Department of Biochemistry and Microbiology

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Open Access
A metagenomic snapshot of selected wastewater treatment plants in Vhembe Region, Limpopo, South Africa: Investigating the resistome
(2024-09-06) Jacobs, Damien; Potgieter, N.; Traore, A. N.
Background: Water is crucial for human life. Rural communities often rely on natural water sources which may become polluted by wastewater due to various activities such as domestic use and agriculture. Antibiotic resistance genes (ARGs) may be transferred from wastewater to the environment and pose a global challenge they affect both human and animal-related sectors. Studying antibiotic resistance in wastewater treatment plants within Vhembe offers a representation of antibiotic resistance genes from entire communities. Knowledge of antibiotic resistance circulating in Vhembe has been sparsely studied. Metagenomics approaches allow for a broad overview of the resistome and the bacterial communities within environmental samples. Aim: To perform wastewater surveillance of antibiotic resistance genes and associated bacteria within Vhembe, Limpopo, South Africa, using a metagenomics approach. Method: A total of 32 sample duplicates were collected from the influents (n=18) and the effluents (n = 14) from nine wastewater treatment plants (WWTPs) around the Vhembe region, Limpopo, South Africa. One hundred milliliter was filtered using sterile cotton gauze and Wattman filter paper to remove debris and membrane filtered through 0.22um membrane filters to capture the bacteria within each sample. DNA was extracted directly from the 0.22 μm filters using a DNA miniprep kit. DNA was quantified using a spectrophotometer. Shotgun 18 metagenomic sequencing was performed on DNA extracts. Open-source bioinformatics pipelines were used to process and analyze raw sequence data, uncovering information of the bacterial community composition and associated ARGs in wastewater. Results: Site observations reveal animal and human activities within and near the sites. ARG analysis revealed an overall number 0f 220 ARGs detected across the WWTPs. Thirty-six genes were common to influent samples and 16 in within effluent samples, encoding predominantly against macrolides, sulfonamides and tetracyclines, beta-lactamases, and aminoglycosides. Some unique ARGS were detected at sites near South African borders. Bacterial Diversity showed the predominance of some genera, such as Arcobacter, Aeromonas, Pseudomonas and Acinetobacter. Pathogens were predominantly enteric and pulmonary, with some being linked to animals in past studies. A notable increase in some members of Mycobactericeae, among other bacteria, was noted in effluents.
Open Access
Antibacterial activity of the crude extract and fractions of spirostachys africana against multi-drug resistant bacteria
(2019-05) Ajmal, Antoinette Alliya; Traore, A. N.; Potgieter, N.; Tshisikhawe, P.
Background: The high on-going incidences of infectious diseases, specifically those caused by multi-drug resistant bacteria in the last decade has made it a necessity to investigate a variety of antimicrobial drug sources, such as plants. Medicinal plants have played a significant role in drug discovery for western pharmaceuticals recently and have also been used successfully by traditional healers and herbalists to treat various infectious diseases for centuries. Currently, a few medicinal plants are commercialized, reason being most medicinal plants phytochemicals have not been studied yet, although they have been traditionally used by healers. Due to the constant development of multi-drug resistance of bacteria to antibiotics, S. africana extracts can provide an opportunity to finding new antibacterial compounds that can be used as the foundation for formulating new antimicrobial drugs. Objectives: The aim of this study was to screen antibacterial activity of the crude extract and fractions of S. africana against multi-drug resistant bacteria and to also evaluate other biological properties. Methods: Preliminary screening of phytochemical constituents of S. africana and fractions was done using standard qualitative and quantitative methods. Antibacterial activity of the extracts was evaluated using the agar well diffusion method and the microdilution assay against MDR bacterial strains. Antioxidant activities of the MCE and its fractions were measured by DPPH and reducing power assays, and the toxicity of the MCE and its fractions was tested on Vero cells using Cell-based high content screening assay. Results: Phytochemical analysis of the MCE and fractions obtained in this study showed the presence of phenolics, flavonoids, alkaloids, steroids, saponins, cardiac glycosides and terpenoids in most of S. african’s test samples. Fraction F1 and F2 both lacked alkaloids and saponins. The micro-plate dilution assay demonstrated that the MCE and all its fractions can inhibit the growth of all selected MDR bacterial strains tested against at different concentrations (0.1mg/ml to >12.5mg/ml), wherein the lowest MIC averages were obtained from fractions F3 and F6, with 0.59 mg/ml and 0.71 mg/ml MIC averages respectively. Contrary to the micro-plate dilution assay, the well diffusion assay demonstrated that MCE and all its fractions were not active against all the selected MDR bacterial strains tested against, as no inhibition was shown against the growth of K. pneumonia by any of S. african’s test samples. For DPPH assay, the IC50 of S. african’s test samples ranged between 0.01 ±0.34 mg/ml to 0.62 ± 0.05 mg/ml, whiles for the reducing power assay, EC50 measured ranged between 0.61 ± 0.01 mg/ml and 11.30 ± 0.04 mg/ml. The MCE and fraction F2 exhibited the highest toxicity to Vero cells. Conclusion: The MCE and fractions of the plant S. africana have antibacterial activity against MDR bacterial strains, beneficial biological properties and contains potential antibacterial compounds that may be valuable in the discovery of new potential drugs for treatment of infectious diseases
Open Access
Antibiogram and molecular characterization of staphylococcus aureus isolated from gym equipment in public fitness centres in Thohoyandou, Vhembe District, Limpopo Province
(2016-05) Mashau, Thendo Precious; Musie, E. M.; Masebe, T.
See the attached abstract below
Open Access
Antimicrobial, cytotoxic and prelimenary phytochemical analysis of four medicinal plants and their formulation
(2018-05-18) Mboweni, Hlayisa Fredah; Samie, A.; Tshikalange, T. E.
