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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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Stress responses of cag promoters.A. Heat-shock response of the Pcag promoters. Primer extension analyses were performed on total RNA extracted from bacterial cultures of H. pylori wild type strain grown to exponentially phase and maintained at 37°C or exposed to 42°C for 30 min. B. Iron-dependent regulation of the PcagA promoter. Liquid cultures of wild type and Δfur strains were grown to OD600 = 0.5 or OD600 = 1.7 and treated for 30 min with 1 mM (NH4)2Fe(SO4)2 (Fe+), or 100 µM 2,2-dipyridyl (Fe-). mRNA levels at the PcagA promoter were assayed by quantitative primer extension on the total RNA extracted. C. In vitro binding of Fur protein to the PcagB-PcagA promoter region in a DNaseI footprinting assay. Concentrations of Fur dimer, 0 nM (lane 1), 21 nM (lane 2), 42 nM (lane 3), 84 nM (lane 4), 168 nM (lane 5) and 336 nM (lane 6). The binding reaction was performed in a final volume of 50 µL in presence of divalent iron ions as cofactors (150 µM (NH4)2Fe(SO4)2). The vertical grey and black bars on the right of the panel (I-IV) indicate the areas of partial and complete DNaseI protection, respectively, resulting from binding of Fur on the probe. Fur binds to nucleotide positions +2 to -14 (I), -20 to -63 (II), -108 to -115 (III) and -145 to -188 (IV) with respect to the +1 transcriptional start site (TSS) of PcagA. An hypersensitivity band that appears at high concentrations of Fur is indicated by a black triangle. On the left side of the panel, the TSS downstream PcagB and PcagA are indicated with bent arrows, while the relative position of the -10 and -35 regions of the 2 promoters are indicated as vertical black boxes. G+A: G+A sequencing reaction ladder. D. DNaseI footprinting of Fur protein to the PcagB-PcagA promoter region without the supplement of iron ions and with 150 µM 2,2-dipyridyl used to sequester the Fe2+ ions. The experimental conditions used for the footprinting assay are the same as described in panel C.
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pone-0098416-g003: Stress responses of cag promoters.A. Heat-shock response of the Pcag promoters. Primer extension analyses were performed on total RNA extracted from bacterial cultures of H. pylori wild type strain grown to exponentially phase and maintained at 37°C or exposed to 42°C for 30 min. B. Iron-dependent regulation of the PcagA promoter. Liquid cultures of wild type and Δfur strains were grown to OD600 = 0.5 or OD600 = 1.7 and treated for 30 min with 1 mM (NH4)2Fe(SO4)2 (Fe+), or 100 µM 2,2-dipyridyl (Fe-). mRNA levels at the PcagA promoter were assayed by quantitative primer extension on the total RNA extracted. C. In vitro binding of Fur protein to the PcagB-PcagA promoter region in a DNaseI footprinting assay. Concentrations of Fur dimer, 0 nM (lane 1), 21 nM (lane 2), 42 nM (lane 3), 84 nM (lane 4), 168 nM (lane 5) and 336 nM (lane 6). The binding reaction was performed in a final volume of 50 µL in presence of divalent iron ions as cofactors (150 µM (NH4)2Fe(SO4)2). The vertical grey and black bars on the right of the panel (I-IV) indicate the areas of partial and complete DNaseI protection, respectively, resulting from binding of Fur on the probe. Fur binds to nucleotide positions +2 to -14 (I), -20 to -63 (II), -108 to -115 (III) and -145 to -188 (IV) with respect to the +1 transcriptional start site (TSS) of PcagA. An hypersensitivity band that appears at high concentrations of Fur is indicated by a black triangle. On the left side of the panel, the TSS downstream PcagB and PcagA are indicated with bent arrows, while the relative position of the -10 and -35 regions of the 2 promoters are indicated as vertical black boxes. G+A: G+A sequencing reaction ladder. D. DNaseI footprinting of Fur protein to the PcagB-PcagA promoter region without the supplement of iron ions and with 150 µM 2,2-dipyridyl used to sequester the Fe2+ ions. The experimental conditions used for the footprinting assay are the same as described in panel C.

Mentions: To study the transcriptional regulation of cag promoters in response to environmental changes, we exposed exponentially growing cultures of H. pylori G27 strain to various stress conditions that challenge the bacterial metabolism or fitness. Total RNA was extracted from treated and untreated samples and transcript levels at the cag promoters were assayed by quantitative primer extensions with cag-specific oligonucleotides (Table 2). In bacterial cultures exposed to heat shock (30 min at 42°C) we observed a 6- to 40-fold reduction of mRNA levels at most cag promoters (Fig. 3A). Exceptions to this finding were at the Pcagζ and PcagA promoters that showed unchanged transcript levels (Fig. 3A). Subsequently, we assayed the mRNA levels at all cag promoters in H. pylori strains deleted of the heat shock transcriptional regulatory genes hspR and hrcA[39]. In comparison to the wild type strain, the knock-out ΔhspR and ΔhrcA mutants grown in normal conditions or exposed to heat shock treatment showed similar mRNA levels at cag promoters (data not shown). Thus, the observed variation in the mRNA levels after heat shock is not under the direct control of HspR or HrcA, likely reflecting a pleiotropic effect on transcription or mRNA stability.


