Dual species transcriptomics reveals conserved metabolic and immunologic processes in interactions between human neutrophils and Neisseria gonorrhoeae

Abstract

Neisseria gonorrhoeae (the gonococcus, Gc) causes the sexually transmitted infection gonorrhea. Gc is a prominent threat to human health by causing severe lifelong sequelae, including infertility and chronic pelvic pain, which is amplified by the emergence of "superbug" strains resistant to all current antibiotics. Gc is highly adapted to colonize human mucosal surfaces, where it survives despite initiating a robust inflammatory response and influx of polymorphonuclear leukocytes (PMNs, neutrophils) that typically clear bacteria. Here, dual-species RNA-sequencing was used to define Gc and PMN transcriptional profiles alone and after infection. Core host and bacterial responses were assessed for two strains of Gc and three human donors' PMNs. Comparative analysis of Gc transcripts revealed overlap between Gc responses to PMNs, iron, and hydrogen peroxide; 98 transcripts were differentially expressed across both Gc strains in response to PMN co-culture, including iron-responsive and oxidative stress response genes. We experimentally determined that the iron-dependent TbpB is suppressed by PMN co-culture, and iron-limited Gc have a survival advantage when cultured with PMNs. Analysis of PMN transcripts modulated by Gc infection revealed differential expression of genes driving cell adhesion, migration, inflammatory responses, and inflammation resolution pathways. Production of pro-inflammatory cytokines, including IL1B and IL8, the adhesion factor ICAM1, and prostaglandin PGE2 were induced in PMNs in response to Gc. Together, this study represents a comprehensive and experimentally validated dual-species transcriptomic analysis of two isolates of Gc and primary human PMNs that gives insight into how this bacterium survives innate immune onslaught to cause disease.

Fig 1. Principal Component Analyses (PCA) of Gc and PMN transcriptomes.

A) PCA of Gc transcriptomes based on 1582 core genes. PC1 separates the 130 and H041 strains, and PC2 separates Gc infected PMN samples from Gc in culture alone. B) PCA of PMN transcriptomes. PC1 separates infected PMN samples from uninfected PMNs, and PC2 separates the different PMN donors with D55 trending towards the top, D60 primarily in the middle, and D6 towards the bottom.

Fig 2. Venn diagram of differentially expressed (DE) Gc genes (Dataset S3) and Z-scored heatmaps of selected Gc regulons showing expression patterns for DE genes shared by strains FA1090 Opaless 130 and H041.

Colors indicate Z-score normalized expression values. The 98 shared DE genes (i.e. DE in 130 and H041, grouped using a one-to-one orthology) classified into major Gc regulons and heatmaps are presented for both species. Heatmaps of strain FA1090 Opaless 130 and H041 specific DE genes are shown in S6 and S7 Figs.

Fig 3. Comparison of Gc differentially expressed (DE) genes identified in this study to other transcriptomics datasets (Dataset S3).

Total lengths of bars represent the number of genes identified in this study. Light grey bars represent genes that were only identified in the Gc response to PMNs (this study) and the indicated dataset. Dark grey bars represent genes that were identified in multiple datasets. The black bar represents genes that were not identified in other studies.

Fig 4. Gc exposed to PMNs exhibit production of an iron-regulated metal acquisition protein and increased survival in iron-deplete conditions.

A) Gc strain FA1090 Opaless 130 was inoculated onto IL8-treated, adherent human PMNs or into media without PMNs, and incubated for 1 h. PMNs were treated with saponin, and Gc were collected and processed for Western blotting. Gc lysates were separated by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and stained with rabbit anti-TbpB polyclonal antisera. The intensity of TbpB is reported relative to the loading control Zwf, which was recognized with rabbit anti-Zwf antiserum. A representative blot from a single experiment is shown. B) Quantification of TbpB/Zwf ratio from 3 independent experiments, using PMNs from 3 unrelated individuals. Error bars indicate SEM. Significance was determined by one-way ANOVA with Holm–Sidak correction for multiple comparisons, * indicates p < 0.05, ** p < 0.01 and *** p < 0.001. C and D) Strain FA1090 Opaless 130 was iron starved in media containing 10 μM deferoxamine (DFO) ± 10 μM Fe(NO₃)₃ for 2.5 hours then was inoculated onto IL8-treated, adherent human PMNs or into media without PMNs. Percent Gc survival was calculated by enumerating colony-forming units (CFU) from PMN lysates or media control at 3h post-infection as the percent of CFU for that strain at 0 min. Shapes are replicates from different donors’ PMNs. n = 4 independent experiments. C) Significance was determined by two-way ANOVA with Holm–Sidak correction for multiple comparisons. ** indicates p < 0.005. ns indicates not significant. D) Data is reported as the change in percent survival for Gc exposed to PMNs compared to Gc in media without PMNs from panel C. Significance was determined by one-way ANOVA with Holm–Sidak correction for multiple comparisons. * indicates p < 0.05.

Fig 5. Ingenuity Pathway Analysis (IPA)-enriched Disease and Functions.

Utilizing neutrophil cell specific parameters, resulting functions of genes DE in Gc-PMN co-cultures vs PMN mono-cultures at 1h are shown. Blue: co-culture with FA1090 Opaless 130; red: H041. The IPA determined Z-scores of Disease and Function enrichment by DE genes are shown (>0 reveals activation, while <0 reveals inhibition).

Fig 6. Venn diagram of differentially expressed (DE) PMN genes (Dataset S4) and a Z-scored heatmap of the top 50 PMN genes with the highest expression among DE genes, arranged by infection condition.

Highest expression was based on average VST counts across all samples. Boxes on the heatmap correspond to the different Venn intersections to which they belong. Bold and italicized genes were examined in subsequent experiments.

Fig 7. PMNs increase production of pro-inflammatory cytokines and adhesion molecules upon Gc infection.

A-B) Scatter plot of Log₂(Fold Change) (L2FC) of all transcripts vs the -Log₁₀(adj pval) for PMNs infected with (A) FA1090 Opaless 130 or (B) H041. Targets that were selected for additional analysis are labeled. C) Supernatants were collected from uninfected and FA1090 Opaless 130-infected adherent PMNs after 4h and quantified by multiplex cytokine analysis (“Protein”). Selected targets identified from PMN DE genes are displayed. The Log₂(Fold Change) (L2FC) in detected protein for 130 infected PMNs at 4h vs uninfected PMNs at 4h is reported. The corresponding L2FC transcript levels identified by RNA-seq for 130+PMN_1h vs PMN_1h is also indicated (“Transcript”). D and E) Supernatants were collected from PMNs left uninfected or exposed to FA1090 Opaless 130 or H041 at the indicated timepoints. IL8 (D) and PGE2 (E) concentrations were measured by ELISA. Bars represent the mean ± SEM. n = 3 independent experiments. F and G) PMNs were collected and assessed by flow cytometry at the indicated timepoints. ICAM1 (F) and ITGAX (CD11c) (G) surface expression was assessed by Median Fluorescent Intensity (MFI) of PE-ICAM1 and APC-CD11c on CD11b+ /CD14- /CD16+ /CD49- cells (i.e., neutrophils). Bars represent the mean MFI ± SEM. n = 4 independent experiments. D-G) Shapes are data points from different donors’ PMNs. Significance was determined by two-way ANOVA with Holm–Sidak correction for multiple comparisons, *indicates p < 0.05.