BACKGROUND: Medicinal plants form an important part of the Southern African cultural heritage. Indigenous populations, for example the Vha-Venda people, tend to use medicinal plants in formulations rather than western medicines for health and survival. In order to certify and give scientific credibility to the use of medicinal plants formulations used by Vha-Venda people for the treatment of diseases, several assays were carried out. The present study was aimed at assessing phytochemical content, antimicrobial, antioxidant and cytotoxic activities of four indigenous Venda medicinal plants in a formulation and compare their activity with each plant used individually. METHODS: Peltophorum africanum (roots), Pterocarpus angolensis (bark), Terminalia sericea (roots) and Ximenia caffra (roots) were collected from the Thohoyandou area. The collected plant parts were extracted with methanol and water respectively. Individual plant extracts and Five designed formulations were tested for their antimicrobial activity against Staphylococcus aureus ATCC 25923 (Methicillin Resistant), Staphylococcus aureus ATCC 33591(Methicillin Susceptible), beta lactamase producing Klebsiella pneumonia (ATCC 700603) and extended spectrum beta lactamase producing E. coli (ATCC 35218), four clinical isolates of Candida spp and Cryptococcus neoformans using the Broth dilution method. Minimum bactericidal concentration (MBC) of the extracts was determined by culturing the contents of minimum inhibitory concentration (MIC) on nutrient agar. Similarly, minimum fungicidal concentration (MFC) was also determined by culturing contents of MIC in sabouraud dextrose agar (SDA). Extracts were further assessed for their total phenolic content, total flavonoid content and Qualitative phytochemical analysis. The antioxidant ability of the plants extracts and formulations to scavenge free radical DPPH was also determined. The plant formulations were assessed for their anti-HIV activity using the reverse transcriptase colorimetric assay kit. Cytotoxicity against human lymphatic endothelial cells (HLEC) was determined using MTT assay. RESULTS: Methanolic and aqueous extracts of T. sericea exhibited the best antifungal and antibacterial activities whilst P. angolensis and X. caffra showed poor activities. Methanolic plant formulations showed good activities compared to aqueous formulations. However, Fractional Inhibition Concentration Index showed that there was 1 synergistic interaction, 25 additive interactions and 14 antagonistic interactions between the plant extracts. The methanolic formulation 3 showed the best overall phenolic content at 11.85±0.109 mgGAE/g whilst aqueous X. caffra extract showed the least content at 4.546±0.104 mgGAE/g. Higher total flavonoid contents were seen in methanolic formulation 4 at 2.75±0.02 mgQE/g. Qualitative phytochemical analysis revealed the presence of flavonoids, phenolics, terpenoids, tannins, saponins and steroids in 80% of the tested plant extracts and formulations. All plant extracts and formulations exhibited good antioxidant activity against DPPH. The methanolic formulation showed the best antioxidant activity with IC50 of 0.094 ± 0.33μg/ml. For anti- HIV inhibition, all formulations at 200μg/ml exhibited higher percentage of HIV-1 reverse transcriptase inhibition with methanolic mixture 3 being the best overall at 97.5% activity whilst aqueous mixture5 was the least active with 63.03% inhibition activity. Moreover, the best anti-HIV activity at 100μg/ml was exhibited by methanolic mixture 3 at 71% inhibition. Furthermore, aqueous X. caffra, mixture 2 inhibited 26% and 51% at 12.5mg/ml and 3.125mg/ml respectively. Peltophorum africanum and mixture 5 inhibited 34%, 54% and 43% at 3.125mg/ml, 6.25mg/ml and 12.5 mg/ml respectively of Human Lymphatic Endothelial cells growth. CONCLUSIONS: The results from the study indicated that most of the commonly used traditional medicinal Plants in the Venda region when mixed together have merit for use in traditional medical practice as they have shown good antimicrobial activities, good antioxidant xviii activities, good phytochemical activities and good cell proliferation activity. However some formulations showed antagonistic interaction against bacteria. Some Individual medicinal plants showed toxicity at higher concentrations against immune cells. Whereas formulations promoted cell proliferation, therefore, the use of such individual plants in the treatment of infections should be highly monitored as they may pose a health threat to normal immune cells. Generally, plants are potential pharmacological agents which needs to be preserved and harvested with care.
Open Access
Application of cloning in the detection of HIV-1 and drug resistant minority populations
(2015-01-14) Hatyoka, Luiza Miyanda; Bessong, T. M.; Masebe, Tracy. M.
Open Access
Assessment of knowledge, attitude and practices of small household farmers towards heartwater disease and molecular characterization of Ehrlichia ruminantium in Limpopo province, South Africa
(2023-10-05) Mathebula, Doris; Samie, A.; Sigidi, M. T.
Background: Heartwater is a disease spread by ticks that is brought on by the obligatory intracellular bacterium Ehrlichia ruminantium. Heartwater disease is one of the major obstacles to livestock production as it affects many livestock animals including domesticated animals such as goats, cattle, and sheep as well as wild ruminants. In Southern Africa, it is spread by Amblyomma hebraeum ticks, and in the rest of Sub-Saharan Africa, it is spread by A. variegatum ticks. In this study, epidemiological and molecular features of Ehrlichia ruminantium in Mopani and Vhembe Districts, from ticks isolated from cattle, goats and sheep were investigated. Method: A total of 121 small household farmers from different villages in the Vhembe and Mopani Districts were recruited in the study after they have signed an informed consent. The participants were interviewed using a questionnaire to collect data related to their knowledge, attitude and practices towards heartwater disease. Ticks were collected from cattle, goats, and sheep belonging to 48 households, yielding a total of 244 ticks. Genomic Deoxyribonucleic acid was extracted from the ticks and analysed using real-time qPCR assay targeting a 226bp fragment of the PCS20 gene. Finally, a number of samples were sent for sequencing to identify different strains circulating in the region. Neighbor-joining method was used to infer phylogenetic positions on the basis of 16S rRNA gene. Results: According to the findings of the questionnaire evaluation, only about 23.1% of the participants had some knowledge of heartwater disease. Furthermore, the highest proportion of the study population (76%) associated heartwater with air-borne transmission and 77.7% of the participants failed to identify the season in which heartwater commonly occurs. About 69% of the respondents associated ticks with animal diseases while 49.6 % correctly highlighted that, ticks are disease carriers. Farmers had a positive attitude towards control and treatment of heartwater by stating that they would use prescribed medicine 23.9%, vaccines 7.4% or consulting a specialist 2.5%. Few farmers indicated that they use homemade mixtures (0.8%), dipping and spraying (0.8%) to manage animal diseases. The most preferred method of tick control used by farmers was spraying of acaricide treatment 63.6%. On account of the poor animal services reported among the visited rural communities, some farmers opted for removing ticks by hand 28.9% as their supplementary tick control method. Majority of the farmers fed their livestock at the bush 68.6% which was the most contributing factor to increased tick infestation as reported by farmers. Several plants used as medicine to treat various animal diseases were mentioned. These included: Melia azedarach, Albizia adianthifolia, Gymnosporia senegalensis, Dicerocarryum senecioides etc. The type of disease affecting the livestock mentioned by the participants was diarrhoea (43.0%) which is among the list of heartwater symptoms. The study demonstrates that respondents had inadequate knowledge about heartwater disease. Animal services need to be upgraded in order to help the farmers improve the quality of their produce. The results of the PCR test showed that 56.2% of the household farms had E. ruminantium infection. The distribution of E. ruminantium by source of ticks and animal type revealed that cattle are more prone to tick distribution 43.5% as compared to other animal sources of ticks. Nzhelele municipality had the highest prevalence of E. ruminantium (37.0%) compared to the other municipalities. Prevalence of E. ruminantium by gender of farmers was found to be high from males 36.7% than females 25.0%. Farmers with household income > R20000 had the highest prevalence of E. ruminantium (50.0%) than farmers with household income < R500, (14.3%). The age of the farmers 15 – 20 years old revealed the highest prevalence 46.7 % of E. ruminantium infection. However, there were no infection among the ticks obtained from animals belonging to farmers above 60 years old. Farmers who had tertiary level of education showed the highest prevalence 46.8% probably because of limited time to attend to their livestock. Many clades were identified by phylogenetic analysis of the E. ruminantium PCS20 genotypes. Conclusion: This study showed that there is need to further educate small farmers on heartwater disease. It also showed that indigenous knowledge still contributes significantly to the management of animal diseases in most rural communities. The application of real time PCR showed a high prevalence of E. ruminantium infected A. hebraeum ticks from livestock and should be considered in the continuous monitoring of the animal population in order to avoid heartwater disease outbreak in the communities which could be detrimental for the local economy. Furthermore, future vaccine development against E. ruminantium should consider the diversity observed in the present study. That could be helpful in managing heartwater disease in the areas that were investigated.
