Functional characterization of gonococcal toxin-antitoxin systems
Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection, gonorrhoea. Gonorrhoea is a global health problem and one of the most common sexually transmitted infections in the world. Antibiotic resistance in N. gonorrhoeae is a growing problem, and in order to overcome this,...
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| Format: | Thesis (University of Nottingham only) |
| Language: | English |
| Published: |
2024
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| Online Access: | https://eprints.nottingham.ac.uk/78707/ |
| _version_ | 1848801096361836544 |
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| author | Bagabas, Salwa S. |
| author_facet | Bagabas, Salwa S. |
| author_sort | Bagabas, Salwa S. |
| building | Nottingham Research Data Repository |
| collection | Online Access |
| description | Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection, gonorrhoea. Gonorrhoea is a global health problem and one of the most common sexually transmitted infections in the world. Antibiotic resistance in N. gonorrhoeae is a growing problem, and in order to overcome this, it is necessary to develop preventative vaccines or novel treatments. Bacteria have evolved toxin-antitoxin (TA) systems to protect themselves from stressful conditions. TA systems have been linked to various functions including persister cell formation, biofilm formation, phage resistance and stabilisation of genetic elements. Previous work using the Toxin-Antitoxin DataBase (TADB) revealed the presence of three chromosomally-encoded type II TA systems (HicAB, MazEF, VapBC/FitAB) in N. gonorrhoeae strain FA1090. These were hypothesised to play an important role in the lifestyle of this human-restricted pathogen. In addition, exploitation of one or more of gonococcus TA systems could lead to novel treatment interventions.
In this study, further bioinformatics analysis using PubMLST.org examined the prevalence and conservation of the TA systems across isolates. The FitAB, MazEF (and to a lesser extent the HicAB) TA systems were very highly prevalent and conserved across over 5000 N. gonorrhoeae isolates examined. To provide experimental confirmation that the predicted TA systems could influence bacterial growth, DNA fragments corresponding to fitA, fitB and both genes were amplified from N. gonorrhoeae FA1090, ligated into an arabinose-inducible expression plasmid and transformed into E. coli. Qualitative and quantitative growth assays utilizing these strains, along with previously engineered strains harbouring hicAB and mazEF constructs, revealed a lack of FitB or MazF-mediated toxicity in E. coli. In contrast, a HicA-mediated growth arrest effect, which could be abolished by co-expression of HicB, was detected, thus providing confirmation that the N. gonorrhoeae FA1090 genes, NGO1627/1628, encode a functional HicAB TA system. Subsequently, the HicA residues His24, His29, or His40, respectively, were changed to alanine residues by site-directed mutagenesis. Quantitative growth analysis confirmed that the His24 and His29 residues, but not His40 of gonococcal HicA are required for toxicity.
Mutagenesis of the three TA systems was undertaken where both toxin and antitoxin genes were deleted and replaced by a kanamycin cassette in N. gonorrhoeae FA1090. Furthermore, complemented derivatives of FA1090ΔhicAB that expressed IPTG-inducible HicA, HicB or HicAB, respectively, were also generated. The growth characteristics of the complemented strains (FA1090ΔhicAB:hicA, FA1090ΔhicAB:hicB and FA1090ΔhicAB:hicAB) were examined in comparison to the wild-type and mutant strain. The results confirmed that all strains grew as the wild-type, either with induction with IPTG or not, with the exception of FA1090ΔhicAB:hicA induced with IPTG which exhibited growth arrest as judged by OD measurements.
