Trypanosoma brucei: Protein Expression Microarrays and Circulating miRNA during Infection

The last decades have been galvanized by efforts to reduce the ever widening gap that exist between functional genomics and proteomics. Assigning molecular functions to, and breaking through the complex networks in which each and every individual protein of the cellular proteome is involved is the breathtaking task that needs to be overcome in order to understand the molecular and physiological basis underlying health and disease. System-wide analysis is an approach that can permit a better understanding of the proteome, and there is need for robust and reliable platforms for such analysis to be developed. To contribute to current efforts, I have developed and optimized methods for the production of functional protein microarrays in a miniaturized form from cDNA products and genomic DNA as template source. I have further shown that such arrays are very useful and reliable in various applications such as protein-protein interactions, protein-RNA interactions, Kinase substrate identification and antibody selection. I have validated some data obtained from these arrays in vivo in a model organism, Trypanosoma brucei. This confirms that the platform can well contribute to the already existing proteomic tools in generating reliable biological data. Moreover, using cDNA products allow for the analysis of disease-related and rare transcripts as well as other spliced variants. Current diagnostic tools for human African trypanosomiases are very invasive and in some cases are not sensitive enough, and this is compounded by a highly heterogeneous seropositive patient population that is difficult to classify. To address this, I have equally analyzed the miRNA and mRNA expression pattern in the peripheral blood of patients in search for new markers. Thirteen differentially expressed miRNAs were identified, three of which (miRNA-199a-3p, miRNA-27b and miRNA-126*) were highly selective (>95%). These miRNAs have also been reported to be differentially regulated in other diseases and miRNA-199a-3p for example is used as a diagnostic biomarker. They are therefore not suitable as specific biomarkers in sleeping sickness. I have however shown that there is deregulation of miRNA expression following T. brucei infection, and most of the differentially regulated miRNAs are related to immune responses to infectious agents and other inflammatory responses influencing disease outcome

An atypical DYRK kinase connects quorum-sensing with posttranscriptional gene regulation in Trypanosoma brucei.

The sleeping sickness parasite, Trypanosoma brucei, uses quorum sensing (QS) to balance proliferation and transmission potential in the mammal bloodstream. A signal transduction cascade regulates this process, a component of which is a divergent member of the DYRK family of protein kinases, TbDYRK. Phylogenetic and mutational analysis in combination with activity and phenotypic assays revealed that TbDYRK exhibits a pre-activated conformation and an atypical HxY activation loop motif, unlike DYRK kinases in other eukaryotes. Phosphoproteomic comparison of TbDYRK null mutants with wild-type parasites identified molecules that operate on both the inhibitory 'slender retainer' and activatory 'stumpy inducer' arms of the QS control pathway. One of these molecules, the RNA-regulator TbZC3H20, regulates parasite QS, this being dependent on the integrity of its TbDYRK phosphorylation site. This analysis reveals fundamental differences to conventional DYRK family regulation and links trypanosome environmental sensing, signal transduction and developmental gene expression in a coherent pathway

A hub and spoke nuclear lamina architecture in trypanosomes

The nuclear lamina supports many functions, including maintaining nuclear structure and gene expression control, and correct spatio-temporal assembly is vital to meet these activities. Recently, multiple lamina systems have been described that, despite independent evolutionary origins, share analogous functions. In trypanosomatids the two known lamina proteins, NUP-1 and NUP-2, have molecular masses of 450 and 170 kDa, respectively, which demands a distinct architecture from the ∼60 kDa lamin-based system of metazoa and other lineages. To uncover organizational principles for the trypanosome lamina we generated NUP-1 deletion mutants to identify domains and their arrangements responsible for oligomerization. We found that both the N- and C-termini act as interaction hubs, and that perturbation of these interactions impacts additional components of the lamina and nuclear envelope. Furthermore, the assembly of NUP-1 terminal domains suggests intrinsic organizational capacity. Remarkably, there is little impact on silencing of telomeric variant surface glycoprotein genes. We suggest that both terminal domains of NUP-1 have roles in assembling the trypanosome lamina and propose a novel architecture based on a hub-and-spoke configuration

Personalised proteome analysis by means of protein microarrays made from individual patient samples