Open Access
Assessment of microbial quality and safety of ground beef/product sold in differrent retailers around Thohoyandou Area, Vhembe District, Limpopo
(2023-05-19) Mukosi, Fulufhelo Valentine; Musie, E. M.; Traore, A. N.
Title: Assessment of microbial quality and safety of ground beef/product obtained from selected retailers around Thohoyandou area; Vhembe district; Limpopo. Background: It has been proven that animal products are easily contaminated with microorganisms, and this supports microbial growth if not properly handled, processed, and preserved. Ground beef and its wholesale products are becoming popular because of the demand for rapid meal preparation and services, especially in the fast-food industry. Despite the control measures in place, foodborne infections continue to be an immense problem, with millions of cases occurring annually worldwide. In South Africa, illnesses and deaths related to food consumption continue to be reported. In addition to the misery caused, the financial loss associated with meat spoilage and illnesses is enormous. Therefore, this study aimed to assess ground beef's microbial quality and safety in different retailers around Thohoyandou area, Vhembe District. Methodology: A total of 160 ground beef/product samples was randomly purchased from various retailers in Thohoyandou and transported on ice to the University of Venda microbiology laboratory for analysis. The potential microbes were cultured in enrichment media (peptone buffered water) for 5 minutes in room temperature. The culture was then sub-cultured in different plates containing selective media (e.g., EMB for E. coli, SS for salmonella and Shigella, MSA for Staphylococcus spp.) using the spread plate technique. Isolates were then identified by the Gram staining technique and biochemical tests such as Catalase, Urease, Citrate, Kligler Iron Agar, and VITEK system. Moreover, the antibiogram activity of isolated pathogens was screened against medically used and commercially available antibiotics. Furthermore, the DNA of the isolates was extracted, and multiplex PCR was conducted to determine different virulence genes and pathotypes. Hemolysin test was done in blood agar plates to identify virulence characteristics of E. coli isolates. Results: Out of 160 samples analyzed, E. coli was detected in 80 (50%), Staphylococcus spp. in 117 (73.12%), Salmonella in 60 (37.5%), and Shigella species in 108 (67.5%). Most Enterobacteriaceae (E. coli, Salmonella and Shigella) isolates were resistant to Ampicillin and Cefoxitin. Staphylococcus isolates showed high resistance to Cefoxitin (93.33%) and Oxacillin (93.33%). Out of 30 E. coli isolates subjected to mPCR assay, 23 isolates were of different pathotypes with EPEC (53.33%) being the most prevalent pathotype. Asta with 73.33% was the dominant virulence gene obtained. Thirty (30) E. coli isolates were tested for hemolysin activity and Alpha hemolytic activity was observed in 76.6% isolates, while beta hemolytic activity observed in 10% isolates. Some of the isolates presented non-hemolytic strains (13.3%). Conclusion: It was concluded that ground beef/products from established retailers were contaminated with pathogenic bacteria, and microbial quality was thus inadequate.
Open Access
Biophysical characterization of KO513, a protein expressed in human invasive glioblastoma
(2020-06-26) Nemukondeni, Ndivhuwo; Burger, A.; Zininga, T.
Glioblastoma multiforme (GBM) is the deadliest brain tumour. GBM is associated with poor prognosis, with its patients having a very short median survival and poor response to chemotherapy. GBM is a World Health Organization (WHO) grade IV glioma and accounts for up to 78% of all brain tumours. This phenotype harbours a series of mutations that provide cells with selective growth advantages that promote survival and proliferation in a hostile and hypoxic environment. KIAA0513 is one of the genes identified to be upregulated in GBM phenotype through gene expression profiling. KIAA0513 gene codes for K0513 protein. KIAA0513 is ubiquitously expressed and is enriched in the cerebral area of the brain. It is predicted to be involved in signalling pathways including neuroplasticity, cytoskeletal regulation and apoptosis. The main objective of the current study was to perform biophysical characterization of K0513. Characterization of K0513 will allow structure-function annotation which may serve as a basis for establishment of K0513 as a potential biomarker for GBM. Using bioinformatics analysis, a potentially functional SBF2 domain was identified. The threedimensional homology model shows that K0513 is a globular protein, with the identified SBF2 domain and transmembrane region in proximity to one another. Predicted interacting partners includes members of the Rab GTPase family, membrane proteins, transcription factors and neurotransmitters. The overall in silico analysis suggest that K0513 may possess nucleotide exchange factor (NEF) activity for Rab3a. Recombinant proteins, non-codon harmonized (K0513W) and codon harmonized (K0513H) were expressed in E. coli XL1-Blue cells. The recombinant K0513H was successfully purified using nickel-affinity chromatography, this is the first study to report on the recombinant expression and purification of K0513. Using tryptophan fluorescence assay and limited proteolysis, nucleotides were found to have no significant effect on the tertiary structural conformation of K0513. Pull down assay shows promising interactors of K0513 from fibrosarcoma cell line; future studies will identify the interactors using Liquid chromatography-mass spectrometry.