Another study finding was confirmation of hicAB gene expression in wild-type FA1090 during in vitro growth by extraction of total RNA and reverse transcription polymerase chain reaction (RT-PCR) analysis to detect specific mRNA transcripts. The use of specific hicA and hicB primers confirmed expression of both genes, and the combination of a hicA forward and hicB reverse primer provided evidence that both genes are co-transcribed. A final finding came when the total RNA preps were examined using an Agilent Bioanalyser. Notably, a doublet 16S rRNA peak was apparent in total RNA prepared from FA1090ΔhicAB:hicA strain induced with IPTG, but not the uninduced strain, suggesting interaction (or cleavage) of 16S rRNA by HicA, and potentially giving an insight into the mechanism by which gonococcal HicA influences bacterial growth and viability. |
| first_indexed | 2025-11-14T21:02:01Z |
| format | Thesis (University of Nottingham only) |
| id | nottingham-78707 |
| institution | University of Nottingham Malaysia Campus |
| institution_category | Local University |
| language | English |
| last_indexed | 2025-11-14T21:02:01Z |
| publishDate | 2024 |
| recordtype | eprints |
| repository_type | Digital Repository |
| spelling | nottingham-787072024-12-11T04:40:09Z https://eprints.nottingham.ac.uk/78707/ Functional characterization of gonococcal toxin-antitoxin systems Bagabas, Salwa S. Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection, gonorrhoea. Gonorrhoea is a global health problem and one of the most common sexually transmitted infections in the world. Antibiotic resistance in N. gonorrhoeae is a growing problem, and in order to overcome this, it is necessary to develop preventative vaccines or novel treatments. Bacteria have evolved toxin-antitoxin (TA) systems to protect themselves from stressful conditions. TA systems have been linked to various functions including persister cell formation, biofilm formation, phage resistance and stabilisation of genetic elements. Previous work using the Toxin-Antitoxin DataBase (TADB) revealed the presence of three chromosomally-encoded type II TA systems (HicAB, MazEF, VapBC/FitAB) in N. gonorrhoeae strain FA1090. These were hypothesised to play an important role in the lifestyle of this human-restricted pathogen. In addition, exploitation of one or more of gonococcus TA systems could lead to novel treatment interventions. In this study, further bioinformatics analysis using PubMLST.org examined the prevalence and conservation of the TA systems across isolates. The FitAB, MazEF (and to a lesser extent the HicAB) TA systems were very highly prevalent and conserved across over 5000 N. gonorrhoeae isolates examined. To provide experimental confirmation that the predicted TA systems could influence bacterial growth, DNA fragments corresponding to fitA, fitB and both genes were amplified from N. gonorrhoeae FA1090, ligated into an arabinose-inducible expression plasmid and transformed into E. coli. Qualitative and quantitative growth assays utilizing these strains, along with previously engineered strains harbouring hicAB and mazEF constructs, revealed a lack of FitB or MazF-mediated toxicity in E. coli. In contrast, a HicA-mediated growth arrest effect, which could be abolished by co-expression of HicB, was detected, thus providing confirmation that the N. gonorrhoeae FA1090 genes, NGO1627/1628, encode a functional HicAB TA system. Subsequently, the HicA residues His24, His29, or His40, respectively, were changed to alanine residues by site-directed mutagenesis. Quantitative growth analysis confirmed that the His24 and His29 residues, but not His40 of gonococcal HicA are required for toxicity. Mutagenesis of the three TA systems was undertaken where both toxin and antitoxin genes were deleted and replaced by a kanamycin cassette in N. gonorrhoeae FA1090. Furthermore, complemented derivatives of FA1090ΔhicAB that expressed IPTG-inducible HicA, HicB or HicAB, respectively, were also generated. The growth characteristics of the complemented strains (FA1090ΔhicAB:hicA, FA1090ΔhicAB:hicB and FA1090ΔhicAB:hicAB) were examined in comparison to the wild-type and mutant strain. The results confirmed that all strains grew as the wild-type, either with induction with IPTG or not, with the exception of FA1090ΔhicAB:hicA induced with IPTG which exhibited growth arrest as judged by OD measurements. Another study finding was confirmation of hicAB gene expression in wild-type FA1090 during in vitro growth by extraction of total RNA and reverse transcription polymerase chain reaction (RT-PCR) analysis to detect specific mRNA transcripts. The use of specific hicA and hicB primers confirmed expression of both genes, and the combination of a hicA forward and hicB reverse primer provided evidence that both genes are co-transcribed. A final finding came when the total RNA preps were examined using an Agilent Bioanalyser. Notably, a doublet 16S rRNA peak was apparent in total RNA prepared from FA1090ΔhicAB:hicA strain induced with IPTG, but not the uninduced strain, suggesting interaction (or cleavage) of 16S rRNA by HicA, and potentially giving an insight into the mechanism by which gonococcal HicA influences bacterial growth and viability. 2024-12-11 Thesis (University of Nottingham only) NonPeerReviewed application/pdf en cc_by https://eprints.nottingham.ac.uk/78707/1/Bagabas%2C%20Salwa%2C%2020166040%2C%20final%20version.pdf Bagabas, Salwa S. (2024) Functional characterization of gonococcal toxin-antitoxin systems. PhD thesis, University of Nottingham. Toxin-antitoxin systems; Bacterial growth; Mutagenesis; Gonococcal HicA |
| spellingShingle | Toxin-antitoxin systems; Bacterial growth; Mutagenesis; Gonococcal HicA Bagabas, Salwa S. Functional characterization of gonococcal toxin-antitoxin systems |
| title | Functional characterization of gonococcal toxin-antitoxin systems |
| title_full | Functional characterization of gonococcal toxin-antitoxin systems |
| title_fullStr | Functional characterization of gonococcal toxin-antitoxin systems |
| title_full_unstemmed | Functional characterization of gonococcal toxin-antitoxin systems |
| title_short | Functional characterization of gonococcal toxin-antitoxin systems |
| title_sort | functional characterization of gonococcal toxin-antitoxin systems |
| topic | Toxin-antitoxin systems; Bacterial growth; Mutagenesis; Gonococcal HicA |
| url | https://eprints.nottingham.ac.uk/78707/ |