Investigating the host-binding properties of Neisseria gonorrhoeae and newer therapeutics against this pathogen.

Abstract

Background With 80 million annual cases, gonorrhoea is a common sexually transmitted disease caused by Neisseria gonorrhoeae (N. gonorrhoeae). The current study investigated a reverse vaccinology approach (determining the CEACAM binding patterns of N. gonorrhoeae) and alternative therapeutics (nanoemulsion and plant extracts) to combat this disease as well as evaluated cost-effective assays that target virulence proteins such as the opa gene (in-house opa-based real-time PCR assay). Methodology This was a retrospective laboratory-based study using stored bacterial isolates and primary genital swabs collected from larger studies. For the binding assays, we investigated the host-pathogen associations by determining the CEACAM binding patterns of N. gonorrhoeae isolated from symptomatic (G180) versus asymptomatic (G136) pregnant women. To identify the complete repertoire of G136 and G180 Opa proteins, DNA was isolated and a PCR with primers targeting the conserved regions of opa genes was performed. Thereafter, opa amplicons were ligated into pCR Blunt II TOPO, and single clones from this opa amplicon library were sequenced to identify the respective unique opa genes. The Opa proteins identified in this study were subjected to test expression and Western blotting was performed to verify these Opa proteins with a monoclonal antibody against neisserial Opa proteins. Thereafter, binding assays were performed to analyse the host-binding properties of N. gonorrhoeae. For the objective which involved the development and evaluation of the performance of an in-house opa-based real-time PCR assay, three primer sets targeting the opa gene of N. gonorrhoeae were designed and evaluated against published opa gene primers (comparator assay). The in-house and published primers were tested against laboratory and clinical isolates of N. gonorrhoeae as well as non-gonococcal Neisseria control isolates. The antimicrobial properties of Ocimum tenuiflorum (“holy basil”), Moringa oleifera, and Azadirachta indica plants against N. gonorrhoeae were then explored. The plants were collected from the Botanical Gardens, Durban, South Africa. Upon collection and transport to the laboratory, the plants were left to dry naturally from sunlight for about 4 to 5 days and then used in the preparation of the aqueous extracts. The nanoemulsions were produced according to published methods. Different concentrations of the extracts (1000 μM, 100 μM, 10 μM, and 1 μM) were tested against N. gonorrhoeae isolates using the disk diffusion method. The extracts were also tested for their toxicity against human erythrocytes. Results In this study, were able to identify nine distinct Opa proteins from G136 and ten distinct Opa proteins from G180. For isolate G136 (asymptomatic patient), 66.7% of the Opa proteins bound to CEACAM3, 55.6% bound to CEACAM1, and 88.9% bound to CEACAM5. For isolate G180 (symptomatic patient), 30% of the Opa proteins bound to CEACAM3, 80% bound to CEACAM1, and 70% bound to CEACAM5. In this study, it was shown that the N. gonorrhoeae Opa proteins from a symptomatic patient bound at a higher frequency to CEACAM1 and 5 which causes the pathogen to invade the host cell and cause infections. However, N. gonorrhoeae Opa proteins from the asymptomatic patient bound at a higher frequency to CEACAM3. According to the real-time PCR evaluation assays, the opa 1 primer performed better than opa 2, opa 3, and the comparator opa primer sets. The opa 1 assay produced positive amplification for the five WHO and the six N. gonorrhoeae clinical isolates, whereas the comparator assay amplified 90.9% of the samples. For the endocervical DNA samples, 82.8% were amplified with the opa 1 assay, while the comparator assay had only amplified 27.6% of the samples. For the vaginal DNA samples, the opa 1 assay amplified 95.0% of the samples, whereas the comparator assay amplified 25.0% of the samples. All eleven (100%) urine DNA samples were amplified with the opa 1 assay, in contrast to 36.4% with the comparator assay. The opa 1 assay showed no cross-reactivity with non-gonococcal isolates, whilst cross-reactivity was observed with the comparator assay. The opa 1 assay also had a higher limit of detection when compared to the other assays. In this study, all six gonococcal isolates exhibited clear zones of inhibition when exposed to the 1000μM concentration of all three of the nanoemulsion-based plant extracts investigated. Conversely, no zones of inhibition were detected at extract concentrations of 100μM, 10μM, and 1μM for five of the isolates. Isolate G176, however, displayed zones of inhibition at both 1000μM and 100μM concentrations when exposed to the nanoemulsion plant-based extracts derived from Ocimum tenuiflorum. Additionally, the WHO control strains included in the assay exhibited zones of inhibition at the 1000μM concentration. Notably, the WHO Y strain displayed zones of inhibition at both 1000μM and 100μM concentrations when exposed to nanoemulsion-based extracts from Ocimum tenuiflorum and Azadirachta indica. Importantly, the analysis revealed 0% haemolytic activity against human erythrocytes, indicating the non-toxic nature of the extracts. Conclusion In this study, it was demonstrated that N. gonorrhoeae Opa proteins obtained from symptomatic patients exhibited a higher affinity for CEACAM1 and CEACAM5, facilitating the pathogen's invasion of host cells and subsequent infection. Conversely, Opa proteins from asymptomatic patients displayed a higher binding frequency to CEACAM3. These Opa-CEACAM interactions are believed to potentially restrict the dissemination of gonococci by promoting granulocyte-mediated opsonin-independent phagocytosis. Investigating interactions represents a crucial step forward in the quest for vaccine development against this pathogen. This study contributes valuable insights to the expanding body of knowledge concerning the host-receptor binding profiles of this pathogen. This study demonstrated that the opa 1 primer was the superior primer when compared to opa 2, opa 3, and the comparator opa primers from Verma et al., (2012) study. Therefore, this in-house opa 1 assay can be potentially used and further evaluated for its use as a diagnostic assay. This study also demonstrated that the combination of nanoemulsions with plant extracts (specifically, Ocimum tenuiflorum, Moringa oleifera, and Azadirachta indica) presents a promising alternative to traditional antibiotics for combatting N.gonorrhoeae infections. This strategy capitalizes on the antimicrobial attributes of naturalcompounds and utilizes nanoemulsion technology to optimize their delivery and efficacy. Nevertheless, continuous research and development efforts must be undertaken to validate and enhance the viability of this approach for clinical application.