Open Access
Burden of infection and genetic characterization of human herpes virus type 8 in HIV infected individuals in Northern South Africa
(2019-05-16) Etta, Elizabeth Mashu; Bessong, Pascal Obong; Mavhandu - Ramarumo, Lufuno Grace
Human herpes virus type 8 (HHV-8), also known as Kaposi’s sarcoma associated herpes virus (KSHV), is the etiologic agent of Kaposi’s sarcoma (KS), and AIDS related Kaposi’s sarcoma (AIDS-KS). HHV-8 which is a member of the Herpesviridae family, exhibits extensive genetic diversity globally. In endemic regions, infection with HHV-8 occurs very early on in life, which is an indication of both environmental and vertical routes of transmission. The advent of HIV leads to the classification of an AIDS-KS defining condition in HIV infections. This suggests that in regions where HIV and HHV-8 are endemic, KS may become common in a mature HIV epidemic. Just like the prevalence of HIV in Northern South Africa is generally high as in most regions of the country, as the HIV epidemic matures in South Africa, it is important to understand the burden and distribution of HHV-8 infection, and the likely genotypes infecting the population. The main objective of the thesis was to establish the epidemiology and infecting genotypes of HHV-8 in Northern South Africa (Limpopo Province), where no data exists. First, a systematic review of the literature was carried out for the entire African continent to determine the seroprevalence and genotype distribution of HHV-8 in all African countries (n=53). In this review, Sudan and South Sudan were considered as one country. Articles were searched using the PRISMA guideline and exported using an article grid. More than two-thirds (64%) of the studies reported on seroprevalence, 29.3% on genotypes; and 9.5% were on both seroprevalence and genotypes. About 45% (24/53) of the African countries had data on HHV-8 seroprevalence exclusively, and more than half (53%) had data on either seroprevalence or genotypes. Almost half (47%) of the countries had no data on HHV-8 infection. There was high heterogeneity in the types of tests and interpretation algorithms used in determining HHV-8 seropositivity across the different studies. Generally, seroprevalence ranged from 2.0% in a group of young children in Eritrea to 100% in a small group of individuals with KS in the Central Africa Republic and a larger group of KS in individuals in Morocco. Approximately, 16% of all the studies reported on children. The difference in seroprevalence across the African region was not significant (95% CI, X2 =0.86; p =0.35), although specifically, a relatively significant ETTA MASHU ELIZABETH, PHD IN MICROBIOLOGY|UNIVERSITY OF VENDA, 2019|VIII level of infection was observed in HIV-infected children. About 38% of the countries had data on K1 genotypes A, A5, B, C, F and Z which occurred at frequencies of 5.3%, 26.3%, 42.1%, 18.4%, 5.3% and 2.6% respectively. Twenty-three percent of the countries had data for K15 genotypes, whereas genotypes P, M and N occurred at frequencies of 52.2%, 39.1% and 8.7% respectively. Data on HHV-8 inter-genotype recombinant is scanty. Our finding suggests that HHV-8 is endemic on the entire African continent, and in HIV endemic regions, but there is need for a harmonized testing protocol for better understanding of HHV-8 seropositivity. HHV-8 genotype A5 and B for K1 gene and genotype P and M for K15 gene are the most predominant genotypes in Africa. The review, for the first time, has provided information on HHV-8 burden on the entire African continent, and suggests that vaccine development efforts for Africa should focus on genotypes B and P. The second component of the investigation focused on the burden of HHV-8 in an HIV population in Northern South Africa (Limpopo Province). Plasma from 3501 HIV infected individuals from 5 districts in Limpopo Province were assessed for antibodies to both the lytic antigen (ORFK8.1) and the latent antigen (ORF73). The distribution of infection was analyzed based on demographic, socioeconomic, and immunological parameters. Statistical inferences for significant differences were determined by Chisquare at a confidence interval of 95%. P-values less than 0.05 were considered significant. About 19.0% of the study population was positive for antibodies to either the lytic or latent antigens or both. Prevalence of antibodies to the lytic antigen was significantly higher than prevalence of antibodies to the latent antigen (17.3% vs 4.1%; p=0.0001). Significant differences were observed for age groups, racial population groups, districts and year of sample collection (p=<0.0001, p=<0.0001, p=<0.0001 and p=0.0385) respectively. Associations were found between both antigens in comparison to the different variables such as age group, racial population groups and districts (R2 value ranging between 0.886 and 1.0). The burden of HHV-8 has now been established for the first time in Northern South Africa. The third aspect of the investigation was a meta-analysis of HHV-8 seroprevalence in Southern Africa in order to understand the impact of geographical location (urban vs rural) on infection. The analysis revealed a significant association between urban settings and HHV-8 infection (p=0.0001). ETTA MASHU ELIZABETH, PHD IN MICROBIOLOGY|UNIVERSITY OF VENDA, 2019|IX The fourth component of the thesis examined the detection of HHV-8 antigen through polymerase chain reaction (PCR) in 534 participants in HIV infected and HIV noninfected populations. A selection of mouthwash DNA samples were subjected to Next Generation Sequencing (NGS) for subsequent genotype inference. Mouth wash samples were obtained from each consenting individual before eating or smoking, and their DNA was purified. A 233bp fragment of the ORF26 gene of HHV-8 was amplified by PCR. HHV-8 was detected in 150 of the 534 participants (28.1%). A significant difference in detection was observed for gender, HIV status, district and the level of education (p=0,0003; p=0.0094; p=0.0002 and p=0.0095) respectively. Consensus sequences were derived from NGS reads for 13 samples. The genotyping results revealed that genotype Q, B, E and N are the genotypes predominant in the study population. As such no mixed infections were detected. Therefore, from the investigations foregoing have demonstrated for the first time the following: (1) HHV-8 is endemic in the entire African continent, which suggest a coendemicity in regions already endemic for HIV; (2) HHV-8 is endemic in Northern South Africa; (3) Urban settings in Southern Africa are associated with high HHV-8 infection; (4) HHV-8 genotypes Q, B, E and N may be predominant in Northern South Africa, with B and P common on the entire African continent. Hence, studies should focus on the generation of full length HHV-8 genomes of the common genotypes to support the selection of genes for vaccine design and development.
