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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

Composite comparisons between transcriptomes of N. gonorrhoeae in natural infection vs. growth in CDM.(A) Log values of expression (RPKM) levels are indicated. Expression levels in all 4 cervico-lavage samples were averaged and compared to the average expression levels of the three isolated N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration). Genes in blue had q-values < 0.05. (B) Functional enrichment of genes showing changes in expression when examining gonococcal transcriptomes during natural infection compared to growth in CDM supplemented with ferric nitrate (100 μM final concentration). Availability of several subject samples permitted statistical analysis and genes were considered to be differentially expressed between two samples if the q value was < 0.05 and the fold change > 2. Blue bars indicate functional enrichment of genes showing increased expression during natural infection in vivo compared to growth in CDM. Red bars indicate functional enrichment of genes showing decreased expression during natural infection in vivo compared to growth in CDM.
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pone.0133982.g004: Composite comparisons between transcriptomes of N. gonorrhoeae in natural infection vs. growth in CDM.(A) Log values of expression (RPKM) levels are indicated. Expression levels in all 4 cervico-lavage samples were averaged and compared to the average expression levels of the three isolated N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration). Genes in blue had q-values < 0.05. (B) Functional enrichment of genes showing changes in expression when examining gonococcal transcriptomes during natural infection compared to growth in CDM supplemented with ferric nitrate (100 μM final concentration). Availability of several subject samples permitted statistical analysis and genes were considered to be differentially expressed between two samples if the q value was < 0.05 and the fold change > 2. Blue bars indicate functional enrichment of genes showing increased expression during natural infection in vivo compared to growth in CDM. Red bars indicate functional enrichment of genes showing decreased expression during natural infection in vivo compared to growth in CDM.

Mentions: Several gonococcal genes were regulated in a similar fashion in each subject, suggesting that a common response of the gonococcus may be dependent on intrinsic features of the human female genital tract. Thus, we next examined the combined results from the 4 cervico-lavage samples to define an ‘average’ expression of each gonococcal gene during infection (S3 Table). As a comparison, we also combined the data resulting from the in vitro growth of the 3 available infecting strains, and lastly, used these averages to compare in vivo and in vitro gene expression. We identified 305 genes with statistically significant changes in expression, defined as a q-value of <0.05 and > than a 2-fold change in expression. A total of 140 genes showed increased expression and 165 decreased expression during in vivo vs. in vitro growth (S4 Table; and Fig 4A). Functional enrichment analysis was performed to define how the gonococcus alters gene expression during infection (Fig 4B and 4C). We observed that genes involved in membrane composition/function, iron scavenging, transposases and tRNAs were strongly enriched among genes with increased expression in vivo compared to growth in CDM. In contrast, several housekeeping genes were enriched among genes showing decreased expression, suggesting changes in growth and replication rates of N. gonorrhoeae during infection compared to growth in CDM (Fig 4B and 4C). Other categories such as pilin and lipoprotein genes also were enriched among genes showing decreased expression despite the fact that when examining subjects individually these genes were sometimes found to be enriched among genes showing increased expression. Interestingly, 20 genes encoding phage-associated proteins showed statistically significant regulation, with seven of these genes showing increased expression during infection as compared to growth in CDM (S4 Table). Phage-associated genes were also slightly enriched among genes showing decreased expression in vivo. The expression levels of all genes from cervico-vaginal lavage samples compared to strains grown in CDM are shown in S3 Table.


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)

Composite comparisons between transcriptomes of N. gonorrhoeae in natural infection vs. growth in CDM.(A) Log values of expression (RPKM) levels are indicated. Expression levels in all 4 cervico-lavage samples were averaged and compared to the average expression levels of the three isolated N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration). Genes in blue had q-values < 0.05. (B) Functional enrichment of genes showing changes in expression when examining gonococcal transcriptomes during natural infection compared to growth in CDM supplemented with ferric nitrate (100 μM final concentration). Availability of several subject samples permitted statistical analysis and genes were considered to be differentially expressed between two samples if the q value was < 0.05 and the fold change > 2. Blue bars indicate functional enrichment of genes showing increased expression during natural infection in vivo compared to growth in CDM. Red bars indicate functional enrichment of genes showing decreased expression during natural infection in vivo compared to growth in CDM.
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pone.0133982.g004: Composite comparisons between transcriptomes of N. gonorrhoeae in natural infection vs. growth in CDM.(A) Log values of expression (RPKM) levels are indicated. Expression levels in all 4 cervico-lavage samples were averaged and compared to the average expression levels of the three isolated N. gonorrhoeae strains grown in vitro in CDM supplemented with ferric nitrate (100 μM final concentration). Genes in blue had q-values < 0.05. (B) Functional enrichment of genes showing changes in expression when examining gonococcal transcriptomes during natural infection compared to growth in CDM supplemented with ferric nitrate (100 μM final concentration). Availability of several subject samples permitted statistical analysis and genes were considered to be differentially expressed between two samples if the q value was < 0.05 and the fold change > 2. Blue bars indicate functional enrichment of genes showing increased expression during natural infection in vivo compared to growth in CDM. Red bars indicate functional enrichment of genes showing decreased expression during natural infection in vivo compared to growth in CDM.
Mentions: Several gonococcal genes were regulated in a similar fashion in each subject, suggesting that a common response of the gonococcus may be dependent on intrinsic features of the human female genital tract. Thus, we next examined the combined results from the 4 cervico-lavage samples to define an ‘average’ expression of each gonococcal gene during infection (S3 Table). As a comparison, we also combined the data resulting from the in vitro growth of the 3 available infecting strains, and lastly, used these averages to compare in vivo and in vitro gene expression. We identified 305 genes with statistically significant changes in expression, defined as a q-value of <0.05 and > than a 2-fold change in expression. A total of 140 genes showed increased expression and 165 decreased expression during in vivo vs. in vitro growth (S4 Table; and Fig 4A). Functional enrichment analysis was performed to define how the gonococcus alters gene expression during infection (Fig 4B and 4C). We observed that genes involved in membrane composition/function, iron scavenging, transposases and tRNAs were strongly enriched among genes with increased expression in vivo compared to growth in CDM. In contrast, several housekeeping genes were enriched among genes showing decreased expression, suggesting changes in growth and replication rates of N. gonorrhoeae during infection compared to growth in CDM (Fig 4B and 4C). Other categories such as pilin and lipoprotein genes also were enriched among genes showing decreased expression despite the fact that when examining subjects individually these genes were sometimes found to be enriched among genes showing increased expression. Interestingly, 20 genes encoding phage-associated proteins showed statistically significant regulation, with seven of these genes showing increased expression during infection as compared to growth in CDM (S4 Table). Phage-associated genes were also slightly enriched among genes showing decreased expression in vivo. The expression levels of all genes from cervico-vaginal lavage samples compared to strains grown in CDM are shown in S3 Table.

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