ADDENDUM Gut Microbes 6:6, 377--381; November/December 2015; © 2015 Taylor & Francis Group, LLC
Helicobacter pylori and CagA under conditions of iron deficiency Jennifer M Noto and Richard M Peek Jr* Division of Gastroenterology, Department of Medicine; Vanderbilt University Medical Center; Nashville, TN USA
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Keywords: CagA, cag type IV secretion system, gastric cancer, Helicobacter pylori, iron deficiency *Correspondence to: Richard M Peek; Email: richard. [email protected] Submitted: 09/11/2015 Accepted: 10/05/2015 http://dx.doi.org/10.1080/19490976.2015.1105426 Addendum to: Noto JM, Lee JY, Gaddy JA, Cover TL, Amieva MR, Peek RM Jr. Regulation of Helicobacter pylori Virulence Within the Context of Iron Deficiency. J Infect Dis 2015; 211 (11):1790-4
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ron deficiency is the most common nutritional deficiency worldwide and compelling evidence has demonstrated that this condition heightens the risk of gastric cancer. Infection with Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. Recent work has demonstrated that, under conditions of iron deficiency, H. pylori-induced gastric carcinogenesis is augmented through increased formation of the strain-specific cag type IV secretion system and enhanced delivery of the bacterial oncoprotein CagA into host cells. Although CagA is a potent virulence factor that promotes oncogenic responses, additional studies have now demonstrated that CagA modulates host cell iron homeostasis in vitro and fundamental metabolic functions of the bacterial cell in vivo. Here we discuss these findings and describe working models by which CagA exerts its effects on gastric epithelial cells, with particular emphasis on its potential role in modulation of host iron homeostasis.
More than 14 million new cancer cases and over 8 million cancer-related deaths occur annually worldwide.2,3 Similar to the incidence of iron deficiency, cancer predominantly affects individuals residing in developing countries. Gastrointestinal malignancies represent a significant subset of the collective cancer burden, accounting for nearly 2 million deaths annually.2,3 Gastric cancer exerts the greatest global impact and represents the fifth most common malignancy and the third leading cause of cancer-related death worldwide, contributing to greater than 700,000 deaths each year.2,3 Epidemiologic studies throughout the world have demonstrated significant relationships between diet and gastrointestinal cancer risk. In particular, iron has been shown to contribute to cancer risk. However, iron represents a double-edged sword in that iron excess is associated with chronic diseases, such as cardiovascular disease, type 2 diabetes, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, neurodegenerative diseases, and cancer,1,4,5 while iron deficiency is also associated with an increased risk of gastric malignancy.6-11
Iron Deficiency and Gastric Cancer
Helicobacter pylori
Iron deficiency is the most common nutritional deficiency throughout the world. In addition to affecting large populations of people in developing countries, this deficiency is prevalent in industrialized countries as well. It is estimated that iron deficiency is present in more than 2 billon people worldwide, affecting more people (30% of the world’s population) than any other condition and making it a public health concern of epidemic proportions.1 Gut Microbes
Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. Intestinal-type gastric adenocarcinoma involves progression of normal gastric mucosa to chronic superficial gastritis, followed by the development of lesions with higher premalignant potential, including atrophic gastritis, intestinal metaplasia, dysplasia, and finally adenocarcinoma.12,13 Although the majority of colonized persons with chronic gastritis remain asymptomatic, chronic gastric inflammation is the primary inflection point in the carcinogenic cascade 377
disrupts adherens junctions (AJ) leading to aberrant activation of b-catenin (b) and an overall loss of barrier function and cellular polarity (Fig. 1),31-33 alterations that play an important role in carcinogenesis. Additionally, CagA can lead to activation of NF-kB, which is followed by induction of proinflammatory immune responses, such as the production of IL-8 (Fig. 1),34-38 critical steps to the development of gastritis.
