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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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Related in: MedlinePlus

Mapping of cag promoters.A. Genomic organization of the cag pathogenicity island in the H. pylori G27 strain. (1) Transcriptional units mapped in this study; black: plus strand; grey: minus strand. (2) Annotated cag genes and ORFs; white block arrow: ORF encoding effector toxin CagA; dark grey block arrows: ORFs encoding structural T4SS components; light grey block arrows: ORFs encoding components with ancillary or unknown function. (3) Alphabetic classification of cag genes. (4) Numeric classification of cag genes. (5) Annotation of cag genes in reference strain 26695. (6) Transcriptional start sites mapped in [20]. (7) Transcriptional units identified in [17], [33]. (8) Transcriptional units identified by unbiased promoter-trapping experiments in [30]B. Summary of relevant features within the nucleotide sequences of the Pcag promoters mapped in this study. The TSSs (+1) are boxed in black boxes and showed in boldface. Sequences corresponding to -10 regions, the extended TGn elements and recognizable -35 region are enlightened in grey boxes. C. Comparison of the transcript levels at the Pcag promoters fused with lux reporter genes. mRNA levels of the Pcag-lux constructs were assayed by primer extension analysis using the oligonucleotide VSluxC1 and quantified with a phospoimager. The mean values from three independent experiments are reported as arbitrary units of 32P counts.
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pone-0098416-g001: Mapping of cag promoters.A. Genomic organization of the cag pathogenicity island in the H. pylori G27 strain. (1) Transcriptional units mapped in this study; black: plus strand; grey: minus strand. (2) Annotated cag genes and ORFs; white block arrow: ORF encoding effector toxin CagA; dark grey block arrows: ORFs encoding structural T4SS components; light grey block arrows: ORFs encoding components with ancillary or unknown function. (3) Alphabetic classification of cag genes. (4) Numeric classification of cag genes. (5) Annotation of cag genes in reference strain 26695. (6) Transcriptional start sites mapped in [20]. (7) Transcriptional units identified in [17], [33]. (8) Transcriptional units identified by unbiased promoter-trapping experiments in [30]B. Summary of relevant features within the nucleotide sequences of the Pcag promoters mapped in this study. The TSSs (+1) are boxed in black boxes and showed in boldface. Sequences corresponding to -10 regions, the extended TGn elements and recognizable -35 region are enlightened in grey boxes. C. Comparison of the transcript levels at the Pcag promoters fused with lux reporter genes. mRNA levels of the Pcag-lux constructs were assayed by primer extension analysis using the oligonucleotide VSluxC1 and quantified with a phospoimager. The mean values from three independent experiments are reported as arbitrary units of 32P counts.

Mentions: Recent studies have provided insights on the transcriptional organization of the H. pylori cag pathogenicity island, with the mapping of transcriptional start sites (TSS) and the identification of putative promoter regions. In strain 26695, out of 40 putative 5′-end of RNA transcripts identified [20], 14 map within the 300 bp upstream of annotated ORFs, and are predicted to contain the promoter regions of Cag protein coding sequences. These results were recently confirmed in different strains by promoter-trap and reverse transcription analyses of ORFs and intergenic regions [30]. The positions of the 5′ end of RNA transcripts and transcriptional units identified are schematically represented reported in Fig. 1A. To study their regulation we set out to map the 5′ end of these transcripts by primer extension analyses on total RNA extracted from H. pylori strain G27 grown to mid-log phase using oligonucleotides mapping downstream of the 14 aforementioned predicted promoters (Fig. 1A, Table 2). The remaining 26 internal and antisense TSSs deserve more dedicated studies and have been deliberately excluded from the current study.


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)

Mapping of cag promoters.A. Genomic organization of the cag pathogenicity island in the H. pylori G27 strain. (1) Transcriptional units mapped in this study; black: plus strand; grey: minus strand. (2) Annotated cag genes and ORFs; white block arrow: ORF encoding effector toxin CagA; dark grey block arrows: ORFs encoding structural T4SS components; light grey block arrows: ORFs encoding components with ancillary or unknown function. (3) Alphabetic classification of cag genes. (4) Numeric classification of cag genes. (5) Annotation of cag genes in reference strain 26695. (6) Transcriptional start sites mapped in [20]. (7) Transcriptional units identified in [17], [33]. (8) Transcriptional units identified by unbiased promoter-trapping experiments in [30]B. Summary of relevant features within the nucleotide sequences of the Pcag promoters mapped in this study. The TSSs (+1) are boxed in black boxes and showed in boldface. Sequences corresponding to -10 regions, the extended TGn elements and recognizable -35 region are enlightened in grey boxes. C. Comparison of the transcript levels at the Pcag promoters fused with lux reporter genes. mRNA levels of the Pcag-lux constructs were assayed by primer extension analysis using the oligonucleotide VSluxC1 and quantified with a phospoimager. The mean values from three independent experiments are reported as arbitrary units of 32P counts.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098416-g001: Mapping of cag promoters.A. Genomic organization of the cag pathogenicity island in the H. pylori G27 strain. (1) Transcriptional units mapped in this study; black: plus strand; grey: minus strand. (2) Annotated cag genes and ORFs; white block arrow: ORF encoding effector toxin CagA; dark grey block arrows: ORFs encoding structural T4SS components; light grey block arrows: ORFs encoding components with ancillary or unknown function. (3) Alphabetic classification of cag genes. (4) Numeric classification of cag genes. (5) Annotation of cag genes in reference strain 26695. (6) Transcriptional start sites mapped in [20]. (7) Transcriptional units identified in [17], [33]. (8) Transcriptional units identified by unbiased promoter-trapping experiments in [30]B. Summary of relevant features within the nucleotide sequences of the Pcag promoters mapped in this study. The TSSs (+1) are boxed in black boxes and showed in boldface. Sequences corresponding to -10 regions, the extended TGn elements and recognizable -35 region are enlightened in grey boxes. C. Comparison of the transcript levels at the Pcag promoters fused with lux reporter genes. mRNA levels of the Pcag-lux constructs were assayed by primer extension analysis using the oligonucleotide VSluxC1 and quantified with a phospoimager. The mean values from three independent experiments are reported as arbitrary units of 32P counts.
Mentions: Recent studies have provided insights on the transcriptional organization of the H. pylori cag pathogenicity island, with the mapping of transcriptional start sites (TSS) and the identification of putative promoter regions. In strain 26695, out of 40 putative 5′-end of RNA transcripts identified [20], 14 map within the 300 bp upstream of annotated ORFs, and are predicted to contain the promoter regions of Cag protein coding sequences. These results were recently confirmed in different strains by promoter-trap and reverse transcription analyses of ORFs and intergenic regions [30]. The positions of the 5′ end of RNA transcripts and transcriptional units identified are schematically represented reported in Fig. 1A. To study their regulation we set out to map the 5′ end of these transcripts by primer extension analyses on total RNA extracted from H. pylori strain G27 grown to mid-log phase using oligonucleotides mapping downstream of the 14 aforementioned predicted promoters (Fig. 1A, Table 2). The remaining 26 internal and antisense TSSs deserve more dedicated studies and have been deliberately excluded from the current study.

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