Project description:Mechanisms regulating gene expression in malaria parasites are not well understood. Little is known about how this parasite regulates its gene expression during transition from one developmental stage to another and in response to various environmental conditions. Parasites in a diseased host face environments which differ from the static, well adapted in vitro conditions in culture. Parasites thus need to adapt quickly and effectively to these conditions by establishing transcriptional states which is best suited for better survival. With the discovery of natural antisense transcripts (NATs) in this parasite and considering the various proposed mechanisms by which NATs might regulate gene expression, it has been speculated that these might be playing a critical role in gene regulation. We report here the diversity of NATs that exist in this parasite, using isolates taken directly from patients with differing clinical symptoms caused by malaria infection. A total of 797 NATs targeted against annotated loci have been detected using a custom designed strand specific microarray. Out of these, 545 are unique to this study. The majority of NATs were positively correlated with the expression pattern of the sense transcript. Many were found to be differentially regulated in response to disease conditions. Antisense transcripts mapped to a wide variety of biochemical/ metabolic pathways, especially pathways pertaining to the central carbon metabolism and stress related pathways. Our data strongly suggests that a large group of NATs detected here are unannotated transcription units antisense to annotated gene models. The results reveal a previously unknown set of NATs that prevails in this parasite, their differential regulation in disease conditions and mapping to functionally well annotated genes. The results detailed here call for studies to deduce the possible mechanism of action, which would help in understanding the in vivo pathological adaptations of these parasites.

Project description:Mechanisms regulating gene expression in malaria parasites are not well understood. Little is known about how this parasite regulates its gene expression during transition from one developmental stage to another and in response to various environmental conditions. Parasites in a diseased host face environments which differ from the static, well adapted in vitro conditions in culture. Parasites thus need to adapt quickly and effectively to these conditions by establishing transcriptional states which is best suited for better survival. With the discovery of natural antisense transcripts (NATs) in this parasite and considering the various proposed mechanisms by which NATs might regulate gene expression, it has been speculated that these might be playing a critical role in gene regulation. We report here the diversity of NATs that exist in this parasite, using isolates taken directly from patients with differing clinical symptoms caused by malaria infection. A total of 797 NATs targeted against annotated loci have been detected using a custom designed strand specific microarray. Out of these, 545 are unique to this study. The majority of NATs were positively correlated with the expression pattern of the sense transcript. Many were found to be differentially regulated in response to disease conditions. Antisense transcripts mapped to a wide variety of biochemical/ metabolic pathways, especially pathways pertaining to the central carbon metabolism and stress related pathways. Our data strongly suggests that a large group of NATs detected here are unannotated transcription units antisense to annotated gene models. The results reveal a previously unknown set of NATs that prevails in this parasite, their differential regulation in disease conditions and mapping to functionally well annotated genes. The results detailed here call for studies to deduce the possible mechanism of action, which would help in understanding the in vivo pathological adaptations of these parasites. Plasmodium falciparum isolates were collected from patients (n = 11) with differing clinical conditions.The patients exhibited symptoms categorized as un-complicated (n =2) or complicated malaria (n = 9). Criteria for determination of complicated disease were based on World Health Organization year 2000 guidelines. Microarray array based transcriptional profiling was carried out to detect prevalence of natural antisense transcript.

Project description:Plasmodium falciparum merozoite surface protein MSPDBL2 is a highly polymorphic target of naturally acquired immune responses encoded by a single copy gene under strong balancing selection in natural populations. The MSPDBL2 protein is expressed in only a minority of mature schizonts of any cultured parasite line, and mspdbl2 gene expression in culture is increased in response to overexpression of the gametocyte development inducer GDV1 in an engineered parasite clone, so there is a need to characterise its natural expression variation. In this study, MSPDBL2 in mature schizonts was analyzed in the first ex vivo culture cycle of 102 clinical isolates from four endemic populations in different West African countries, by immunofluorescence microscopy with antibodies against a conserved region of the protein. In most isolates, less than 1% of mature schizonts were positive for MSPDBL2 (median of 0.6% overall), but the frequency distribution was highly skewed as eleven isolates had more than 3% schizonts positive and one had 73% positive. The expression frequencies were similar across the four endemic populations, and there was no significant difference between the clinical isolates overall and frequencies of positive schizonts previously seen in cultured P. falciparum laboratory lines. To explore whether expression of other gene loci correlated with MSPDBL2 expression, whole transcriptome sequencing was performed on schizont-enriched material from 17 of the clinical isolates with a wide range of proportions of schizonts positive. Transcripts of particular parasite genes were highly significantly positively correlated with MSPDBP2 positivity in schizonts as well as with mspdbl2 gene transcript levels, with overrepresentation of many genes previously implicated as likely to be involved in gametocytogenesis, but not including other genes including the gametocytogenesis master regulator ap2g. Although MSPDBL2 expression occurs in a highly variable proportion of schizonts in clinical isolates, and correlation with expression of some gametocytogenesis-related genes is consistent with regulation by GDV1, it is not apparently a direct marker of sexual commitment and its function in the parasite remains to be determined.

