Abstract
Structural modification of peptidoglycan (PG) is one of the mechanisms that pathogenic bacteria use to evade the host innate immune system. For the noninvasive human gastric pathogen Helicobacter pylori, PG delivery to the host cells is one trigger of the immune response. H. pylori HP310 was markedly up-expressed upon cell exposure to oxidative stress. However, disruption of HP310 did not produce a phenotype distinguishable from the parent, including oxidative stress resistance characteristics. HP310 shows very weak homology to a known gene pgdA encoding PG deacetylase in Streptococcous pneumoniae. PGs from wild type H. pylori and the HP310 mutant were purified and analyzed by matrix-assisted laser desorption ionization time-of-flight and high pressure liquid chromatography. The parent strain PG is partially deacetylated, whereas several major PG-deacetylated muropeptides are absent or significantly reduced in the HP310 mutant. PG deacetylase activity was directly demonstrated by use of pure PG and HP310 protein by measuring the release of acetic acid. The Gram-negative bacterium H. pylori is highly resistant to lysozyme (up to 50 mg/ml), but the HP310 mutant is less resistant to lysozyme compared with the parent strain. Complementation of an hp310 strain with the wild type gene restored lysozyme resistance. The purified PG from the mutant is more susceptible to lysozyme (0.3 mg/ml) digestion than the wild type PG. The PG deacetylation appears to confer lysozyme resistance to escape immune detection. HP310 is representative of a new subfamily of bacterial PG deacetylases.
Highlights
Peptidoglycan (PG) was found in recent years to be an important factor involved in virulence by pathogenic bacteria [3]
The major oxidative stress-related proteins, such as catalase (KatA), alkyl-hydroperoxide reductase (AhpC), and the neutrophil-activating protein (NapA), are abundantly expressed in H. pylori, and they can be identified by one-dimensional SDS-PAGE [23]
During studies on H. pylori adaptation to oxidative stress, we noticed that HP310 was greatly up-expressed when cells are exposed to an elevated oxygen condition
Summary
Bacterial Strains and Growth Conditions—H. pylori strain 43504 was used as the wild type. The php310: kan construct was created via insertion of a kanamycin resistance cassette into the BamHI site within the HP310 fragment and confirmed by restriction analysis This plasmid was used for natural transformation of H. pylori, and the hp310:kan mutant strain was obtained by incubating cells on blood agar plates with kanamycin at 5% O2. Isolation and Preparation of Peptidoglycan—H. pylori cells were harvested and washed with ice-cold 20 mM sodium acetate (pH 5), centrifuged at 8,000 ϫ g for 30 min at 4 °C, and resuspended in 10 ml of the same buffer. Analysis of Lysozyme Digestion Products of PG by Mass Spectrometry—Portions of the reaction mixtures resulting from lysozyme digestion were reduced with borodeuteride as described above, and desalted using a Superdex-Peptide HR 10/30 FPLC column (Amersham Biosciences) eluted with 50 mM ammonium acetate (pH 6.0), collecting 0.4-ml fractions. After addition of 0.3 mg/ml lysozyme, the absorbance at 600 nm was monitored every hour
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