Open Access
Characterization of cervicovaginal HPV virome and bacteriome in HIV -i infected women in Northern South Africa
(2023-05-19) Ratshilindela, Mpho; Bessong, Pascal Obong; Tebit, Denis
BACKGROUND: The presence of a highly diverse bacterial species in the cervicovaginal niche is linked to a higher risk of contracting human immunodeficiency virus (HIV), persistent infection with human papillomavirus (HPV) and consequently cervical cancer. It is unknown how cervicovaginal bacterial species associate and interact with other viruses. This study hypothesized that HIV/HPV co-infected women have an increased diversity of cervicovaginal virome and bacteriome. OBJECTIVES: The study’s primary objective was to characterize cervicovaginal HPV virome and bacteriome in HIV-infected women from selected health care facilities in Northern South Africa. Specific objectives were to determine the presence of HPV virome in HIV-infected women and HIV-noninfected women, to determine the presence of bacteriome in HIV-infected women and HIV-noninfected women, and to determine the relational occurrence of virome and bacteriome according to HIV status. METHODOLOGY: Cervical swabs from 50 HIV/HPV co-infected women; 50 HIV-infected, HPV-noninfected women; and from 50 HPV-infected, HIV-noninfected women were used to extract total deoxyribonucleic acid (DNA). To determine HPV virome, total DNA was enriched by rolling circle amplification (RCA) using an illustra TempliPhi amplification kit. To determine bacteriome, a two-round polymerase chain reaction (PCR) was employed targeting a bacterial 16S rRNA gene. Amplification products obtained from RCA and PCR were purified and sequenced by Next Generation Sequencing (NGS) using a MiniSeq platform (Illumina). The quality of the sequences was validated using the FastQC program. Sequences of good quality were assigned to viral family and genera using the Dragen metagenomics online tool with BaseSpace Sequence Hub. Bacterial sequences were assigned and categorised into vaginal community state types (CSTs) using the Dragen metagenomics online tool with BaseSpace Sequence Hub. The relational occurrence of virome and bacteriome according to HIV status was assessed through Chi-square statistical analysis available in GraphPad Prism version 9.3.1. Differences in occurrence were expressed as probability (P)-values. RESULTS: A diverse group of viral families was observed among HIV/HPV co-infected women. Papillomaviridae (14/34; 41%) was the most prevalent (P<0.0001), followed by Herpesviridae and Phycodnaviridae (12/34; 35%) each, Poxviridae (10/34; 29%), Mimiviridae (7/34; 20%), Maiseilleviridae (3/34; 9%) and Anelloviridae (2/34; 6%) in order of decreasing prevalence. A highly diverse group of bacteriophages was observed, with Myoviridae (31/34, 91%) being the most prevalent (P<0.0001). Among HPV-infected HIV-noninfected women, v Papillomaviridae (8/26; 31%) was the most prevalent (P<0.0001), followed by Anelloviridae (4/26; 15%), Phycodnaviridae (4/26; 15%), Poxviridae and Herpesviridae (2/26; 8%) each in order of decreasing prevalence. Myoviridae (6/26; 23%) was the most prevalent bacteriophage family detected (P=0.0005). Among HIV-infected HPV-noninfected women, a small group of viral families was observed, with Myoviridae (3/11,27%) and Siphoviridae (3/11, 27%) being the most prevalent viral families detected (P=0.0005 each). HPV 16 was the most common high risk (hr) HPV type in HIV-infected women and HIV-noninfected women of this study. This genotype co-existed with other hrHPV or probable hr types including HPV 54, HPV 53, and HPV 26. Overall, hrHPV types were more prevalent in HIV-infected women than in HIV-noninfected women, although this difference was not statistically significant (P=0.2832). When the occurrence of hrHPV types were considered individually, HPV 54 occurred more significantly in HIV-noninfected women than in HIV-infected women (P<0.0003). Bacteriome revealed the presence of community state types (CSTs) one, two, three, four and five among study participants. Among HIV/HPV co-infected women, CST one (L. crispatus), CST three (L. iners) and CST four (high bacterial diversity) were observed, with CST four being the most prevalent. Among HPV-infected, HIV-noninfected women, CST one, three, four and five (L. jensenni) were observed, with CST four, also being the most prevalent. Among HIV-infected, HPV noninfected women, CST three and four were observed. CST three and four were associated with viral infections. Gardnerella vaginalis (75%), Pretovella spp (54%), Atopobium spp (50%), Bifidobacterium spp (27%), Porphyromonas spp (53%), Pseudomonas spp (50%), Faecalibacterium prausnitzii (47%) and Bacteroides spp (35%) were the most prevalent anaerobic bacterial species detected in CST four. A diverse group of bacterial families occurred more significantly among HIV-infected women as compared to HIV-noninfected women for NGS data of RCA products (P<0.0001) and NGS data of 16S amplification products (P<0.0001). CONCLUSION: In conclusion, this study showed a higher diversity of cervicovaginal virome and bacteriome in women who were HIV/HPV co-infected than in those singly infected with HIV or HPV. The relationship between viral infections and a high diversity of bacterial species (CST four) observed herein may be a useful indicator of the individual’s disease state, indicating the likelihood of developing cervical lesions and cervical cancer. As a result, additional research is necessary to uncover the association between viral infections and CST four with disease state. Vaccine development and antiviral research should also target compounds that boost the cervicovaginal environment and maintain vaginal homeostasis.
Open Access
Characterization of cholesterol 25-hydroxylase expression in human macrophages
(2019-09-20) Magoro, Tshifhiwa; Bessong, Pascal O.; Hahn, Young Shin; Jennelle, Lucas
Background Conversion of Cholesterol to 25-HydroxyCholesterol (25HC) by Cholesterol 25-hydroxylase (CH25H) has been shown to exert broad antiviral properties. Given its antiviral activities, CH25H is part of an increasingly appreciated connection between type I interferon (IFN-I) and lipid metabolism. Moreover, the details of this connection appear to differ in mouse and human cells. Nevertheless, the molecular basis for the induction of CH25H in humans is not known. Objective Elucidation of signaling and transcriptional events for induction of CH25H expression is critical to design therapeutic antiviral agents. Materials and methods: Wildtype THP-1 monocytic cell-line or THP-1 MyD88 Knockout cell-line were treated with PMA for 72 hours for differentiation into macrophages. Differentiated macrophages or Microglial cells were stimulated with either TLR-agonists, pro-inflammatory cytokine, or interferons, and CH25H mRNAs expression levels were measured by qPCR. Results In this study, we show that CH25H is induced by Zika virus infection or TLR stimulation. Interestingly, CH25H is induced by pro-inflammatory cytokines including 1L- 1, TNF-, and IL-6, and this induction depends on STAT-1 transcription factor. Additionally, we have observed that ATF3 weakly binds to the CH25H promoter, suggesting co-operation with STAT-1. However, ZIKV induced CH25H was independent of type I interferon. Conclusion This study has demonstrated for the first time that pro-inflammatory cytokines such as 1L-1, TNF-, and IL-6 induce CH25H expression. Moreover, this provides further understanding to the connection between innate immunity and sterol metabolism and encourages the exploration of cytokines in antiviral immunity.
Open Access
Characterization of diarrhoeagenic escherichia coli and human norovirus from daycare centres in the Vhembe Region of the Limpopo Province
(2020-03) Munzhedzi, Lutendo; Potgieter, N.; Traore, A. N.; Kabue, J. P.
