Identification and determination of genetic diversity in simian malaria parasites among wild long-tailed monkey populations from various regions of Peninsular Malaysia

Malaria still remains a major cause of mankind death globally in spite of a century of research. It is clear that, understanding and accurate measurement of malaria incidence as one of the most critical tropical diseases have significant role to control and prevent this lethal infection.Since the probability of P. knowlesizoonotic transmission to humanswas proven, the necessity of study on simian malaria parasites isincreasingly obvious. Simian malaria parasites are readily infectious to long-tailed (Macaca facsicularis) and pigtailed (Macaca nemestrina) monkeys. The chance of this pathogenic species switching to humans as their desired host is not ignorable due to theincrease of human populations in recent years, as well asecological alterations which are causedby pollution or deforestation. In fact macaques are preferred host for Anopheles mosquitoes but human may change this situation by alteration on the natural habitat of macaques and mosquitoes. In this study, the distribution of five Plasmodium species namely: P. knowlesi, P. inui, P. cynomolgi, P. fieldi and P. coatneyiamong the wild populations of M. fascicularisin six states in Peninsular Malaysia were determined using highly specific (nested-PCR) assays. The advantage of this method lies in its ability to detect very low numbers of parasites. Monkey blood samples provided by the Department of Wild Life and National Parks Malaysia (PERHILITAN) were collected on Flinders Technical Association cards (FTA cards). FTA cardsare strongly recommended for collecting fluid samples for epidemiological studies particularly when sampling is being done in areas far from the main laboratory or during a long sampling trip. The prevalence of these five simian Plasmodiumspecieswas determined in 13 different locations of Peninsular Malaysia.Geographic distribution of the collected samples provided by PERHILITAN ranged from Northwest [Penang (island), Penang (mainland) and Jerejak Island], West (Selangor, Perak), Southwest (Negeri Sembilan), East (Pahang) and Northeastern (Kelantan) of Peninsular Malaysia. DNA was extracted from the blood spots on FTA Cards. All five simian Plasmodium species were successfully detected using nested PCR assay. Among the five species, P. knowlesi had the highest prevalence (34.3%), followed by P. inui (33.2%),P. cynomolgi (27.9%), P. fieldi (27.6%) and P. coatneyi(16.6%). Co-infections of macaques with multiple species of Plasmodium parasites were also observed. Kelantan had the highest prevalencerate among the states for all five simian malaria species. The incidence rate of three Plasmodiums pecies which are P. inui, P. fieldiand P. knowlesi were higher than 50% among the samples obtained from this state. Twenty positive DNA samples with single infection of P. knowlesi(12 samples) andP.cynomolgi (8 samples)as well as 20 uninfected monkey DNA samples were chosen to investigate the genetic diversity of these parasites using 26 different ISSR (inter simple sequence repeat)markers. A total of 103 ISSR loci for infected samples and 95 for uninfected samples were generated. The analyses of the infected and uninfected samples using ISSR markers confirmed the efficiency of both the markers and the clustering methods. By these methods, the samples not only were separated according to their geographical distribution, but the samples were grouped into two distinct clusters according to the species of the malaria parasite. Overall, this study shows the importance of research aboutmalaria parasite species which are infectious toM. fascicularis and the necessity of preventive and control plans to decrease the chance of host- switchoccurrence.Thisstudy also provides information for further investigations to design and develop diagnostic microsatellite markers for the macaques in the future

Genetic diversity and population structure of long-tailed macaque (Macaca fascicularis) populations in Peninsular Malaysia

Background: The genetic diversity and structure of long-tailed macaques (Macaca fascicularis) in Peninsular Malaysia, a widely used non-human primate species in biomedical research, have not been thoroughly characterized. Methods: Thirteen sites of wild populations of long-tailed macaques representing six states were sampled and analyzed with 18 STR markers. Results: The Sunggala and Penang Island populations showed the highest genetic diversity estimates, while the Jerejak Island population was the most genetically discrete due to isolation from the mainland shelf. Concordant with pairwise Fst estimates, STRUCTURE analyses of the seven PCA-correlated clusters revealed low to moderate differentiation among the sampling sites. No association between geographic and genetic distances exists, suggesting that the study sites, including island study sites, are genetically if not geographically contiguous. Conclusions: The status of the genetic structure and composition of long-tailed macaque populations require further scrutiny to develop this species as an important animal model in biomedical research

