Transcriptional and Translational Responsiveness of the Neisseria gonorrhoeae Type IV Secretion System to Conditions of Host Infections

Abstract

The type IV secretion system of Neisseria gonorrhoeae translocates single-stranded DNA into the extracellular space, facilitating horizontal gene transfer and initiating biofilm formation. Expression of this system has been observed to be low under laboratory conditions, and multiple levels of regulation have been identified. We used a translational fusion of lacZ to traD, the gene for the type IV secretion system coupling protein, to screen for increased type IV secretion system expression. We identified several physiologically relevant conditions, including surface adherence, decreased manganese or iron, and increased zinc or copper, which increase gonococcal type IV secretion system protein levels through transcriptional and/or translational mechanisms. These metal treatments are reminiscent of the conditions in the macrophage phagosome. The ferric uptake regulator, Fur, was found to repress traD transcript levels but to also have a second role, acting to allow TraD protein levels to increase only in the absence of iron. To better understand type IV secretion system regulation during infection, we examined transcriptomic data from active urethral infection samples from five men. The data demonstrated differential expression of 20 of 21 type IV secretion system genes during infection, indicating upregulation of genes necessary for DNA secretion during host infection.

Keywords: Neisseria gonorrhoeae; biofilms; copper; iron regulation; phagosomes; transcriptional regulation; transcriptome; type IV secretion; urethral infection; zinc.

FIG 1

Gonococcal genetic island (GGI) and traD::lacZ reporter construct. (A) Map of the GGI. Genes necessary for secretion are shown in green. Transcripts measured in qRT-PCR experiments are indicated with stars. Promoters (green arrows) are marked for operons necessary for secretion only. tra and trb genes, as well as ltgX, are homologous to genes found on the E. coli F-plasmid. To date, no putative regulators have been identified. Blue brackets indicate the 4.4-kb region from exp1-ydcA. (B) Gene map of traD::lacZ translational fusion reporter construct. The origin of transfer (oriT) is indicated by a stem-loop.

FIG 2

Iron sensing plays a regulatory role in T4S. (A) β-Galactosidase assay detecting TraD-LacZ expression from strains wild type (WT) for fur versus the fur-1 mutant in both iron-replete and iron-depleted growth media. *, P < 0.05 in Student’s t test comparing sample to wild-type fur in iron-replete media. Data are averages of three experiments; error bars show SEM. (B) Representative Western blot against the FLAG tag in TraD-FLAG3 strains with wild-type fur and fur-1 in iron-replete media. Arrow indicates the TraD-FLAG3 band. (C) qRT-PCR comparing GGI transcripts from the wild type and the fur-1 mutant in both iron-replete [cGCBL + 1.2 μM Fe(NO₃)₃] and iron-depleted (GCBL plus 100 μM deferoxamine) growth media. *, P < 0.05 in Student’s t test comparing ΔC_T values to those of the wild type in iron-replete medium. Data are averages of three experiments; error bars show 95% confidence intervals. Transcripts were normalized to rmp. (D) Quantification of DNA in supernatants from the wild type (MS11), a ΔGGI strain (ND500), and the fur-1 mutant. Values were corrected for extracellular DNA resultant from lysis by subtracting ΔGGI strain (ND500) values in each experimental replicate. Student’s t test determined no significant differences from MS11. Data are averages of three experiments; error bars show SEM.

FIG 3

Zinc and copper both alter TraD expression. (A and B) β-Galactosidase assays of traD::lacZ gonococci with various concentrations of ZnSO₄ (A) or CuSO₄ (B). Significance was determined by one-way analysis of variance (ANOVA) and Dunnett’s posttest. Data are averages of three experiments; error bars show SEM. (C and D) qRT-PCR of GGI transcripts in response to 100 μM ZnSO₄ (C) or 500 μM CuSO₄ (D). Significance was determined by Student’s t test comparing ΔC_T values. Data are averages of three experiments; error bars show 95% confidence interval. Transcripts were normalized to rpoB. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

FIG 4

Semicooperative upregulation of TraD by zinc, copper, and iron chelation using 100 μM deferoxamine, 250 μM ZnSO₄, and 500 μM CuSO₄. (A) β-Galactosidase assay detecting TraD-LacZ expression. Significance was determined by Student’s t test comparing samples to untreated cultures. *, P < 0.05; **, P < 0.01; ****, P < 0.0001. Data are averages of three experiments; error bars show SEM. (B) Representative Western blot against the FLAG epitope. Native expression of traD::FLAG3 (top) and induced overexpression of traK::FLAG3 (bottom) are shown. The WT has no FLAG tag. (C) qRT-PCR showing no significant differences between all tested GGI transcripts by Student’s t test comparing ΔC_T values. Data are averages of four experiments; bars show 95% confidence interval. Transcripts were normalized to rpoB.

FIG 5

Manganese represses T4S. (A) Representative Western blot for TraD-FLAG3 demonstrates a visible decrease in TraD in cultures treated with 100 μM MnCl₂. Arrow indicates the TraD-FLAG3 band. (B) Extracellular DNA quantification shows less DNA is secreted in manganese-treated cultures. Values were corrected for extracellular DNA resultant from lysis by subtracting ΔGGI strain (ND500) values in each experimental replicate. Data are averages of three experiments; error bars show SEM. (C) qRT-PCR showing decreased traD and traK transcript with 100 μM MnCl₂. **, P < 0.01 in Student’s t test comparing ΔC_T values. Data are averages of three experiments; error bars show 95% confidence interval. Transcripts were normalized to rpoB.

FIG 6

The GGI is differentially expressed during natural urethral infection in humans. Shown is a heat map of GGI gene expression in laboratory-grown gonococcal isolates and urethral swab samples. Brackets on the left indicate genes necessary for DNA secretion in laboratory culture. n = 5. *, q value < 0.05.

FIG 7

GGI transcript levels are elevated in surface-adhered cells. Shown is a qRT-PCR analysis of representative GGI genes. #, P < 0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.001 in Student’s t test comparing ΔC_T values. Data are averages of four experiments; error bars show 95% confidence interval. Transcripts were normalized to rpoB.

FIG 8

Model of TraD regulation by metals. When iron (orange circles) availability is high, Fur represses expression of the type IV secretion system coupling protein, TraD, through an unknown intermediate (yellow, depicted here as either an sRNA or a protein). In the phagosome, iron sequestration releases Fur-mediated TraD repression, resulting in increased TraD protein levels. Residual apo-Fur binding may happen transiently at the traD promoter, with small effects on traD transcript levels. High levels of manganese (pink rhombuses) repress traD transcription, but the phagosome pumps manganese away from intracellular gonococci, allowing transcription to proceed. Zinc (purple squares) and copper (blue triangles) contribute to the upregulation of TraD in an undefined manner; here we depict several points at which this regulation could occur: repression of a repressor or protease, direct stabilization of TraD, or enhancement of a TraD chaperone.