Construction and Characterization of a Derivative of Neisseria gonorrhoeae Strain MS11 Devoid of All opa Genes

Abstract

bTo better understand the role of Opa in gonococcal infections, we created and characterized a derivative of MS11 (MS11⌬opa) that had the coding sequence for all 11 Opa proteins deleted. The MS11⌬opa bacterium lost the ability to bind to purified lipooligosaccharide (LOS). While nonpiliated MS11⌬opa and nonpiliated Opa-expressing MS11 cells grew at the same rate, nonpiliated MS11⌬opa cells rarely formed clumps of more than four bacteria when grown in broth with vigorous shaking. Using flow cytometry analysis, we demonstrated that MS11⌬opa produced a homogeneous population of bacteria that failed to bind monoclonal antibody (MAb) 4B12, a MAb specific for Opa. Opa-expressing MS11 cells consisted of two predominant populations, where ϳ85% bound MAb 4B12 to a significant level and the other population bound little if any MAb. Approximately 90% of bacteria isolated from a phenotypically Opa-negative colony (a colony that does not refract light) failed to bind MAb 4B12; the remaining 10% bound MAb to various degrees. Piliated MS11⌬opa cells formed dispersed microcolonies on ME180 cells which were visually distinct from those of piliated Opa-expressing MS11 cells. When Opa expression was reintroduced into MS11⌬opa, the adherence ability of the strain recovered to wild-type levels. These data indicate that Opa contributes to both bacterium-bacterium and bacterium-host cell interactions.

Constitutively Opa-Expressing and Opa-Deficient Neisseria gonorrhoeae Strains Differentially Stimulate and Survive Exposure to Human Neutrophils

“…In contrast, the Opaless FA1090 derivative described in this work grew with similar kinetics to the opa-intact parent. While it is possible that we selected for a mutation that allows for Gc growth in the absence of all Opa expression, opa-deficient derivatives of strain MS11 Gc and strain H44/76 Neisseria meningitidis also show no in vitro growth defect (19,59). Each of the 11 transformation events that yielded Opaless occurred at a frequency of Ն0.02 per CFU, which is similar to the frequency we measure for transformation of non-opa constructs (our unpublished observations).…”

“…A subset of Opa proteins bind heparan sulfate proteoglycans on epithelial cells or indirectly engage integrins by binding vitronectin (14)(15)(16)(17). Opa proteins can also recognize lacto-N-neotetraose moieties on LOS (18,19). Opa expression is phase variable due to slipped-strand mispairing at pentameric repeats in the 5= end of opa genes (20,21).…”

Neisseria gonorrhoeae uses cellular proteins CXCL10 and IL8 to enhance HIV‐1 transmission across cervical mucosa

“…This NG strain was routinely grown in 5% CO 2 at 37°C on gonococcal medium base (GCB; Difco) or in chocolate agar plates (Remel) for 18‐24 hours . This was then selected for Opa‐positive colonies by choosing the opaque phenotype when the cultured colonies were observed with oblique light under a dissecting microscope . The working cultures of each bacteria were generated with two to three colonies from each culture types from the plate, suspended in 10% RPMI with the absorbance adjusted to a concentration of 1 × 10 7 cfu/mL for each experiments.…”

The Genetics of Neisseria Species

“…However, there is no correlation between orientation or distance of these three opa genes from the origin of replication and their expression levels. Strains containing deletions in all opa genes have been created in both N. meningitidis (134) and N. gonorrhoeae (8,95), and strains constitutively expressing specific opa genes have been constructed (32,93) to facilitate studies examining the contribution of specific proteins to pathogenesis without the issue of phase variation confounding the results.…”