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The Gonococcal Transcriptome during Infection of the Lower Genital Tract in Women.

McClure R, Nudel K, Massari P, Tjaden B, Su X, Rice PA, Genco CA - PLoS ONE (2015)

Bottom Line: Gonorrhea is a highly prevalent disease resulting in significant morbidity worldwide, with an estimated 106 cases reported annually.A total of 140 genes were increased in expression during natural infection compared to growth in CDM, and 165 genes were decreased in expression.Large differences were found in gene expression profiles under each condition, particularly with genes involved in DNA and RNA processing, iron, transposase, pilin and lipoproteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, MA, United States of America; Department of Microbiology, Boston University School of Medicine, Boston, MA, United States of America.

ABSTRACT
Gonorrhea is a highly prevalent disease resulting in significant morbidity worldwide, with an estimated 106 cases reported annually. Neisseria gonorrhoeae, the causative agent of gonorrhea, colonizes and infects the human genital tract and often evades host immune mechanisms until successful antibiotic treatment is used. The alarming increase in antibiotic-resistant strains of N. gonorrhoeae, the often asymptomatic nature of this disease in women and the lack of a vaccine directed at crucial virulence determinants have prompted us to perform transcriptome analysis to understand gonococcal gene expression patterns during natural infection. We sequenced RNA extracted from cervico-vaginal lavage samples collected from women recently exposed to infected male partners and determined the complete N. gonorrhoeae transcriptome during infection of the lower genital tract in women. On average, 3.19% of total RNA isolated from female samples aligned to the N. gonorrhoeae NCCP11945 genome and 1750 gonococcal ORFs (65% of all protein-coding genes) were transcribed. High expression in vivo was observed in genes encoding antimicrobial efflux pumps, iron response, phage production, pilin structure, outer membrane structures and hypothetical proteins. A parallel analysis was performed using the same strains grown in vitro in a chemically defined media (CDM). A total of 140 genes were increased in expression during natural infection compared to growth in CDM, and 165 genes were decreased in expression. Large differences were found in gene expression profiles under each condition, particularly with genes involved in DNA and RNA processing, iron, transposase, pilin and lipoproteins. We specifically interrogated genes encoding DNA binding regulators and iron-scavenging proteins, and identified increased expression of several iron-regulated genes, including tbpAB and fbpAB, during infection in women as compared to growth in vitro, suggesting that during infection of the genital tract in women, the gonococcus is exposed to an iron deplete environment. Collectively, we demonstrate that a large portion of the gonococcal genome is expressed and regulated during mucosal infection including genes involved in regulatory functions and iron scavenging.

No MeSH data available.


Related in: MedlinePlus

Expression of Gonococcal fbp and tbp genes.(A) Expression levels of fbpC, (blue) fbpB (red), lbpB (green), lbpA (purple), hbpA (cyan), hbpB (orange), hbpC (light blue), tbpA (pink), tbpB (light green), hpuB (light purple), and hpuA (teal) genes in RNA isolated from individual cervico-lavage samples compared to RNA isolated from the corresponding infecting N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration). (B) Expression levels of fbpA (blue) and hpbD (red) genes in RNA isolated from individual cervico-lavage samples compared to RNA isolated from the corresponding infecting N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration). These genes were expressed at significantly higher levels and were placed in a separate graph for ease of viewing. (C) Average expression levels of fbpC, fbpB, lbpB, lbpA, hbpA, hbpB, hbpC, tbpA, tbpB, hpuB, and hpuA gene expression in RNA isolated from 4 cervico-lavage samples (red bars) and average expression levels from the 3 isolated N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration) (blue bars). Expression levels (RPKM) are shown on the x-axis and represent averages of all cervico-vaginal lavage samples or all isolated strains grown in CDM. * Indicates a q-value of < 0.0001. (D) Average expression levels of fbpA and hpbD gene expression combined in RNA isolated from 4 cervico-lavage samples (red bars) and average expression levels from the 3 isolated N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration) (blue bars). Expression levels (RPKM) are shown on the x-axis and represent averages of all cervico-vaginal lavage samples or all isolated strains grown in CDM. * Indicates a q-value of < 0.0001.
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pone.0133982.g005: Expression of Gonococcal fbp and tbp genes.(A) Expression levels of fbpC, (blue) fbpB (red), lbpB (green), lbpA (purple), hbpA (cyan), hbpB (orange), hbpC (light blue), tbpA (pink), tbpB (light green), hpuB (light purple), and hpuA (teal) genes in RNA isolated from individual cervico-lavage samples compared to RNA isolated from the corresponding infecting N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration). (B) Expression levels of fbpA (blue) and hpbD (red) genes in RNA isolated from individual cervico-lavage samples compared to RNA isolated from the corresponding infecting N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration). These genes were expressed at significantly higher levels and were placed in a separate graph for ease of viewing. (C) Average expression levels of fbpC, fbpB, lbpB, lbpA, hbpA, hbpB, hbpC, tbpA, tbpB, hpuB, and hpuA gene expression in RNA isolated from 4 cervico-lavage samples (red bars) and average expression levels from the 3 isolated N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration) (blue bars). Expression levels (RPKM) are shown on the x-axis and represent averages of all cervico-vaginal lavage samples or all isolated strains grown in CDM. * Indicates a q-value of < 0.0001. (D) Average expression levels of fbpA and hpbD gene expression combined in RNA isolated from 4 cervico-lavage samples (red bars) and average expression levels from the 3 isolated N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration) (blue bars). Expression levels (RPKM) are shown on the x-axis and represent averages of all cervico-vaginal lavage samples or all isolated strains grown in CDM. * Indicates a q-value of < 0.0001.