BACKGROUND: Child daycare centers (DCCs) host a variety of pathogenic enteric microorganisms which may be found in various areas within the compound. Enteric pathogens are the most common cause of diarrhoea worldwide in children under the age of 5 years and are a burden in developing countries with high numbers of diarrhoeal diseases leading to a high mortality rate. OBJECTIVE: To perform a pilot study to characterize diarrhoeagenic E. coli and Norovirus strains from child daycare centers in the Vhembe Region in Limpopo Province, South Africa. MATERIALS AND METHODS: Two daycare centers were randomly selected in the Vhembe District for this study. A total of 83 samples were collected from various areas within the daycare centers. For E. coli, the environmental and handwash samples were analysed using the Colilert®/ Quanti-tray® 2000 technique and stool samples were cultured on Eosin Methylene Blue agar: presumptive E. coli isolates were confirmed using three biochemical tests (Kliglers Iron Agar test, Indole test and Urease test). Total nucleic acid was extracted from presumptive E. coli isolates using a semiautomated method and DNA was used for further m-PCR confirmation. For human norovirus identification, environmental and handwash samples were subjected to membrane filtration and RNA was extracted using the semi-automated system. RNA was extracted from stool samples (clinical samples) using the Allprep® Powerfecal® RNA Kit. For detection of Norovirus, RNA extracts were subjected to the Seegene Allplex™ Gastrointestinal Full Panel Assay. Positive samples for Norovirus were amplified using a One-step Ahead RT-PCR followed by Sanger sequencing. Phylogenetic analysis was done using MEGA 7 software. RESULTS: E. coli was isolated from the environmental samples (10%), stool samples (100%), handwash samples of daycare workers (40%) and handwash samples of children (13.6%); several bacterial co-infections among pathotypes were observed. Norovirus prevalence in this study, was detected in 5% of handwash samples of the children, 10% from handwash samples of the workers and 27% were stool specimens. All detected samples were co-infection between bacterial pathotypes (90.4%) as well xi as bacterial-viral co-infections (9.6%). Norovirus genotype GII.1 was shown through sequencing of one positive sample. CONCLUSION: The study findings showed genetic diversity of E. coli in these settings. The phylogenetic analysis revealed NoV genotype GII.I capsid sequenced to share a common ancestor with previously reported strains associated with outbreaks of NoV globally. The high prevalence of E. coli and NoV detected in this study will aid in setting guidelines for hygiene in the DCC environment.
Open Access
Characterization of E. Coli and Staphylococcus aureus isolated from Clinical and Subclinical cases of bovine mastitis in the Limpopo Dairy Farm (Limpopo, South Africa)
(2020-03) Badugela, Ndivhuwo; Musie, E.; Sigidi, M. T.; Traore, A. N.
Background: Staphylococcus species and Eschericia Coli has been predominantly found to cause mastitis in dairy farms. Milk harbor various pathogenic microorganisms that causes foodborne and intramammary infections. The aim of this study was to characterize Staphylococcus spp. and Eschericia coli spp. isolated from clinical and subclinical cases of bovine mastitis in the Limpopo dairy farm. Methods: Semi structured questionnaire was used prior milk sampling to acquire farm management strategies. A total of 253 milk samples were collected from the dairy farm between 2018 and 2019. California mastitis test was done to screen for mastitis and culture methods were used for the isolation and identification of E. coli and Staphylococcus species. Further identification and biochemical confirmation for bacterial isolates were performed using API test kit and automated VITEK® 2 system. Eschericia coli isolates were characterized using a multiplex PCR. Automated VITEK® 2 system and Kirby Bauer disc diffusion method were also used to determine antibiotic susceptibility of the isolates. Results: The study reported fair farm practices and management system with low mastitis burden. California mastitis test revealed an overall mastitis on 94/250 (37%) of the samples. Of 94 samples cultured, a total of 32 (34%) were positive for E. coli strains and 48 (51%) were positive for Staphylococcus spp. [Staphylococcus sciuri 19 (40%) and Staphylococcus xylosus 10 (21%)]. Out of 32 Escherichia coli isolates 27 (93%) and 19 (66%) were detected with astA gene and sta which encodes for enteroaggregative E. coli respectively. Most Staphylococcus species isolates were highly resistant to Erythromycin (93%); Nalixidic acid (86%). The presence of pathogenic E. coli and Staphylococcus species in milk may pose health risks or problem and improving sanitary conditions may reduce the burden of mastitis. For future studies, further analysis of both E. coli and Staphylococcus species to determine virulence and resistant genotyping in order to investigate possible mutations is recommended.
Open Access
Characterization of E. coli strains from rural communities in the Vhembe District (Limpopo South Africa)
(2019-09-20) Banda, Ntshunxeko Thelma; Potgieter, N.; Traore, A. N.
Background: Escherichia coli is a facultative anaerobic bacterium that forms part of the gut microbiota. It is used as an indicator that confirms recent faecal contamination. E. coli have been identified amongst the pathogens that are mostly responsible for moderate to severe diarrheal outbreaks in the low and middle-income countries. With South Africa facing an issue in water scarcity, issues concern poor sanitation and hygiene practices results in serious public health problems and allows E. coli to be transmitted from infected human or animal faeces to a new susceptible host using environmental reservoirs such as soil, water, hands as the transmission pathway. Objective: The primary objective of the study was to characterize E. coli strains from rural communities of Vhembe district, Limpopo, South Africa. Methodology: Households of 7 villages in the Vhembe district were randomly selected. A total of 81 households (HHs) were part of the study. In each household, a structured questionnaire was used to background information on WASH practices. Samples taken from each HH included toilet seat swabs, floor swabs, child and mother handwash samples, stored water samples and running tap water samples. A total of 399 samples were analysed using Colilert® Quanti-trays®/2000 method to detect the presence of Escherichia coli. Positive E. coli samples were further identified using multiplex polymerase chain reaction (m-PCR) to determine the pathogenic strains of E. coli. Transmission pathways were established using identified strains. Results: Data from the structured questionnaires showed common problems of availability of running tap water; lack of provision of sanitation; open practice on defaecation and very little hand hygiene practices. A total of 91 (22.81%) samples tested positive for E. coli with the Colilert® Quanti-trays®/2000 method. The mothers’ handwash samples had the most E. coli prevalence followed by stored water samples. The most prevalent E. coli pathotype was EPEC with the virulence gene eae. Atypical EPEC (60.44%) outnumbered the typical EPEC (5.49%). The pathotype ETEC was detected in 41.76% samples and EHEC in 9.89% samples. Transmission pathway was observed from the different households; with eae gene (aEPEC) being the most detected from samples looking at the LT gene (ETEC). v | P a g e Discussion: All 7 villages are facing common issues such as lacking running water, poor sanitation and improper hand hygiene practices. The mothers were the most contaminated and it was observed that its due to the daily activities that they perform around the house. It is of importance for them to practice proper hand hygiene to prevent transmission of pathogenic E. coli to the children via direct or indirect transmission route. The pathogenic E. coli was detected from all different samples collected from the households including the floor and toilet seat samples. EPEC was detected the most, and studies have shown that this strain is known to cause diarrheal infections in young children from developing countries. Conclusion: The members of the study village households were aware of the WASH services and its importance. However, proper implementation into their day-to-day life is lacking due to the high number of TC and E. coli detected from handwash samples and stored water samples from the households. Recommendation: Appropriate WASH strategies should be established to ensure good health at the village households. Further studies should be done to check possible transmission pathways from more village households.
Open Access
Characterization of heat shock protein 70-z (PfHsp70-z) from plasmodium falciparium
(2015) Zininga, Tawanda; Shonhai, Addmore; Burger, A.
