Azurin of pathogenic Neisseria spp. is involved in defense against hydrogen peroxide and survival within cervical epithelial cells

Abstract

Laz, a lipid-modified azurin of the human pathogens Neisseria gonorrhoeae and Neisseria meningitidis, is involved in defense against oxidative stress and copper toxicity; laz mutant strains are hypersensitive to hydrogen peroxide and copper. The N. gonorrhoeae laz mutant also has decreased survival in an ex vivo primary human ectocervical epithelial assay.

FIG. 1.

Restriction endonuclease, plasmid, and open reading frame (ORF) maps of the laz region. The line labeled FA1090 and MC58 represents the restriction endonuclease map of a region of the N. gonorrhoeae strain FA1090 genome sequencing project (accession number AE004969) and a region of the N. meningitidis strain MC58 genome sequencing project (accession number AE002447). The open arrow above the line indicates the orientation and the location of the azurin ORF. Below the FA1090-MC58 line, the thick black lines represent the plasmids constructed during this work. The vector, pGEM T-Easy (Promega), of each of these plasmids is represented by black boxes. The restriction endonuclease sites shown indicate where the kanamycin resistance cassette (KAN) was inserted. The rectangular box at the top represents the PCR products used as probes in this study. The laz probe was constructed from PCR products utilizing primers azurinfor and H8inrev.

FIG. 2.

(A) H₂O₂ survival test of N. gonorrhoeae strain 1291 wild type (WT) and laz mutant. In this assay, cells were grown on BHI agar ± 100 μM MnSO₄ and exposed to 40 mM H₂O₂. (B) H₂O₂ survival test of N. meningitidis strain MC58 wild type (WT) and laz mutant. In this assay, cells were grown on BHI agar and exposed to 10 mM or 15 mM H₂O₂. Experiments were performed in triplicate. The y axis error bars indicate ±1 standard deviation of the mean.

FIG. 3.

(A) Copper sensitivity test of N. gonorrhoeae strain 1291 wild type (WT) and laz mutant or (B) N. meningitidis strain MC58 wild type (WT) and laz mutant. Sensitivity was measured as the diameter (mm) of growth inhibition around a disk with 0, 0.5, or 1 M CuSO₄. Experiments were performed in triplicate. The y axis error bars indicate ±1 standard deviation of the mean. There is a statistically significant difference in the mean percent survival of the laz mutant strain relative to WT in all cases (P ≤ 0.05, as determined using a Student t test).