Mentions: Previously, we reported that the gonococcal iron and Fur-regulated genes were expressed in urethral and cervico-vaginal lavage samples obtained from men and women with gonococcal infections [16,17]. While these studies confirmed expression of tbp and fbp genes during infection, they did not address differential expression relative to corresponding infecting strains grown in vitro. In the current study we examined expression of tbp and fbp genes in the three culture-positive subject samples and compared this expression to the corresponding strains grown in vitro. Predominantly, increased expression of iron regulated tbpA and tbpB genes was seen in samples from each subject as compared to expression in vitro, although expression in individual samples varied. We also observed increased expression of fbpABC genes in vivo as compared to expression in vitro (Fig 5). Analysis of average expression of tbpAB and fbpAC genes from all cervico-vaginal lavage samples also revealed statistically significant increased levels compared to organisms growth in CDM with ferric nitrate (q < 0.0001; Fig 5). Consistent with these results, the repressor of these genes, Fur, was expressed at lower levels during natural infection compared to growth in CDM with ferric nitrate (Table 3 and S3 Table). Other iron-regulated genes also showed regulation; both hpuA and hpuB were highly expressed in vivo as compared to in vitro, and two out of four heme utilization proteins also showed a similar pattern (although not statistically significant). In contrast, lactoferrin binding proteins showed higher expression in vitro compared to in vivo (Fig 5). Because in vitro growth was performed under iron-replete conditions (when iron scavenging genes exhibit low expression), the high in vivo expression of tbp, fbp, and hpu genes, as well as NrrF, combined with the low expression of Fur suggests that gonococci encounter an iron-deplete environment in the female genital tract.


The Gonococcal Transcriptome during Infection of the Lower Genital Tract in Women.

McClure R, Nudel K, Massari P, Tjaden B, Su X, Rice PA, Genco CA - PLoS ONE (2015)

Expression of Gonococcal fbp and tbp genes.(A) Expression levels of fbpC, (blue) fbpB (red), lbpB (green), lbpA (purple), hbpA (cyan), hbpB (orange), hbpC (light blue), tbpA (pink), tbpB (light green), hpuB (light purple), and hpuA (teal) genes in RNA isolated from individual cervico-lavage samples compared to RNA isolated from the corresponding infecting N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration). (B) Expression levels of fbpA (blue) and hpbD (red) genes in RNA isolated from individual cervico-lavage samples compared to RNA isolated from the corresponding infecting N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration). These genes were expressed at significantly higher levels and were placed in a separate graph for ease of viewing. (C) Average expression levels of fbpC, fbpB, lbpB, lbpA, hbpA, hbpB, hbpC, tbpA, tbpB, hpuB, and hpuA gene expression in RNA isolated from 4 cervico-lavage samples (red bars) and average expression levels from the 3 isolated N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration) (blue bars). Expression levels (RPKM) are shown on the x-axis and represent averages of all cervico-vaginal lavage samples or all isolated strains grown in CDM. * Indicates a q-value of < 0.0001. (D) Average expression levels of fbpA and hpbD gene expression combined in RNA isolated from 4 cervico-lavage samples (red bars) and average expression levels from the 3 isolated N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration) (blue bars). Expression levels (RPKM) are shown on the x-axis and represent averages of all cervico-vaginal lavage samples or all isolated strains grown in CDM. * Indicates a q-value of < 0.0001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4526530&req=5

