Type IV secretion machinery promotes ton-independent intracellular survival of Neisseria gonorrhoeae within cervical epithelial cells

Abstract

Survival of Neisseria gonorrhoeae within host epithelial cells is expected to be important in the pathogenesis of gonococcal disease. We previously demonstrated that strain FA1090 derives iron from a host cell in a process that requires the Ton complex and a putative TonB-dependent transporter, TdfF. FA1090, however, lacks the gonococcal genetic island (GGI) that is present in the majority of strains. The GGI in strain MS11 has been partially characterized, and it encodes a type IV secretion system (T4SS) involved in DNA release. In this study we investigated the role of iron acquisition and GGI-encoded gene products in gonococcal survival within cervical epithelial cells. We demonstrated that intracellular survival of MS11 was dependent on acquisition of iron from the host cell, but unlike the findings for FA1090, expression of the Ton complex was not required. Survival was not dependent on a putative TonB-like protein encoded in the GGI but instead was directly linked to T4SS structural components in a manner independent of the ability to release or internalize DNA. These data suggest that expression of selected GGI-encoded open reading frames confers an advantage during cervical cell infection. This study provides the first link between expression of the T4SS apparatus and intracellular survival of gonococci.

FIG. 1.

Invasion of ME180 cells by MS11: transmission electron microscopy image of ME180 cells following 4 h of invasion with piliated MS11.

FIG. 2.

Expression of a functional Ton system is not critical for the survival of gonococcal strain MS11 within ME180 cells. (A) Intracellular survival of FA1090 and the isogenic tonB mutant within ME180 cells as assessed by the gentamicin protection assay. (B) Intracellular survival of MS11 and the isogenic tonB mutant within ME180 cells as assessed by the gentamicin protection assay. The numbers of intracellular bacteria per well (IC CFU/well) were monitored for 24 h. Each bar indicates the geometric mean of four independent experiments. *, P < 0.05; **, P < 0.01.

FIG. 3.

Desferal treatment inhibits replication of gonococcal strain MS11 within ME180 cells, as assessed by the gentamicin protection assay. Cultures were either not treated or treated with 100 μM Desferal during the replication phase of the experiment. Desferal chelates ferric iron both from the medium and from within the ME180 cellular compartments. The numbers of intracellular bacteria per well (IC CFU/well) were monitored for 24 h. Each bar indicates the geometric mean of three independent experiments. *, P < 0.05.

FIG. 4.

GGI increases iron-dependent, Ton-independent intracellular survival of strain MS11. (A) Intracellular survival of wild-type strain MS11 (WT) and the isogenic tonB, ΔGGI, and ΔGGI tonB mutants as assessed by the gentamicin protection assay. (B) The defect in intracellular survival of the ΔGGI tonB double mutant was overcome when the cultures were supplemented with 24 μM Fe(NO₃)₃. The intracellular survival of the wild-type strain and the ΔGGI tonB double mutant was compared to the intracellular survival of parallel cultures of the wild-type strain and the ΔGGI tonB double mutant that were supplemented with iron (+Fe). Each bar indicates the geometric mean of three independent experiments. IC, intracellular bacteria. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

FIG. 5.

Expression of Yfd is not critical for the intracellular survival of MS11 within ME180 cells. The mechanism of Ton-independent intracellular survival encoded by the GGI was not dependent on expression of Yfd. The intracellular survival of wild-type strain MS11 (WT), the tonB mutant, and the yfd tonB double mutant within ME180 cells was determined. Each bar indicates the geometric mean of three independent experiments. IC, intracellular bacteria. *, P < 0.05; **, P < 0.01.

FIG. 6.

The mechanism of Ton-independent intracellular survival afforded by the GGI requires components of the T4SS. The intracellular survival of the following gonococcal strains was assessed by using the gentamicin protection assay: wild-type strain MS11 (WT) and the tonB, ΔGGI tonB, traN tonB, traN tonB/traN⁺, traH tonB, and traH tonB/traH⁺ mutants. Each bar indicates the geometric mean of at least three independent experiments. IC, intracellular bacteria. *, P < 0.05; **, P < 0.01.

FIG. 7.

Ton-independent intracellular survival promoted by the GGI is not dependent on DNA secretion and/or uptake. The intracellular survival of the following gonococcal strains was assessed by the gentamicin protection assay: wild-type strain MS11 (WT), the ΔGGI tonB double mutant, the parA tonB double mutant, and the pilT tonB double mutant. Each bar indicates the geometric mean of at least three independent experiments. IC, intracellular bacteria. *, P < 0.05; **, P < 0.01.