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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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Reporter assays with the Pcag-lux strains in host cell co-cultures.Liquid cultures of Pcagζ-, PcagQ- and PcagB-lux strains were added at a multiplicity of infection of 5 to 24-wells plates containing human gastric adenocarcinoma (AGS) cells or with the medium only. The luminescence emitted by the reporter strains was recorded by a multilabel reader at regular intervals. Signals were normalized on the samples without AGS cells and averaged. Standard errors were calculated from four independent experiments (each in four technical replicates).
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pone-0098416-g006: Reporter assays with the Pcag-lux strains in host cell co-cultures.Liquid cultures of Pcagζ-, PcagQ- and PcagB-lux strains were added at a multiplicity of infection of 5 to 24-wells plates containing human gastric adenocarcinoma (AGS) cells or with the medium only. The luminescence emitted by the reporter strains was recorded by a multilabel reader at regular intervals. Signals were normalized on the samples without AGS cells and averaged. Standard errors were calculated from four independent experiments (each in four technical replicates).

Mentions: Host cell contacts are potent elicitors of secretion system gene expression in pathogenic bacteria [15]. To assess the possible in vivo effects exerted by bacterium-host contacts on the transcription of the cag promoters, we used co-cultures of AGS cells and H. pylori G27-derived strains carrying the Pcag-lux transcriptional fusions. Bacterial cultures were grown to mid-log phase and used to infect AGS cells cultured in 24-well plates (AGS+ sample), while same amounts of bacterial cultures were added to plates containing only the medium (AGS- sample). During a time-course experiment, we measured the luminescence of the samples at regular time intervals, and for each time point we calculated the ratio of the signal from the bacteria grown in presence or absence of AGS cells (AGS+/AGS- ratio). The Pcagζ-lux strain exhibited a significant increase of luminescence when co-cultured in the presence of AGS cells, with an AGS+/AGS- ratio that increased over time (Fig. 6). In contrast, the other Pcag-lux strains showed no significant differences between samples cultured with or without the AGS cells, with an AGS+/AGS- ratio unchanged during the experiment, as exemplified by the PcagQ- and PcagB-lux strains (Fig. 6). We can conclude that under the experimental conditions tested, the interaction of H. pylori with its host cells exerts a positive transcriptional effect only on expression levels at the Pcagζ promoter. Previous studies indicated that contact of H. pylori with host cells provokes the increase of visible T4SS pili extruding from the bacterium at the host-pathogen interaction surface [47], and that the protein composition of the cag-T4SS pili differs if bacteria are grown planktonically or in co-culture with AGS host cells [48]. The finding that the interaction with host cells rapidly induces the transcription of the Pcagζ promoter is, therefore, particularly striking. In fact, the cagζεδγ operon encompasses cagδ, a gene that codes for a factor bridging the periplasm across the inner and outer membrane, essential for the stabilization of the T4SS core, as well as cagγ, encoding the transglycosylase involved in the local hydrolyzation of the murein layer important for the formation and extrusion of the assembling T4SS. Interestingly, Kim and colleagues reported similar variations of cagδ expression in H. pylori 69a strain, co-cultured with AGS cells for 1 hour [25]. These evidences suggest a conserved regulation of the operon, likely due to the modulation of the Pcagζ promoter activity. Together, the results indicate that Pcagζ induction may modulate the number of pili, their distribution on the bacterial cell surface and their composition after host cell contact. Previous observations of the AGS-induced regulation of other cag promoters (e.g. cagA, cagP and cagS) [20], [49], were not confirmed in this study, possibly due to strain-specific responses to host-cell contact, or due to the different reporter system used to monitor the responses.


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)

Reporter assays with the Pcag-lux strains in host cell co-cultures.Liquid cultures of Pcagζ-, PcagQ- and PcagB-lux strains were added at a multiplicity of infection of 5 to 24-wells plates containing human gastric adenocarcinoma (AGS) cells or with the medium only. The luminescence emitted by the reporter strains was recorded by a multilabel reader at regular intervals. Signals were normalized on the samples without AGS cells and averaged. Standard errors were calculated from four independent experiments (each in four technical replicates).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098416-g006: Reporter assays with the Pcag-lux strains in host cell co-cultures.Liquid cultures of Pcagζ-, PcagQ- and PcagB-lux strains were added at a multiplicity of infection of 5 to 24-wells plates containing human gastric adenocarcinoma (AGS) cells or with the medium only. The luminescence emitted by the reporter strains was recorded by a multilabel reader at regular intervals. Signals were normalized on the samples without AGS cells and averaged. Standard errors were calculated from four independent experiments (each in four technical replicates).
Mentions: Host cell contacts are potent elicitors of secretion system gene expression in pathogenic bacteria [15]. To assess the possible in vivo effects exerted by bacterium-host contacts on the transcription of the cag promoters, we used co-cultures of AGS cells and H. pylori G27-derived strains carrying the Pcag-lux transcriptional fusions. Bacterial cultures were grown to mid-log phase and used to infect AGS cells cultured in 24-well plates (AGS+ sample), while same amounts of bacterial cultures were added to plates containing only the medium (AGS- sample). During a time-course experiment, we measured the luminescence of the samples at regular time intervals, and for each time point we calculated the ratio of the signal from the bacteria grown in presence or absence of AGS cells (AGS+/AGS- ratio). The Pcagζ-lux strain exhibited a significant increase of luminescence when co-cultured in the presence of AGS cells, with an AGS+/AGS- ratio that increased over time (Fig. 6). In contrast, the other Pcag-lux strains showed no significant differences between samples cultured with or without the AGS cells, with an AGS+/AGS- ratio unchanged during the experiment, as exemplified by the PcagQ- and PcagB-lux strains (Fig. 6). We can conclude that under the experimental conditions tested, the interaction of H. pylori with its host cells exerts a positive transcriptional effect only on expression levels at the Pcagζ promoter. Previous studies indicated that contact of H. pylori with host cells provokes the increase of visible T4SS pili extruding from the bacterium at the host-pathogen interaction surface [47], and that the protein composition of the cag-T4SS pili differs if bacteria are grown planktonically or in co-culture with AGS host cells [48]. The finding that the interaction with host cells rapidly induces the transcription of the Pcagζ promoter is, therefore, particularly striking. In fact, the cagζεδγ operon encompasses cagδ, a gene that codes for a factor bridging the periplasm across the inner and outer membrane, essential for the stabilization of the T4SS core, as well as cagγ, encoding the transglycosylase involved in the local hydrolyzation of the murein layer important for the formation and extrusion of the assembling T4SS. Interestingly, Kim and colleagues reported similar variations of cagδ expression in H. pylori 69a strain, co-cultured with AGS cells for 1 hour [25]. These evidences suggest a conserved regulation of the operon, likely due to the modulation of the Pcagζ promoter activity. Together, the results indicate that Pcagζ induction may modulate the number of pili, their distribution on the bacterial cell surface and their composition after host cell contact. Previous observations of the AGS-induced regulation of other cag promoters (e.g. cagA, cagP and cagS) [20], [49], were not confirmed in this study, possibly due to strain-specific responses to host-cell contact, or due to the different reporter system used to monitor the responses.

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