Malaria is a parasitic disease that accounts for more than 660 thousand deaths annually, mainly in children. Malaria is caused by five Plasmodium species P. ovale, P. vivax, P. malariae, P. falciparum and P. knowlesi. The most lethal cause of cerebral malaria is P. falciparum. The parasites have been shown to up-regulate some of their heat shock proteins (Hsp) in response to stress. Heat shock protein 70 (called DnaK in prokaryotes) is one of the most prominent groups of chaperones whose role is central to protein homeostasis and determines the fate of proteins. Six Hsp70 genes are represented on the genome of P. falciparum. The Hsp70 genes encode for proteins that are localised in different sub-cellular compartments. Of these two occur in the cytosol, PfHsp70-z and PfHsp70-1; two occur in the endoplasmic reticulum, PfHsp70-2 and PfHsp70-y; one in the mitochondria, PfHsp70-3 and one exported to the red blood cell cytosol, PfHsp70-x. PfHsp70-1 is a well characterized canonical Hsp70 involved in prevention of protein aggregation and facilitates protein folding. Little is known about PfHsp70-z. PfHsp70-z was previously shown to be an essential protein implicated in the folding of proteins possessing asparagine rich repeats. However, based on structural evidence PfHsp70-z belongs to the Hsp110 family of proteins and is thought to serve as a nucleotide exchange factor (NEF) of PfHsp70-1. The main aim of this study is to elucidate the functional roles of PfHsp70-z as a chaperone and its interaction with PfHsp70-1. In the current study, PfHsp70-z was cloned and expressed in E. coli JM109 cells. This was followed by its purification using nickel chromatography. The expression of PfHsp70-z in parasites cultured in vitro was investigated and its association with PfHsp70-1 was explored using a co-immuno precipitation assay. PfHsp70-z expression in malaria parasites is up regulated by heat stress and the protein is heat stable based on investigations conducted using Circular Dichroism. Furthermore, the direct interaction between recombinant forms of PfHsp70-z and PfHsp70-1 were investigated using slot blot and surface plasmon resonance assays. PfHsp70-z was observed to exhibit ATPase activity. In addition, the direct interaction between PfHsp70-z and PfHsp70-1 is promoted by ATP. Based on limited proteolysis and tryptophan fluorescence analyses, PfHsp70-z binds ATP to assume a unique structural conformation compared to the conformation of the protein bound to ADP or in nucleotide-free state. PfHsp70-z was able to suppress the heat-induced aggregation of malate dehydrogenase and luciferase in vitro. Interestingly, while ATP appears to modulate the conformation of PfHsp70-z, the chaperone function of PfHsp70-z was not influenced by ATP. Altogether, these findings suggest that Characterization of Heat Shock Protein 70-z (PfHsp70-z) from Plasmodium falciparum iii PfHsp70-z serves as an effective peptide substrate holding chaperone. In addition, PfHsp70-z may also serve as the sole nucleotide exchange factor of PfHsp70-1. The broad spectrum of functions of this protein, could explain this PfHsp70-z is an essential protein in malaria parasite survival. This is the first study to show that PfHsp70-z possess independent chaperone activity and that it interacts with its cytosolic counterpart, PfHsp70-1 in a nucleotide dependent fashion. Furthermore, the study shows that PfHsp70-z is a heat stable molecule and that it is capable of forming high order oligomers.
Open Access
Characterization of HIV-1 drug resistance mutations from plasma and peripheral mononuclear cells in patients failing antiretroviral treatment in Bela-Bela, South Africa
(2015-09-16) Etta, Elisabeth Mashu; Bessong, Pascal Obong; Tebit Denis Manga
Open Access
Characterization of human astrovirus in pediatric patients with diarrhea from rural communities of Limpopo South Africa
(2020-03) Khumela, Ronewa; Potgieter, N.; Traore, A. N.; Kabue, J. P.
Background: Globally, approximately 7,600,000 children under the age of 5 die annually due to diarrhea caused by viruses, bacteria and parasites. Human Astrovirus (HAstVs) has been identified as a causative agent of diarrheal disease worldwide especially in young children under five years of age. Recent reports in South Africa demonstrated HAstVs as a potential pathogen associated with diarrhea. The aim of this study was therefore to investigate the genetic characteristics of HAstVs in young children with diarrhea in rural communities of Vhembe District, Limpopo Province. Methodology: A total of 141 archived RNA, extracted from stool samples, were retrieved from -20°C storage. Using questionnaire, clinical data useful in the analysis of results were captured. The RIDA®GENE Viral Stool Panel I (PG1325) multiplex real-time RT-PCR assay was used for the detection of Astrovirus. Positive Astrovirus extracts were amplified by one-step ahead RT-PCR (cat no: 220213, (QIAGEN)) and one-step RT-PCR kit (cat no: 210212, (QIAGEN)) using specific primers targeting the viral capsid and polymerase regions. Amplified fragments were sequenced, and phylogenetic trees constructed by the neighbor-joining method using MEGA X (10.0.5) software. Results: HAstVs was detected in 10 (7%) of the 141 stool samples. A total of 9/10 (90%) HAstVs cases were from outpatients. The sequence analyses revealed HAstV genotype 1 and 2. A putative recombinant strain was found. Phylogenetic analysis revealed strain’s relatedness to others circulating in the African continent. Conclusion: This is the first study to characterize HAstVs from pediatric stool sample in the Vhembe district of Limpopo/South Africa. The study findings revealed the presence of HAstV type 1 and 2 in young children in the rural communities of the Vhembe District. Human Astrovirus genotype 1 and 2 are globally associated with diarrhea. Systematic surveillance to monitor HAstV strains circulation will help to understand the role of the pathogen in the study area.
Open Access
Characterization of PFF1010c, a type IV Plasmodium fasciparum heat shock protein 40
(2016) Mutavhatsindi, Hygon; Shonhai, A.; Burger, A.
Malaria is caused by protozoa of the genus Plasmodium. Malaria accounts for approximately more than half a million deaths yearly. Of the five species of Plasmodium, P. falciparum accounts for the most deadly form of the disease. P. falciparum survives under various physiological conditions during its life cycle. The parasite employs its molecular chaperones machinery particularly heat shock proteins (Hsps) to protect its protein constituents during physiological stress. Hsps are conserved molecules that constitute a major part of the cell’s molecular chaperone system. P. falciparum Hsps play an important cyto-protective role guaranteeing that the malarial parasite survives under the severe conditions that prevail in the host environment. PFF1010c is a type IV P. falciparum heat shock protein 40. PFF1010c is predicted to be expressed only at the gametocyte stage of the malarial parasite’s life cycle. The aim of the current study was to investigate the expression PFF1010c by parasites and the gametocyte stage as well as characterize the structure-function features of the protein. PFF1010c was successfully expressed in E. coli cells. Despite successful expression of the protein, its purification proved problematic. The attempt to purify PFF1010c was carried out under both native and denaturing conditions. Far Western blot analysis to investigate direct interaction between PFF1010c and PfHsp70-1 was conducted and no interaction was observed. Malarial parasites were harvested at different stages and total protein was isolated. The expression of PFF1010c was confirmed to occur at the gametocyte stage of the parasite’s development using Western blot analysis. This study confirmed that PFF1010c is only expressed at the gametocyte stage of the malarial parasite. Furthermore, PFF1010c was not expressed at the asexual stage. Possible interactors of PFF1010c were predicted by STRING, a bioinformatics based tool. The expression of PfHsp90, PfHop and PfHsp70-1 at the gametocyte stage was investigated and confirmed by Western blot analyses.