Iron Deficiency and Bacterial Pathogenesis
Figure 1. The H. pylori cag type IV secretion system. The cag pathogenicity island encodes a bacterial type IV secretion system, which facilitates the delivery of CagA into gastric epithelial cells. Once inside the cell, CagA can undergo tyrosine phosphorylation by Src and Abl family kinases, where it then interacts with SHP2 and mediates ERK1/2 signaling and ultimately induces morphologic changes. CagA can also remain unphosphorylated, where it interacts with components of both the tight junctions (TJ) and adherens junctions (AJ), leading to dissociation of junctional complexes. In particular, unphosphorylated CagA can lead to disruption of b-catenin (b) from the adherens junction, leading to b-catenin-dependent transcriptional activation of mitogenic responses. CagA can also lead to activation of NF-kB, which leads to NF-kB-mediated proinflammatory responses, such as the induction of IL-8.
induced by H. pylori, which occurs over the course of decades.14,15 Gastric cancer develops in approximately 1–3% of infected individuals and gastric MALT lymphoma occurs in less than 0.1% of infected individuals. Despite these low frequencies, gastric cancer remains the third leading cause of cancer-related death worldwide and H. pylori remains the strongest known risk factor for this disease. H. pylori currently colonizes up to 80% of world’s population, yet only a fraction of individuals ever develop gastric cancer, indicating that a multitude of factors, including host genetics, strain-specific bacterial determinants, and environmental factors, cumulatively contribute to increased gastric cancer risk.
cag Type IV Secretion System One strain-specific bacterial virulence determinant that augments risk of gastric cancer is the cag pathogenicity island. The cag island is present in up to 70% of all Western H. pylori strains and virtually 100% of East-Asian strains.16-19 The cag island encodes a bacterial type IV
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secretion system that delivers the bacterial oncoprotein, CagA, into host cells (Fig. 1). Following its injection into host gastric epithelial cells, CagA exerts procarcinogenic effects. CagA can undergo tyrosine phosphorylation by members of the Abl and Src family kinases (Fig. 1).2025 Phosphorylated-CagA activates a eukaryotic phosphatase (SHP-2) and extracellular signal-regulated kinase 1 and 2 (Erk1/2), leading to robust actin cytoskeletal reorganization and other morphologic changes reminiscent of unrestrained stimulation by growth factors (Fig. 1).2029 CagA can also remain in a unphosphorylated form and exert numerous effects within gastric epithelial cells that contribute to pathogenesis. Unphosphorylated CagA leads to disruption of apicaljunctional complexes (Fig. 1), where CagA associates with the epithelial tightjunction scaffolding protein zona occludens 1 (ZO-1) and the transmembrane protein junctional adhesion molecule A (JAM-A), leading to nascent but incomplete assembly of tight junctions (TJ) at ectopic sites of bacterial attachment.30 In addition, unphosphorylated CagA
Gut Microbes
Recent work has demonstrated that, in a rodent model of H. pylori infection, dietary iron deficiency augments and accelerates the development of gastric inflammation and the development of gastric dysplasia and adenocarcinoma.39 This was mediated by the direct effects of iron limitation on H. pylori virulence as opposed to the direct effects of iron deficiency on the host.39 H. pylori strains isolated from iron deficient animals exhibited altered proteomic profiles, with many targets pertinent to bacterial pathogenesis, as well as heightened virulence phenotypes, as compared to H. pylori strains isolated from iron replete animals.39 Further, H. pylori strains isolated from iron deficient animals exhibited enhanced formation and function of the cag type IV secretion system (Fig. 2).39 Interestingly, these enhanced virulence phenotypes were reversible following the reintroduction of iron, whereby enhanced formation of the cag type IV secretion system, CagA translocation into host cells, and induction of proinflammatory responses were abrogated (Fig. 2).39,40 These studies implicate iron deficiency as an environmental factor that augments and accelerates H. pylori-induced gastric carcinogenesis. In terms of clinical ramifications, these studies have identified a measurable biomarker (low iron) that may be used to target individuals within populations at highest risk for disease for H. pylori treatment and eradication therapy, as global test and treat strategies for H. pylori have not been fully embraced due to the relatively low incidence of cancer that develops among infected individuals.