DNA sequencing has advanced to a state that permits studying the genomes of individual patients as nearly a matter of routine. Towards analysing a tissue's protein content in a similar manner, we established a method for the production of microarrays that represent full-length proteins as they are encoded in individual specimens, exhibiting the particular variations, such as mutations or splice variations, present in these samples. From total RNA isolates, each transcript is copied to a specific location on the array by an on-chip polymerase elongation reaction, followed by in situ cell-free transcription and translation. These microarrays permit parallel analyses of variations in protein structure and interaction that are specific to particular samples

Micro RNA expression profiles in peripheral blood cells of rats that were experimentally infected with Trypanosoma congolense and different Trypanosoma brucei subspecies

To identify miRNAs whose expression are differentially regulated during trypanosome infections a microarray targeting more than 600 rat miRNA was used to analyze the miRNA expression profiles between uninfected rats and animals infected by Trypanosoma congolense and Trypanosoma brucei s.l. The potential targets of dysregulated miRNAs as well as their biological pathways and functions were predicted using several bioinformatics software tools. Irrespective of the infecting trypanosome species, eight miRNAs (seven up- and one down-regulated) were dysregulated during infections. Moreover, other miRNAs were differentially regulated in rats infected by specific trypanosome species. Functional analyses of differentially regulated miRNAs indicated their involvement in diverse biological processes. Among these, transcription repressor activity, gene expression control as well as protein transporter activity were predominant. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis of dysregulated miRNAs revealed their involvement in several biological pathways and disease conditions. This suggests possible modulation of such pathways following trypanosome infection; for example, the MAPK signaling pathway which is known to play vital roles in apoptosis, innate immune response and response to viral infections was highly affected. Axon guidance was equally highly impacted and may indicate a cross reactivity between pathogen proteins and guidance molecules representing one pathological mechanism as it has been observed with influenza HA. Furthermore, Ingenuity pathway analyses of dysregulated miRNAs and potential targets indicated strong association with inflammatory responses, cell death and survival as well as infectious diseases. The data generated here provide valuable information to understand the regulatory function of miRNAs during trypanosome infections. They improved our knowledge on host-parasite cross-talks and provide a framework for investigations to understand the development of trypanosomes in their hosts as well as the differences in the clinical and pathological evolutions of the disease

Molecular characterization of lower vaginal swabs for Human papilloma virus in association with Chlamydia trachomatis infection in Cameroonian Women

Human papilloma virus (HPV) infection is an etiological factor for cervical cancer development and Chlamydia trachomatis (Ct) is considered as a cofactor. Understanding the dynamics of HPV and Ct infection could help to explain the incidence of early onset of cervical cancer (CC) observed in Cameroon.Lower vaginal swabs and sera from sexually active women were analyzed for HPV and Ct infection in association with risk factors. Questionnaires were used to document patients’ lifestyle and risk factors.A total of 206 women participated in the study average 28.1 ± 8 years (16–50 years). HPV prevalence was 23.3% with subtypes 16 and 18 at respectively 2.9% and 1%. Ct infection totalised 40.8%, of which 23.8% were HPV- Ct co-infections. HPV infection was inversely associated with age (p = 0.028). We found a positive association between Ct infection and the number of sex partners (p = 0.012) and a negative association with parity (p = 0.032). There was no significant association between HPV and Ct infections.High rates of HPV and Ct infections could be an indicator of cervical cancer risk in the near future. There is therefore an urgent need for sensitization as well as implementation of appropriate preventive measures. Keywords: HPV, Chlamydia trachomatis, Risk factor, Co-infection, Cervical cancer, Cameroo

Population genetic structure of Central African Trypanosoma brucei gambiense isolates using microsatellite DNA markers

Genetic variation of microsatellite loci is a widely used method for the analysis of population genetic structure of microorganisms. Seven microsatellite markers were used here to characterize Trypanosoma brucei gambiense isolates from Central Africa sub-region in order to improve knowledge on the population genetic structure of this subspecies. These markers confirmed the low genetic polymorphism within Central African T. b. gambiense isolates from the same focus and strong differentiation between different foci. The presence of many multilocus genotypes of T. b. gambiense and the excess of heterozygotes found in this study play in favour of a clonal reproduction of this parasite. But some data may be indicative of a unique recombination event in one subsample. The high F-ST value indicates low migration rates between T. b. gambiense subpopulations (foci). Very negative F-IS suggests fairly small clonal population sizes of this pathogen in the different human trypanosomosis foci of Central Africa