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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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Growth phase-dependent regulation of cag promoters.The Pcag promoters are reported according to the variations of the transcript levels during bacterial growth, with promoters induced at late logarithmic phase (A), repressed during bacterial growth (B) and not sensitive to growth phase-dependent (C). An overnight culture of wild type strain was diluted to an OD600 of 0.08 and cultured for 15 hours. Total RNAs were extracted from equal volumes of cultures at different time points corresponding to OD600 of 0.22 (t1), 0.53(t2), 1.06(t3) and 1.75(t4). Results from the primer extension analysis are shown in the upper panels. The normalized average intensities of the bands from three independent experiments are reported in the graphs as the n-fold change, with error bars indicating the standard deviation.
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pone-0098416-g002: Growth phase-dependent regulation of cag promoters.The Pcag promoters are reported according to the variations of the transcript levels during bacterial growth, with promoters induced at late logarithmic phase (A), repressed during bacterial growth (B) and not sensitive to growth phase-dependent (C). An overnight culture of wild type strain was diluted to an OD600 of 0.08 and cultured for 15 hours. Total RNAs were extracted from equal volumes of cultures at different time points corresponding to OD600 of 0.22 (t1), 0.53(t2), 1.06(t3) and 1.75(t4). Results from the primer extension analysis are shown in the upper panels. The normalized average intensities of the bands from three independent experiments are reported in the graphs as the n-fold change, with error bars indicating the standard deviation.

Mentions: To study the transcriptional regulation of the selected cag promoters during growth, we carried out time course experiments. Aliquots of bacterial cultures were collected at different time points and used to extract total RNA for quantitative primer extension experiments at the 11 cag promoters (Fig 2).


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)

Growth phase-dependent regulation of cag promoters.The Pcag promoters are reported according to the variations of the transcript levels during bacterial growth, with promoters induced at late logarithmic phase (A), repressed during bacterial growth (B) and not sensitive to growth phase-dependent (C). An overnight culture of wild type strain was diluted to an OD600 of 0.08 and cultured for 15 hours. Total RNAs were extracted from equal volumes of cultures at different time points corresponding to OD600 of 0.22 (t1), 0.53(t2), 1.06(t3) and 1.75(t4). Results from the primer extension analysis are shown in the upper panels. The normalized average intensities of the bands from three independent experiments are reported in the graphs as the n-fold change, with error bars indicating the standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098416-g002: Growth phase-dependent regulation of cag promoters.The Pcag promoters are reported according to the variations of the transcript levels during bacterial growth, with promoters induced at late logarithmic phase (A), repressed during bacterial growth (B) and not sensitive to growth phase-dependent (C). An overnight culture of wild type strain was diluted to an OD600 of 0.08 and cultured for 15 hours. Total RNAs were extracted from equal volumes of cultures at different time points corresponding to OD600 of 0.22 (t1), 0.53(t2), 1.06(t3) and 1.75(t4). Results from the primer extension analysis are shown in the upper panels. The normalized average intensities of the bands from three independent experiments are reported in the graphs as the n-fold change, with error bars indicating the standard deviation.
Mentions: To study the transcriptional regulation of the selected cag promoters during growth, we carried out time course experiments. Aliquots of bacterial cultures were collected at different time points and used to extract total RNA for quantitative primer extension experiments at the 11 cag promoters (Fig 2).

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