′UTR-lux signal ratio.
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pone-0098416-g007: Comparison of the Pcag-lux and Pcag-5′UTR-lux reporter signals.A. Schematic representation of the Pcag-lux and Pcag-5′UTR-lux fusion constructs, obtained transforming the G27lux acceptor strain with the PVCC vector. The promoter sequences with or without the 5′untranslated regions (5′UTRs) carried by the pVCC vector are inserted upstream the luxCDABE operon by double homologous recombination and selected by cat chloramphenicol resistance. B. Luminescence signals from three independent experiments were normalized according to the optical density of the cultures and the means values were reported in the graph, with Pcag-lux signals on the X-axis and Pcag-5′UTR-lux signals on the Y-axis. Error bars indicate the standard deviation. A dashed line was added to the graph, corresponding to the 1∶1 ratio of the two signals. Grey dots: cag promoters with 1∶1 signal ratio; black dots: cag promoters with altered Pcag-lux/Pcag-5′UTR-lux signal ratio.

Mentions: The analyses of the sequences downstream the transcriptional start sites of the Pcag promoters showed that cag transcripts harbor 5′ untranslated regions (5′UTRs) of different lengths (Fig. 1B). To assess possible post transcriptional effects mediated by the Pcag 5′UTRs, in analogy to similar findings reported in A. tumefaciens[50], we used the aforementioned Pcag-lux reporter fusions and ad hoc Pcag-5′UTR-lux constructs encompassing also the 5'UTRs downstream of the promoters (Fig. 7A). The luminescence emitted by mid-log growing cultures of these reporter constructs was compared to the corresponding Pcag-lux constructs without the 5′UTR region. Except for Pcagζ, PcagM, PcagF and PcagC, the luminescence counts of the 5'UTR-less constructs correlated well with the transcript levels assayed in primer extension analysis (Fig.1C; 7/11 promoters matching). We observed a nearly 1∶1 signal ratio between the constructs with or without the 5′UTR for PcagA, PcagB, PcagC, PcagM, PcagS and PcagP (Fig. 7B), suggesting that most 5′UTRs downstream of cag promoters do not affect the stability or the translational efficiency of the nascent messenger RNAs. Intriguingly, the luminescence of PcagV-5′UTR-lux constructs decreased significantly with respect to the 5′UTR-less construct (Fig. 7B), suggesting that this sequences could contain elements that reduce the translational efficiency or decrease the mRNA abundance.


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)

Comparison of the Pcag-lux and Pcag-5′UTR-lux reporter signals.A. Schematic representation of the Pcag-lux and Pcag-5′UTR-lux fusion constructs, obtained transforming the G27lux acceptor strain with the PVCC vector. The promoter sequences with or without the 5′untranslated regions (5′UTRs) carried by the pVCC vector are inserted upstream the luxCDABE operon by double homologous recombination and selected by cat chloramphenicol resistance. B. Luminescence signals from three independent experiments were normalized according to the optical density of the cultures and the means values were reported in the graph, with Pcag-lux signals on the X-axis and Pcag-5′UTR-lux signals on the Y-axis. Error bars indicate the standard deviation. A dashed line was added to the graph, corresponding to the 1∶1 ratio of the two signals. Grey dots: cag promoters with 1∶1 signal ratio; black dots: cag promoters with altered Pcag-lux/Pcag-5′UTR-lux signal ratio.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4043881&req=5

pone-0098416-g007: Comparison of the Pcag-lux and Pcag-5′UTR-lux reporter signals.A. Schematic representation of the Pcag-lux and Pcag-5′UTR-lux fusion constructs, obtained transforming the G27lux acceptor strain with the PVCC vector. The promoter sequences with or without the 5′untranslated regions (5′UTRs) carried by the pVCC vector are inserted upstream the luxCDABE operon by double homologous recombination and selected by cat chloramphenicol resistance. B. Luminescence signals from three independent experiments were normalized according to the optical density of the cultures and the means values were reported in the graph, with Pcag-lux signals on the X-axis and Pcag-5′UTR-lux signals on the Y-axis. Error bars indicate the standard deviation. A dashed line was added to the graph, corresponding to the 1∶1 ratio of the two signals. Grey dots: cag promoters with 1∶1 signal ratio; black dots: cag promoters with altered Pcag-lux/Pcag-5′UTR-lux signal ratio.
Mentions: The analyses of the sequences downstream the transcriptional start sites of the Pcag promoters showed that cag transcripts harbor 5′ untranslated regions (5′UTRs) of different lengths (Fig. 1B). To assess possible post transcriptional effects mediated by the Pcag 5′UTRs, in analogy to similar findings reported in A. tumefaciens[50], we used the aforementioned Pcag-lux reporter fusions and ad hoc Pcag-5′UTR-lux constructs encompassing also the 5'UTRs downstream of the promoters (Fig. 7A). The luminescence emitted by mid-log growing cultures of these reporter constructs was compared to the corresponding Pcag-lux constructs without the 5′UTR region. Except for Pcagζ, PcagM, PcagF and PcagC, the luminescence counts of the 5'UTR-less constructs correlated well with the transcript levels assayed in primer extension analysis (Fig.1C; 7/11 promoters matching). We observed a nearly 1∶1 signal ratio between the constructs with or without the 5′UTR for PcagA, PcagB, PcagC, PcagM, PcagS and PcagP (Fig. 7B), suggesting that most 5′UTRs downstream of cag promoters do not affect the stability or the translational efficiency of the nascent messenger RNAs. Intriguingly, the luminescence of PcagV-5′UTR-lux constructs decreased significantly with respect to the 5′UTR-less construct (Fig. 7B), suggesting that this sequences could contain elements that reduce the translational efficiency or decrease the mRNA abundance.

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