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The link between CagA and iron homeostasis has stimulated more recent studies to address the role of CagA under iron deficient conditions. Work performed by Manuel Amieva’s group demonstrated that CagA is required for survival and persistence in polarized epithelial cells under iron-limiting conditions. This work demonstrated that H. pylori cagA- deletion mutants were unable to grow and form microcolonies on a polarized epithelium under low iron conditions, which was due to their inability to acquire iron, as growth and microcolony formation were restored following the addition of exogenous iron.41 These investigators also demonstrated that H. pylori had the ability to disrupt host cell iron homeostasis, leading to alterations in transferrin internalization and trafficking throughout the cell from the basolateral surface to the apical surface, a phenomenon that was dependent on the presence of CagA (Fig. 2).41 These findings demonstrate a role for CagA in modulating host iron homeostasis and a mechanism by which bacteria may acquire iron from host cells. Extending this work in vivo, they subsequently demonstrated that loss of cagA under conditions of iron deficiency decreases H. pylori fitness, resulting in significant decreases in bacterial colonization density of the gastric mucosa.41 Other recent work has now demonstrated that, under conditions of iron depletion, loss of cagA promotes conversion of H. pylori from a classical helical morphology to a coccoid morphology both in vitro and in vivo (Fig. 3),40 a response that has been linked with bacterial responses to environmental stress. This response did not occur with wild-type H. pylori under irondepleted conditions, indicating that CagA is required for adaptation to iron deficient conditions.40 Similar to the reversibility of H. pylori virulence phenotypes under conditions of iron deficiency,39 conversion to coccoid morphology under conditions of iron deficiency was also reversible following the addition of exogenous iron.40 Intriguingly, this phenotype was also observed in the absence of host cells.40 The functional status exerted by coccoid H. pylori remains controversial. Data have shown that this morphological manifestation leads to non-viable H. pylori,42 while
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Figure 2. Enhanced formation and function of the cag type IV secretion system under conditions of iron depletion and its role in disruption of host cell iron homeostasis. H. pylori leads to proinflammatory responses, such as the induction of IL-8, and this response is mediated by translocation of CagA into gastric epithelial cells. Recent evidence has demonstrated that H. pylori can facilitate the mislocalization of the transferrin receptor from the basolateral surface to the apical surface in a CagA-dependent manner. Under conditions of iron deficiency, the formation and function of the cag type IV secretion system is augmented, leading to increased CagA translocation into gastric epithelial cells and increased proinflammatory responses, such as IL-8, and potentially increased access to host iron supplies through enhanced disruption and mislocalization of the transferrin receptor.
other results have demonstrated that coccoid forms are viable and maintain cell structure and virulence expression profiles.43,44 Coccoid forms of H. pylori have also been implicated in relapses of infection after antimicrobial treatment.45 Our data have provided fresh insights into these questions, as we have demonstrated that CagA is required for normal growth and spiral morphology under iron-restricted conditions. There has been significant focus on the study of CagA as a key virulence
factor that significantly disrupts host cell signaling and leads to oncogenic effects. These new findings demonstrate that CagA may also be an important factor for bacterial survival and persistence in the host through modulation of host iron metabolism and its role in nutrient acquisition. New results regarding the role of CagA are exciting and open up many questions and avenues of research regarding the role CagA in bacterial metabolism and survival in the absence of host cells.
Figure 3. CagA is required for spiral morphology under conditions of iron deficiency in vivo. Mongolian gerbils were maintained on iron-normal or iron-deficient diets and then challenged with H. pylori cagA¡ isogenic mutants. Gastric tissues were fixed in paraformaldehyde phosphate buffer fixative and processed, as previously described.46 Under normal iron conditions, H. pylori cagA- isogenic mutants exhibited wild-type spiral colony morphology that was characterized by the classical helical shape. However, under conditions of iron deficiency, H. pylori cagA- isogenic mutants exhibited altered bacterial shape, characterized by a transition to a coccoid morphology, suggesting that CagA is required for spiral morphology in vivo.
