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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.


Related in: MedlinePlus

Electron microscopy of WT H. pylori strain X47-2AL (A, B) and its isogenic amiA mutant (C–F). (C) Shows the chaining phenotype of the amiA mutants. Arrows heads highlight flagella located in the middle of a bacterial chain. Examples of higher magnifications of flagella of the amiA mutant are illustrated in (D–F). (D) Shows polar flagella and (E, F) illustrate flagella at division sites. (G–J) Show transmission electron microscopy of the amiA mutant showing evenly spaced septa that failed to separate daugther cells. WT, wild-type.
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f2: Electron microscopy of WT H. pylori strain X47-2AL (A, B) and its isogenic amiA mutant (C–F). (C) Shows the chaining phenotype of the amiA mutants. Arrows heads highlight flagella located in the middle of a bacterial chain. Examples of higher magnifications of flagella of the amiA mutant are illustrated in (D–F). (D) Shows polar flagella and (E, F) illustrate flagella at division sites. (G–J) Show transmission electron microscopy of the amiA mutant showing evenly spaced septa that failed to separate daugther cells. WT, wild-type.

Mentions: Next, we were interested in analyzing the general morphological phenotype of the amiA mutant since amidases have been implicated in daughter cell separation both in Gram-positive and Gram-negative bacteria. As observed for several other bacteria, the inactivation of the amiA gene resulted in a chaining phenotype (Fig. 2). Also, H. pylori is known for undergoing a morphological transition from spiral to coccoid form during entry in stationary phase. We observed that associated with the chaining phenotype, the amiA mutant failed to undergo morphological transition as previously described.7 Since the sequenced strain 26695 lacks flagella, we also constructed several independent clones in other H. pylori backgrounds. Interestingly, when the amiA gene was inactivated in strains that were motile such as X47-2AL (Fig. 2C–F) and B128 (data not shown), the mutants were still able to synthesize at the poles (Fig. 2D) and some division sites intact flagella (Fig. 2E, F). Although some bacterial chains were motile under the optical microscope, the vast majority was not. Using a soft agar mobility assay, all the amiA independent mutant clones were unable to migrate from the site of inoculation in contrast to the WT strain (data not shown).


Role of the N -Acetylmuramoyl- l -Alanyl Amidase, AmiA, of Helicobacter pylori in Peptidoglycan Metabolism, Daughter Cell Separation, and Virulence
Electron microscopy of WT H. pylori strain X47-2AL (A, B) and its isogenic amiA mutant (C–F). (C) Shows the chaining phenotype of the amiA mutants. Arrows heads highlight flagella located in the middle of a bacterial chain. Examples of higher magnifications of flagella of the amiA mutant are illustrated in (D–F). (D) Shows polar flagella and (E, F) illustrate flagella at division sites. (G–J) Show transmission electron microscopy of the amiA mutant showing evenly spaced septa that failed to separate daugther cells. WT, wild-type.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Electron microscopy of WT H. pylori strain X47-2AL (A, B) and its isogenic amiA mutant (C–F). (C) Shows the chaining phenotype of the amiA mutants. Arrows heads highlight flagella located in the middle of a bacterial chain. Examples of higher magnifications of flagella of the amiA mutant are illustrated in (D–F). (D) Shows polar flagella and (E, F) illustrate flagella at division sites. (G–J) Show transmission electron microscopy of the amiA mutant showing evenly spaced septa that failed to separate daugther cells. WT, wild-type.
Mentions: Next, we were interested in analyzing the general morphological phenotype of the amiA mutant since amidases have been implicated in daughter cell separation both in Gram-positive and Gram-negative bacteria. As observed for several other bacteria, the inactivation of the amiA gene resulted in a chaining phenotype (Fig. 2). Also, H. pylori is known for undergoing a morphological transition from spiral to coccoid form during entry in stationary phase. We observed that associated with the chaining phenotype, the amiA mutant failed to undergo morphological transition as previously described.7 Since the sequenced strain 26695 lacks flagella, we also constructed several independent clones in other H. pylori backgrounds. Interestingly, when the amiA gene was inactivated in strains that were motile such as X47-2AL (Fig. 2C–F) and B128 (data not shown), the mutants were still able to synthesize at the poles (Fig. 2D) and some division sites intact flagella (Fig. 2E, F). Although some bacterial chains were motile under the optical microscope, the vast majority was not. Using a soft agar mobility assay, all the amiA independent mutant clones were unable to migrate from the site of inoculation in contrast to the WT strain (data not shown).

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