pone.0133982.g005: Expression of Gonococcal fbp and tbp genes.(A) Expression levels of fbpC, (blue) fbpB (red), lbpB (green), lbpA (purple), hbpA (cyan), hbpB (orange), hbpC (light blue), tbpA (pink), tbpB (light green), hpuB (light purple), and hpuA (teal) genes in RNA isolated from individual cervico-lavage samples compared to RNA isolated from the corresponding infecting N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration). (B) Expression levels of fbpA (blue) and hpbD (red) genes in RNA isolated from individual cervico-lavage samples compared to RNA isolated from the corresponding infecting N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration). These genes were expressed at significantly higher levels and were placed in a separate graph for ease of viewing. (C) Average expression levels of fbpC, fbpB, lbpB, lbpA, hbpA, hbpB, hbpC, tbpA, tbpB, hpuB, and hpuA gene expression in RNA isolated from 4 cervico-lavage samples (red bars) and average expression levels from the 3 isolated N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration) (blue bars). Expression levels (RPKM) are shown on the x-axis and represent averages of all cervico-vaginal lavage samples or all isolated strains grown in CDM. * Indicates a q-value of < 0.0001. (D) Average expression levels of fbpA and hpbD gene expression combined in RNA isolated from 4 cervico-lavage samples (red bars) and average expression levels from the 3 isolated N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration) (blue bars). Expression levels (RPKM) are shown on the x-axis and represent averages of all cervico-vaginal lavage samples or all isolated strains grown in CDM. * Indicates a q-value of < 0.0001.
Mentions: Previously, we reported that the gonococcal iron and Fur-regulated genes were expressed in urethral and cervico-vaginal lavage samples obtained from men and women with gonococcal infections [16,17]. While these studies confirmed expression of tbp and fbp genes during infection, they did not address differential expression relative to corresponding infecting strains grown in vitro. In the current study we examined expression of tbp and fbp genes in the three culture-positive subject samples and compared this expression to the corresponding strains grown in vitro. Predominantly, increased expression of iron regulated tbpA and tbpB genes was seen in samples from each subject as compared to expression in vitro, although expression in individual samples varied. We also observed increased expression of fbpABC genes in vivo as compared to expression in vitro (Fig 5). Analysis of average expression of tbpAB and fbpAC genes from all cervico-vaginal lavage samples also revealed statistically significant increased levels compared to organisms growth in CDM with ferric nitrate (q < 0.0001; Fig 5). Consistent with these results, the repressor of these genes, Fur, was expressed at lower levels during natural infection compared to growth in CDM with ferric nitrate (Table 3 and S3 Table). Other iron-regulated genes also showed regulation; both hpuA and hpuB were highly expressed in vivo as compared to in vitro, and two out of four heme utilization proteins also showed a similar pattern (although not statistically significant). In contrast, lactoferrin binding proteins showed higher expression in vitro compared to in vivo (Fig 5). Because in vitro growth was performed under iron-replete conditions (when iron scavenging genes exhibit low expression), the high in vivo expression of tbp, fbp, and hpu genes, as well as NrrF, combined with the low expression of Fur suggests that gonococci encounter an iron-deplete environment in the female genital tract.

Bottom Line: Gonorrhea is a highly prevalent disease resulting in significant morbidity worldwide, with an estimated 106 cases reported annually.A total of 140 genes were increased in expression during natural infection compared to growth in CDM, and 165 genes were decreased in expression.Large differences were found in gene expression profiles under each condition, particularly with genes involved in DNA and RNA processing, iron, transposase, pilin and lipoproteins.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Section of Infectious Diseases, Boston University School of Medicine, Boston, MA, United States of America; Department of Microbiology, Boston University School of Medicine, Boston, MA, United States of America.

ABSTRACT
Gonorrhea is a highly prevalent disease resulting in significant morbidity worldwide, with an estimated 106 cases reported annually. Neisseria gonorrhoeae, the causative agent of gonorrhea, colonizes and infects the human genital tract and often evades host immune mechanisms until successful antibiotic treatment is used. The alarming increase in antibiotic-resistant strains of N. gonorrhoeae, the often asymptomatic nature of this disease in women and the lack of a vaccine directed at crucial virulence determinants have prompted us to perform transcriptome analysis to understand gonococcal gene expression patterns during natural infection. We sequenced RNA extracted from cervico-vaginal lavage samples collected from women recently exposed to infected male partners and determined the complete N. gonorrhoeae transcriptome during infection of the lower genital tract in women. On average, 3.19% of total RNA isolated from female samples aligned to the N. gonorrhoeae NCCP11945 genome and 1750 gonococcal ORFs (65% of all protein-coding genes) were transcribed. High expression in vivo was observed in genes encoding antimicrobial efflux pumps, iron response, phage production, pilin structure, outer membrane structures and hypothetical proteins. A parallel analysis was performed using the same strains grown in vitro in a chemically defined media (CDM). A total of 140 genes were increased in expression during natural infection compared to growth in CDM, and 165 genes were decreased in expression. Large differences were found in gene expression profiles under each condition, particularly with genes involved in DNA and RNA processing, iron, transposase, pilin and lipoproteins. We specifically interrogated genes encoding DNA binding regulators and iron-scavenging proteins, and identified increased expression of several iron-regulated genes, including tbpAB and fbpAB, during infection in women as compared to growth in vitro, suggesting that during infection of the genital tract in women, the gonococcus is exposed to an iron deplete environment. Collectively, we demonstrate that a large portion of the gonococcal genome is expressed and regulated during mucosal infection including genes involved in regulatory functions and iron scavenging.

No MeSH data available.


Related in: MedlinePlus