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Disclosure of Potential Conflicts of Interest
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43. Azevedo NF, Almeida C, Cerqueira L, Dias S, Keevil CW, Vieira MJ. Coccoid form of Helicobacter pylori as a morphological manifestation of cell adaptation to the environment. Appl Environmen Microbiol 2007; 73:3423-7; http://dx.doi.org/10.1128/AEM.00047-07 44. Monstein HJ, Jonasson J. Differential virulence-gene mRNA expression in coccoid forms of Helicobacter
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Helicobacter pylori and CagA under conditions of iron deficiency Jennifer M Noto and Richard M Peek Jr* Division of Gastroenterology, Department of Medicine; Vanderbilt University Medical Center; Nashville, TN USA
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Keywords: CagA, cag type IV secretion system, gastric cancer, Helicobacter pylori, iron deficiency *Correspondence to: Richard M Peek; Email: richard. [email protected] Submitted: 09/11/2015 Accepted: 10/05/2015 http://dx.doi.org/10.1080/19490976.2015.1105426 Addendum to: Noto JM, Lee JY, Gaddy JA, Cover TL, Amieva MR, Peek RM Jr. Regulation of Helicobacter pylori Virulence Within the Context of Iron Deficiency. J Infect Dis 2015; 211 (11):1790-4
www.tandfonline.com
ron deficiency is the most common nutritional deficiency worldwide and compelling evidence has demonstrated that this condition heightens the risk of gastric cancer. Infection with Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. Recent work has demonstrated that, under conditions of iron deficiency, H. pylori-induced gastric carcinogenesis is augmented through increased formation of the strain-specific cag type IV secretion system and enhanced delivery of the bacterial oncoprotein CagA into host cells. Although CagA is a potent virulence factor that promotes oncogenic responses, additional studies have now demonstrated that CagA modulates host cell iron homeostasis in vitro and fundamental metabolic functions of the bacterial cell in vivo. Here we discuss these findings and describe working models by which CagA exerts its effects on gastric epithelial cells, with particular emphasis on its potential role in modulation of host iron homeostasis.
More than 14 million new cancer cases and over 8 million cancer-related deaths occur annually worldwide.2,3 Similar to the incidence of iron deficiency, cancer predominantly affects individuals residing in developing countries. Gastrointestinal malignancies represent a significant subset of the collective cancer burden, accounting for nearly 2 million deaths annually.2,3 Gastric cancer exerts the greatest global impact and represents the fifth most common malignancy and the third leading cause of cancer-related death worldwide, contributing to greater than 700,000 deaths each year.2,3 Epidemiologic studies throughout the world have demonstrated significant relationships between diet and gastrointestinal cancer risk. In particular, iron has been shown to contribute to cancer risk. However, iron represents a double-edged sword in that iron excess is associated with chronic diseases, such as cardiovascular disease, type 2 diabetes, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, neurodegenerative diseases, and cancer,1,4,5 while iron deficiency is also associated with an increased risk of gastric malignancy.6-11
Iron Deficiency and Gastric Cancer
Helicobacter pylori
Iron deficiency is the most common nutritional deficiency throughout the world. In addition to affecting large populations of people in developing countries, this deficiency is prevalent in industrialized countries as well. It is estimated that iron deficiency is present in more than 2 billon people worldwide, affecting more people (30% of the world’s population) than any other condition and making it a public health concern of epidemic proportions.1 Gut Microbes
Helicobacter pylori is the strongest known risk factor for the development of gastric adenocarcinoma. Intestinal-type gastric adenocarcinoma involves progression of normal gastric mucosa to chronic superficial gastritis, followed by the development of lesions with higher premalignant potential, including atrophic gastritis, intestinal metaplasia, dysplasia, and finally adenocarcinoma.12,13 Although the majority of colonized persons with chronic gastritis remain asymptomatic, chronic gastric inflammation is the primary inflection point in the carcinogenic cascade 377
disrupts adherens junctions (AJ) leading to aberrant activation of b-catenin (b) and an overall loss of barrier function and cellular polarity (Fig. 1),31-33 alterations that play an important role in carcinogenesis. Additionally, CagA can lead to activation of NF-kB, which is followed by induction of proinflammatory immune responses, such as the production of IL-8 (Fig. 1),34-38 critical steps to the development of gastritis.