Project description:IntroductionUropathogenic E. coli is the leading cause of Urinary tract infections (UTIs), contributing to 80-90% of all community-acquired and 30-50% of all hospital-acquired UTIs. Biofilm forming Uropathogenic E. coli are associated with persistent and chronic inflammation leading to complicated and or recurrent UTIs. Biofilms provide an environment for poor antibiotic penetration and horizontal transfer of virulence genes which favors the development of Multidrug-resistant organisms (MDRO). Understanding biofilm formation and antimicrobial resistance determinants of Uropathogenic E. coli strains will provide insight into the development of treatment options for biofilm-associated UTIs. The aim of this study was to determine the biofilm forming capability, presence of virulence genes and antimicrobial susceptibility pattern of Uropathogenic E. coli isolates in Uganda.MethodsThis was a cross-sectional study carried in the Clinical Microbiology and Molecular biology laboratories at the Department of Medical Microbiology, Makerere University College of Health Sciences. We randomly selected 200 Uropathogenic E. coli clinical isolates among the stored isolates collected between January 2018 and December 2018 that had significant bacteriuria (> 105 CFU). All isolates were subjected to biofilm detection using the Congo Red Agar method and Antimicrobial susceptibility testing was performed using the Kirby disk diffusion method. The isolates were later subjected PCR for the detection of Urovirulence genes namely; Pap, Fim, Sfa, Afa, Hly and Cnf, using commercially designed primers.ResultsIn this study, 62.5% (125/200) were positive biofilm formers and 78% (156/200) of these were multi-drug resistant (MDR). The isolates were most resistant to Trimethoprim sulphamethoxazole and Amoxicillin (93%) followed by gentamycin (87%) and the least was imipenem (0.5%). Fim was the most prevalent Urovirulence gene (53.5%) followed by Pap (21%), Sfa (13%), Afa (8%), Cnf (5.5%) and Hyl (0%).ConclusionsWe demonstrate a high prevalence of biofilm-forming Uropathogenic E. coli strains that are highly associated with the MDR phenotype. We recommend routine surveillance of antimicrobial resistance and biofilm formation to understand the antibiotics suitable in the management of biofilm-associated UTIs.

Project description:IntroductionDrug resistance remains a major challenge in malaria treatment, especially after the emergence of resistance to artemisinin-based combined therapies. Plasmodium falciparum Kelch13 gene mutations are implicated in conferring artemisinin resistance. Thus, this study was aimed at determining the occurrence of Kelch13 (K13) propeller resistance gene polymorphism mutations in Bushenyi district, Uganda.MethodsParticipants suspected to have malaria were recruited. P. falciparum was confirmed using antigen histidine-rich protein 2 (HRP2) (Pf) (Access Bio, Inc, USA) and microscopy. Malaria-positive patients were treated with artemeter-lumefantrine (AL). Blood was withdrawn from participants who tested positive for parasites after day 3 and kept in blood filter papers (ET31CHR; Whatman Limited, Kent, UK). DNA was extracted using chelex-suspension method. Nested polymerase chain reaction (PCR) was conducted and the second-round products sequenced using Sanger's method. Sequenced products were analyzed using DNAsp 5.10.01 software and then blasted on to the NCBI for K13-propeller gene sequence identity using the Basic Local Alignment Search Tool (BLAST).ResultsOut of 283 enrolled participants, 194 completed the follow-up schedule. A total of 134 (69%) had no parasites on day 3, while 60 (31%) had parasites on that day. Out of the 60 samples, 40 (62%) were positively amplified as P. falciparum, with polymorphisms in the K13-propeller gene detected in 3 (7.5%) out of the 40 amplicons. Polymorphisms at codon 1929, 1788 and 1801 were detected separately in one sample each. Sequences have been deposited in NCBI with accession numbers PRJNA720348 and PRJNA720800.ConclusionPolymorphisms in the K13-propeller gene previously reported to be associated with artemisinin resistance were not detected in the P. falciparum isolates from Bushenyi district, Uganda. More studies need to be conducted on the new mutations detected so as to understand their association, if any, with ACT resistance.

Dataset Information

Population genomics of virulence genes of Plasmodium falciparum in clinical isolates from Uganda.

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Population genomics of virulence genes of Plasmodium falciparum in clinical isolates from Uganda.

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