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)

Stress responses of cag promoters.A. Heat-shock response of the Pcag promoters. Primer extension analyses were performed on total RNA extracted from bacterial cultures of H. pylori wild type strain grown to exponentially phase and maintained at 37°C or exposed to 42°C for 30 min. B. Iron-dependent regulation of the PcagA promoter. Liquid cultures of wild type and Δfur strains were grown to OD600 = 0.5 or OD600 = 1.7 and treated for 30 min with 1 mM (NH4)2Fe(SO4)2 (Fe+), or 100 µM 2,2-dipyridyl (Fe-). mRNA levels at the PcagA promoter were assayed by quantitative primer extension on the total RNA extracted. C. In vitro binding of Fur protein to the PcagB-PcagA promoter region in a DNaseI footprinting assay. Concentrations of Fur dimer, 0 nM (lane 1), 21 nM (lane 2), 42 nM (lane 3), 84 nM (lane 4), 168 nM (lane 5) and 336 nM (lane 6). The binding reaction was performed in a final volume of 50 µL in presence of divalent iron ions as cofactors (150 µM (NH4)2Fe(SO4)2). The vertical grey and black bars on the right of the panel (I-IV) indicate the areas of partial and complete DNaseI protection, respectively, resulting from binding of Fur on the probe. Fur binds to nucleotide positions +2 to -14 (I), -20 to -63 (II), -108 to -115 (III) and -145 to -188 (IV) with respect to the +1 transcriptional start site (TSS) of PcagA. An hypersensitivity band that appears at high concentrations of Fur is indicated by a black triangle. On the left side of the panel, the TSS downstream PcagB and PcagA are indicated with bent arrows, while the relative position of the -10 and -35 regions of the 2 promoters are indicated as vertical black boxes. G+A: G+A sequencing reaction ladder. D. DNaseI footprinting of Fur protein to the PcagB-PcagA promoter region without the supplement of iron ions and with 150 µM 2,2-dipyridyl used to sequester the Fe2+ ions. The experimental conditions used for the footprinting assay are the same as described in panel C.
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Related In: Results  -  Collection

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pone-0098416-g003: Stress responses of cag promoters.A. Heat-shock response of the Pcag promoters. Primer extension analyses were performed on total RNA extracted from bacterial cultures of H. pylori wild type strain grown to exponentially phase and maintained at 37°C or exposed to 42°C for 30 min. B. Iron-dependent regulation of the PcagA promoter. Liquid cultures of wild type and Δfur strains were grown to OD600 = 0.5 or OD600 = 1.7 and treated for 30 min with 1 mM (NH4)2Fe(SO4)2 (Fe+), or 100 µM 2,2-dipyridyl (Fe-). mRNA levels at the PcagA promoter were assayed by quantitative primer extension on the total RNA extracted. C. In vitro binding of Fur protein to the PcagB-PcagA promoter region in a DNaseI footprinting assay. Concentrations of Fur dimer, 0 nM (lane 1), 21 nM (lane 2), 42 nM (lane 3), 84 nM (lane 4), 168 nM (lane 5) and 336 nM (lane 6). The binding reaction was performed in a final volume of 50 µL in presence of divalent iron ions as cofactors (150 µM (NH4)2Fe(SO4)2). The vertical grey and black bars on the right of the panel (I-IV) indicate the areas of partial and complete DNaseI protection, respectively, resulting from binding of Fur on the probe. Fur binds to nucleotide positions +2 to -14 (I), -20 to -63 (II), -108 to -115 (III) and -145 to -188 (IV) with respect to the +1 transcriptional start site (TSS) of PcagA. An hypersensitivity band that appears at high concentrations of Fur is indicated by a black triangle. On the left side of the panel, the TSS downstream PcagB and PcagA are indicated with bent arrows, while the relative position of the -10 and -35 regions of the 2 promoters are indicated as vertical black boxes. G+A: G+A sequencing reaction ladder. D. DNaseI footprinting of Fur protein to the PcagB-PcagA promoter region without the supplement of iron ions and with 150 µM 2,2-dipyridyl used to sequester the Fe2+ ions. The experimental conditions used for the footprinting assay are the same as described in panel C.
Mentions: To study the transcriptional regulation of cag promoters in response to environmental changes, we exposed exponentially growing cultures of H. pylori G27 strain to various stress conditions that challenge the bacterial metabolism or fitness. Total RNA was extracted from treated and untreated samples and transcript levels at the cag promoters were assayed by quantitative primer extensions with cag-specific oligonucleotides (Table 2). In bacterial cultures exposed to heat shock (30 min at 42°C) we observed a 6- to 40-fold reduction of mRNA levels at most cag promoters (Fig. 3A). Exceptions to this finding were at the Pcagζ and PcagA promoters that showed unchanged transcript levels (Fig. 3A). Subsequently, we assayed the mRNA levels at all cag promoters in H. pylori strains deleted of the heat shock transcriptional regulatory genes hspR and hrcA[39]. In comparison to the wild type strain, the knock-out ΔhspR and ΔhrcA mutants grown in normal conditions or exposed to heat shock treatment showed similar mRNA levels at cag promoters (data not shown). Thus, the observed variation in the mRNA levels after heat shock is not under the direct control of HspR or HrcA, likely reflecting a pleiotropic effect on transcription or mRNA stability.

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