Iron Deficiency and Bacterial Pathogenesis
Figure 1. The H. pylori cag type IV secretion system. The cag pathogenicity island encodes a bacterial type IV secretion system, which facilitates the delivery of CagA into gastric epithelial cells. Once inside the cell, CagA can undergo tyrosine phosphorylation by Src and Abl family kinases, where it then interacts with SHP2 and mediates ERK1/2 signaling and ultimately induces morphologic changes. CagA can also remain unphosphorylated, where it interacts with components of both the tight junctions (TJ) and adherens junctions (AJ), leading to dissociation of junctional complexes. In particular, unphosphorylated CagA can lead to disruption of b-catenin (b) from the adherens junction, leading to b-catenin-dependent transcriptional activation of mitogenic responses. CagA can also lead to activation of NF-kB, which leads to NF-kB-mediated proinflammatory responses, such as the induction of IL-8.
induced by H. pylori, which occurs over the course of decades.14,15 Gastric cancer develops in approximately 1–3% of infected individuals and gastric MALT lymphoma occurs in less than 0.1% of infected individuals. Despite these low frequencies, gastric cancer remains the third leading cause of cancer-related death worldwide and H. pylori remains the strongest known risk factor for this disease. H. pylori currently colonizes up to 80% of world’s population, yet only a fraction of individuals ever develop gastric cancer, indicating that a multitude of factors, including host genetics, strain-specific bacterial determinants, and environmental factors, cumulatively contribute to increased gastric cancer risk.
cag Type IV Secretion System One strain-specific bacterial virulence determinant that augments risk of gastric cancer is the cag pathogenicity island. The cag island is present in up to 70% of all Western H. pylori strains and virtually 100% of East-Asian strains.16-19 The cag island encodes a bacterial type IV
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secretion system that delivers the bacterial oncoprotein, CagA, into host cells (Fig. 1). Following its injection into host gastric epithelial cells, CagA exerts procarcinogenic effects. CagA can undergo tyrosine phosphorylation by members of the Abl and Src family kinases (Fig. 1).2025 Phosphorylated-CagA activates a eukaryotic phosphatase (SHP-2) and extracellular signal-regulated kinase 1 and 2 (Erk1/2), leading to robust actin cytoskeletal reorganization and other morphologic changes reminiscent of unrestrained stimulation by growth factors (Fig. 1).2029 CagA can also remain in a unphosphorylated form and exert numerous effects within gastric epithelial cells that contribute to pathogenesis. Unphosphorylated CagA leads to disruption of apicaljunctional complexes (Fig. 1), where CagA associates with the epithelial tightjunction scaffolding protein zona occludens 1 (ZO-1) and the transmembrane protein junctional adhesion molecule A (JAM-A), leading to nascent but incomplete assembly of tight junctions (TJ) at ectopic sites of bacterial attachment.30 In addition, unphosphorylated CagA
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Recent work has demonstrated that, in a rodent model of H. pylori infection, dietary iron deficiency augments and accelerates the development of gastric inflammation and the development of gastric dysplasia and adenocarcinoma.39 This was mediated by the direct effects of iron limitation on H. pylori virulence as opposed to the direct effects of iron deficiency on the host.39 H. pylori strains isolated from iron deficient animals exhibited altered proteomic profiles, with many targets pertinent to bacterial pathogenesis, as well as heightened virulence phenotypes, as compared to H. pylori strains isolated from iron replete animals.39 Further, H. pylori strains isolated from iron deficient animals exhibited enhanced formation and function of the cag type IV secretion system (Fig. 2).39 Interestingly, these enhanced virulence phenotypes were reversible following the reintroduction of iron, whereby enhanced formation of the cag type IV secretion system, CagA translocation into host cells, and induction of proinflammatory responses were abrogated (Fig. 2).39,40 These studies implicate iron deficiency as an environmental factor that augments and accelerates H. pylori-induced gastric carcinogenesis. In terms of clinical ramifications, these studies have identified a measurable biomarker (low iron) that may be used to target individuals within populations at highest risk for disease for H. pylori treatment and eradication therapy, as global test and treat strategies for H. pylori have not been fully embraced due to the relatively low incidence of cancer that develops among infected individuals.
