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Role of the N -Acetylmuramoyl- l -Alanyl Amidase, AmiA, of Helicobacter pylori in Peptidoglycan Metabolism, Daughter Cell Separation, and Virulence

View Article: PubMed Central - PubMed

ABSTRACT

The human gastric pathogen, Helicobacter pylori, is becoming increasingly resistant to most available antibiotics. Peptidoglycan (PG) metabolism is essential to eubacteria, hence, an excellent target for the development of new therapeutic strategies. However, our knowledge on PG metabolism in H. pylori remains poor. We have further characterized an isogenic mutant of the amiA gene encoding a N-acetylmuramoyl-l-alanyl amidase. The amiA mutant displayed long chains of unseparated cells, an impaired motility despite the presence of intact flagella and a tolerance to amoxicillin. Interestingly, the amiA mutant was impaired in colonizing the mouse stomach suggesting that AmiA is a valid target in H. pylori for the development of new antibiotics. Using reverse phase high-pressure liquid chromatography, we analyzed the PG muropeptide composition and glycan chain length distribution of strain 26695 and its amiA mutant. The analysis showed that H. pylori lacked muropeptides with a degree of cross-linking higher than dimeric muropeptides. The amiA mutant was also characterized by a decrease of muropeptides carrying 1,6-anhydro-N-acetylmuramic acid residues, which represent the ends of the glycan chains. This correlated with an increase of very long glycan strands in the amiA mutant. It is suggested that these longer glycan strands are trademarks of the division site. Taken together, we show that the low redundancy on genes involved in PG maturation supports H. pylori as an actractive alternative model to study PG metabolism and cell shape regulation.

No MeSH data available.


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Mice colonization with WT X47-2AL and its isogenic amiA mutants after 3, 15, and 30 days of infections (A), and with WT B128 and its isogenic amiA mutants 15 days after infection (B). For each experiment, we used an even mixture of three independent clones of the amiA mutants. Since the amiA mutant chains, we considered it was plausible that we were not able to detect colonization of the mutant using a low infectious dose (represented with gray circles). Therefore, a higher dose was also tested (represented with dark triangles). The amiA mutant was still unable to colonize C57/BL6J mice (ND = non detectable). The data were submitted to a Mann–Whitney test (**p < 0.01).
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f3: Mice colonization with WT X47-2AL and its isogenic amiA mutants after 3, 15, and 30 days of infections (A), and with WT B128 and its isogenic amiA mutants 15 days after infection (B). For each experiment, we used an even mixture of three independent clones of the amiA mutants. Since the amiA mutant chains, we considered it was plausible that we were not able to detect colonization of the mutant using a low infectious dose (represented with gray circles). Therefore, a higher dose was also tested (represented with dark triangles). The amiA mutant was still unable to colonize C57/BL6J mice (ND = non detectable). The data were submitted to a Mann–Whitney test (**p < 0.01).

Mentions: Since the amiA mutant had two major cell morphological defects, impaired daughter cell separation and motility, we investigated the impact on the amiA inactivation on H. pylori capability to colonize mice stomachs. We infected C57/BL6J mice with two parental and fully motile strains, X47-2AL (Fig. 3A) and B128 (Fig. 3B), and their isogenic amiA mutants. We then analyzed their ability to colonize the mouse gastric mucosa at different time points (3, 15, and 30 days after infection; see Fig. 3). Note that the infections were done with an even mixture of three independent clones of amiA mutants in each background. Clearly, the amiA mutant was unable to colonize the stomach of C57/BL6J mice under any conditions tested, indicating that the AmiA protein is required for efficient colonization of the stomach.


Role of the N -Acetylmuramoyl- l -Alanyl Amidase, AmiA, of Helicobacter pylori in Peptidoglycan Metabolism, Daughter Cell Separation, and Virulence
Mice colonization with WT X47-2AL and its isogenic amiA mutants after 3, 15, and 30 days of infections (A), and with WT B128 and its isogenic amiA mutants 15 days after infection (B). For each experiment, we used an even mixture of three independent clones of the amiA mutants. Since the amiA mutant chains, we considered it was plausible that we were not able to detect colonization of the mutant using a low infectious dose (represented with gray circles). Therefore, a higher dose was also tested (represented with dark triangles). The amiA mutant was still unable to colonize C57/BL6J mice (ND = non detectable). The data were submitted to a Mann–Whitney test (**p < 0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036311&req=5

f3: Mice colonization with WT X47-2AL and its isogenic amiA mutants after 3, 15, and 30 days of infections (A), and with WT B128 and its isogenic amiA mutants 15 days after infection (B). For each experiment, we used an even mixture of three independent clones of the amiA mutants. Since the amiA mutant chains, we considered it was plausible that we were not able to detect colonization of the mutant using a low infectious dose (represented with gray circles). Therefore, a higher dose was also tested (represented with dark triangles). The amiA mutant was still unable to colonize C57/BL6J mice (ND = non detectable). The data were submitted to a Mann–Whitney test (**p < 0.01).
Mentions: Since the amiA mutant had two major cell morphological defects, impaired daughter cell separation and motility, we investigated the impact on the amiA inactivation on H. pylori capability to colonize mice stomachs. We infected C57/BL6J mice with two parental and fully motile strains, X47-2AL (Fig. 3A) and B128 (Fig. 3B), and their isogenic amiA mutants. We then analyzed their ability to colonize the mouse gastric mucosa at different time points (3, 15, and 30 days after infection; see Fig. 3). Note that the infections were done with an even mixture of three independent clones of amiA mutants in each background. Clearly, the amiA mutant was unable to colonize the stomach of C57/BL6J mice under any conditions tested, indicating that the AmiA protein is required for efficient colonization of the stomach.

View Article: PubMed Central - PubMed

ABSTRACT

The human gastric pathogen, Helicobacter pylori, is becoming increasingly resistant to most available antibiotics. Peptidoglycan (PG) metabolism is essential to eubacteria, hence, an excellent target for the development of new therapeutic strategies. However, our knowledge on PG metabolism in H. pylori remains poor. We have further characterized an isogenic mutant of the amiA gene encoding a N-acetylmuramoyl-l-alanyl amidase. The amiA mutant displayed long chains of unseparated cells, an impaired motility despite the presence of intact flagella and a tolerance to amoxicillin. Interestingly, the amiA mutant was impaired in colonizing the mouse stomach suggesting that AmiA is a valid target in H. pylori for the development of new antibiotics. Using reverse phase high-pressure liquid chromatography, we analyzed the PG muropeptide composition and glycan chain length distribution of strain 26695 and its amiA mutant. The analysis showed that H. pylori lacked muropeptides with a degree of cross-linking higher than dimeric muropeptides. The amiA mutant was also characterized by a decrease of muropeptides carrying 1,6-anhydro-N-acetylmuramic acid residues, which represent the ends of the glycan chains. This correlated with an increase of very long glycan strands in the amiA mutant. It is suggested that these longer glycan strands are trademarks of the division site. Taken together, we show that the low redundancy on genes involved in PG maturation supports H. pylori as an actractive alternative model to study PG metabolism and cell shape regulation.

No MeSH data available.


Related in: MedlinePlus