Open Access
Characterization of Plasmodium falciparum PFF1010c and screening of pyrimidine-quinoline hybrids as inhibitors of HsP70-HsP40 functional partnerships of the Malaria parasite
(2021-05) Mudau, Pertunia Thendo; Shonhai, A.; Zininga, T.
With nearly half the world’s population at risk of malaria infection, 229 million new cases were recorded in 2019 with Sub-Saharan Africa accounting for most of these cases. In 2020, 384 000 malaria deaths were reported in the WHO African region. Plasmodium falciparum is the most virulent species responsible for over 90 % of all malaria infections. The P. falciparum life cycle occurs through multiple stages in humans and mosquitoes. Many advances have been made in the fight against malaria through vector control and treatment against infection by the parasite. However, resistance of P. falciparum to antimalarial medicines/treatment has hindered global efforts to control and eliminate malaria. A multidimensional approach to fighting malaria by targeting is essential protein machinery is needed. Heat shock proteins (Hsp) are molecular chaperones that are upregulated in response to stress and have long been projected as antimalarial drug targets. P. falciparum Hsp70-1 (PfHsp70-1; PF3D7_0818900) is an essential heat shock protein involved in the folding of newly synthesized and misfolded polypeptides and is also implicated in protein trafficking. PfHsp40 (PF3D7_1437900.1) is a type I Hsp40 co-chaperone of PfHsp70-1 that presents substrates to PfHsp70-1 and stimulates its ATPase activity, while PFF1010c (PF3D7_0620700.1) is a type IV Hsp40 found in the gametocytes that is yet to be characterized. The current study explored pyrimidine-quinoline hybrid (PQH1-4) compounds as possible inhibitors of PfHsp70-1 interaction with PfHsp40. Furthermore, this study sought to characterize the structure and function of PFF1010c. Notably, PFF1010c possess SVN residues in place of the HPD motif located in the so-called J domain of this Hsp40 co-chaperone. For this reason, PFF1010c is an atypical Hsp40 which may lack the capability to interact with PfHsp70-1. To characterise the role of PFF1010c, and consequently, its SVN motif, the SVN motif of PFF1010c was switched with the HPD motif of PfHsp40. The structure-function features of the mutants were characterized relative to the wild type Hsp40s. Bioinformatics based analysis was employed to predict the fold of the mutants relative to the wild type Hsp40s. Recombinant proteins were expressed in E. coli cells and purified using nickel affinity chromatography. Fluorescence spectroscopy was used to map out the tertiary structure of the Hsp40 proteins. It was noted that the HPD motif confers thermostability to the proteins. Possible interaction of PFF1010c with PfHsp70-1 was evaluated using ATPase assay and SPR analysis. Furthermore, the same study was repeated for the motif switch mutants. PFF1010c was found to interact with PfHsp70-1, though the interaction would not be as conventional as the one between PfHsp40 and PfHsp70-1 because PFF1010c does not have the HPD motif. SPR analysis was also employed to investigate binding of the pyrimidine-quinoline hybrid compounds to PfHsp70-1. Compounds PQH1 and PQH4 were found to have high binding affinities for PfHsp70-1. The compounds also inhibited binding of PfHsp40 to PfHsp70-1. The study provides the first evidence that PfHsp70-1 interacts with a type IV Hsp40, where PfHsp70-1 and PFF1010c could be a druggable complex to reduce transmission of malaria from humans to mosquito. The pyrimidine-quinoline hybrid compounds would play a role in the proliferation of the malaria parasite by inhibiting the chaperone activities of PfHsp70-1 and PfHsp40.
Open Access
Comparative analysis of a chimeric Hsp70 of E. coli and Plasmodium falciparum origin relative to its wild type forms
(2019-05-18) Lebepe, Charity Mekgwa; Shonhai, A.; Zininga, T.
Sustaining proteostasis is essential for the survival of the cell and altered protein regulation leads to many cellular pathologies. Heat shock proteins (Hsps) are involved in the regulation of the protein quality control. Hsps are a group of molecular chaperones that are upregulated in response to cell stress and some are produced constitutively. The Hsp70 family also known as DnaK in Escherichia coli (E. coli) is the most well-known group of molecular chaperones. Structurally, Hsp70s consist of a nucleotide binding domain (NBD) and a substrate binding domain (SBD) conjugated by a linker sub-domain. ATP binding and hydrolysis is central to the Hsp70 functional cycle. Hsp70s play a role in cytoprotection especially during heat stress in E. coli. Hsp70s from different organisms are thought to exhibit specialized cellular functions. As such E. coli Hsp70 (DnaK) is a molecular chaperone that is central to proteostasis in E. coli. On the other hand, Plasmodium falciparum Hsp70s are structurally amenable to facilitate folding of P. falciparum substrates. The heterologous production of P. falciparum proteins in E. coli towards drug discovery has been a challenge. There is need to develop tools that enhance heterologous expression and proper folding of P. falciparum proteins in an E. coli expression system. To this end, a chimeric Hsp70, KPf consisting of E. coli DnaK NBD and P. falciparum Hsp70-1 (PfHsp70-1) SBD was previously designed. KPf was shown to confer cytoprotection to E. coli DnaK deficient cells that were subjected to heat stress. In this study it was proposed that KPf has an advantage over E. coli DnaK and PfHsp70-1 in its function as a protein folding chaperone. Therefore, the main aim of this study was to characterize the chaperone function of KPf relative to the function of wild type E. coli and P. falciparum Hsp70s. The recombinant forms of KPf, DnaK and PfHsp70-1 proteins were successfully expressed and purified using nickel affinity chromatography. Circular Dichroism (CD) structural study demonstrated that KPf and PfHsp70-1 are predominantly α-helical and are also heat stable. Tertiary structure studies of PfHsp70-1 and KPf using tryptophan fluorescence revealed that both confirmations of recombinant proteins are perturbed by the presence of ATP more than ADP. Interestingly, the substrate binding capabilities of these proteins were comparable both in the absence or presence of nucleotides ATP/ADP. KPf is an independent chaperone, that exhibit nucleotide binding and hydrolysis. The current study has established unique structure-function features of KPf that distinguishes it from its “parental” forms, DnaK and PfHsp70-1.