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The link between CagA and iron homeostasis has stimulated more recent studies to address the role of CagA under iron deficient conditions. Work performed by Manuel Amieva’s group demonstrated that CagA is required for survival and persistence in polarized epithelial cells under iron-limiting conditions. This work demonstrated that H. pylori cagA- deletion mutants were unable to grow and form microcolonies on a polarized epithelium under low iron conditions, which was due to their inability to acquire iron, as growth and microcolony formation were restored following the addition of exogenous iron.41 These investigators also demonstrated that H. pylori had the ability to disrupt host cell iron homeostasis, leading to alterations in transferrin internalization and trafficking throughout the cell from the basolateral surface to the apical surface, a phenomenon that was dependent on the presence of CagA (Fig. 2).41 These findings demonstrate a role for CagA in modulating host iron homeostasis and a mechanism by which bacteria may acquire iron from host cells. Extending this work in vivo, they subsequently demonstrated that loss of cagA under conditions of iron deficiency decreases H. pylori fitness, resulting in significant decreases in bacterial colonization density of the gastric mucosa.41 Other recent work has now demonstrated that, under conditions of iron depletion, loss of cagA promotes conversion of H. pylori from a classical helical morphology to a coccoid morphology both in vitro and in vivo (Fig. 3),40 a response that has been linked with bacterial responses to environmental stress. This response did not occur with wild-type H. pylori under irondepleted conditions, indicating that CagA is required for adaptation to iron deficient conditions.40 Similar to the reversibility of H. pylori virulence phenotypes under conditions of iron deficiency,39 conversion to coccoid morphology under conditions of iron deficiency was also reversible following the addition of exogenous iron.40 Intriguingly, this phenotype was also observed in the absence of host cells.40 The functional status exerted by coccoid H. pylori remains controversial. Data have shown that this morphological manifestation leads to non-viable H. pylori,42 while
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Figure 2. Enhanced formation and function of the cag type IV secretion system under conditions of iron depletion and its role in disruption of host cell iron homeostasis. H. pylori leads to proinflammatory responses, such as the induction of IL-8, and this response is mediated by translocation of CagA into gastric epithelial cells. Recent evidence has demonstrated that H. pylori can facilitate the mislocalization of the transferrin receptor from the basolateral surface to the apical surface in a CagA-dependent manner. Under conditions of iron deficiency, the formation and function of the cag type IV secretion system is augmented, leading to increased CagA translocation into gastric epithelial cells and increased proinflammatory responses, such as IL-8, and potentially increased access to host iron supplies through enhanced disruption and mislocalization of the transferrin receptor.
other results have demonstrated that coccoid forms are viable and maintain cell structure and virulence expression profiles.43,44 Coccoid forms of H. pylori have also been implicated in relapses of infection after antimicrobial treatment.45 Our data have provided fresh insights into these questions, as we have demonstrated that CagA is required for normal growth and spiral morphology under iron-restricted conditions. There has been significant focus on the study of CagA as a key virulence
factor that significantly disrupts host cell signaling and leads to oncogenic effects. These new findings demonstrate that CagA may also be an important factor for bacterial survival and persistence in the host through modulation of host iron metabolism and its role in nutrient acquisition. New results regarding the role of CagA are exciting and open up many questions and avenues of research regarding the role CagA in bacterial metabolism and survival in the absence of host cells.
Figure 3. CagA is required for spiral morphology under conditions of iron deficiency in vivo. Mongolian gerbils were maintained on iron-normal or iron-deficient diets and then challenged with H. pylori cagA¡ isogenic mutants. Gastric tissues were fixed in paraformaldehyde phosphate buffer fixative and processed, as previously described.46 Under normal iron conditions, H. pylori cagA- isogenic mutants exhibited wild-type spiral colony morphology that was characterized by the classical helical shape. However, under conditions of iron deficiency, H. pylori cagA- isogenic mutants exhibited altered bacterial shape, characterized by a transition to a coccoid morphology, suggesting that CagA is required for spiral morphology in vivo.
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Disclosure of Potential Conflicts of Interest
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