Exploring the Evolution and Function of the first Enzyme in Histidine Biosynthesis

Adenosine triphosphate phosphoribosyl transferase (ATP-PRT) catalyses the first committed step of the histidine biosynthetic pathway in archaea, bacteria, fungi, and plants. The enzyme is allosterically inhibited by histidine, as a means of controlling the pathway in response to metabolic demand for this essential amino acid. Two molecular architectures including a homo-hexameric long form (HisGL) and a hetero-octameric short form (HisGs) have been described for ATP-PRT. A single chain of HisGL comprises domains I and II that house the active site while domain III, or the regulatory domain provides the binding site for histidine as an allosteric inhibitor. HisGS shares a highly similar catalytic core to HisGL, but is associated with a second protein, HisZ, to form its active, regulated complex. Recently, a possible third architecture, the “super-long” form, has been revealed. Resembling the non-covalent hetero-octameric complex of the short form, this protein has been studied from Leuconostoc mesenteroides. It appears likely that this protein has evolved via the fusion of the hisG and hisZ genes encoding the short form complex. This thesis characterises a new member of the ATP-PRT short form from the hyperthermophile Aquifex aeolicus (AaeHisZGS), the super-long form from Leuconostoc mesenteroides (LmeHisZGSL) and explores the functional and evolutionary relationship between the long and short form enzymes. Chapter 2 reports the characteristics of the hyperthermophilic AaeHisZGS complex. HisZ regulatory domains of short form ATP-PRTs and histidyl-tRNA synthetases (HisRS) are derived from the same ancestral gene. Specifically, the regulatory AaeHisZ domain of AaeHisZGS shows high similarity to AaeHisRS due to the presence of the C-terminal anti-codon binding domain, not found for all HisZ sequences. The role of this C-terminal anti-codon binding domain in ligand binding is discussed and a comparison between the wild-type enzyme and the truncated AaeHisZ-Y303term is presented. Kinetic studies confirmed that ADP can act as an alternative substrate equivalent to ATP for both AaeHisZGS wild-type and AaeHisZ-Y303term complexes. Furthermore, the truncation of the C-terminal domain of AaeHisZ caused a change in the histidine inhibition characteristics. Firstly, a two-fold increase in the KiHis of the AaeHisZG-Y303term complex was noted, closely matching the value reported for the mesophilic short form HisZGS from Lactococcus lactis, which naturally lacks the C-terminal HisZ domain. Secondly, changes in the thermodynamic behaviour of the complexes upon histidine binding were observed by isothermal titration calorimetry (ITC), which surprisingly suggests a significant involvement of the AaeHisZ C-terminal domain in the inhibitor binding process. Chapter 3 describes the investigation of the thermophilic short form enzyme from Thermotoga maritima (TmaHisZGS) as well as the modularity of short form ATP-PRT complexes. Several mix-and-match combinations of HisG and HisZ proteins from different sources were assessed for complex formation and functionality. Additionally, the successful design and construction of a chimeric protein — by covalent fusion of the regulatory domain of the long form enzyme to the C-terminus of a short form enzyme — was carried out to probe the ability of the regulatory domain to confer both feedback regulation and enzyme function into a contemporary short form HisGS. Chapter 4 reports the characterisation of the novel ATP-PRT structure, the super-long LmeHisZGSL, that combines HisZ and HisGS in a single open reading frame. Several attempts were made to achieve a functional LmeHisZGSL enzyme with ATP-PRT activity, with no success. Also, the covalently linked LmeHisZGSL was split into two separate units LmeHisG and LmeHisZ. The non-covalent complex between the two subunits of the enzyme was purified successfully but the complex was still devoid of ATP-PRT activity. Chapter 5 describes studies undertaken to give insight into the evolution of the ATP-PRT family using directed evolution methods such as error-prone PCR (epPCR). The generation of gene variant libraries for four different inactive/marginally active ATP-PRT constructs using epPCR is detailed, with a heavy focus on introducing mutations in an unbiased fashion, while achieving a reasonable number of mutated copies in each library. To identify and select variants which attained or improved activity, an E. coli ΔhisG knockout strain, auxotrophic to histidine, was used. A mutant (mutant 50) was successfully identified and isolated from the chimeric LlaHisGS-McubACTChimera random mutation library, displaying three substitutions and exhibiting measurable catalytic activity and histidine sensitivity.</p