Abstract

Many Fungi have a well-developed secondary metabolism. The diversity of fungal species and the diversification of biosynthetic gene clusters underscores a nearly limitless potential for metabolic variation and an untapped resource for drug discovery and synthetic biology. Fungal secondary metabolites exhibit biological activities that have been developed into life-saving medicines and agrochemicals.

Toxic metabolites, known as mycotoxins, contaminate human and livestock food and indoor environments. Secondary metabolites are determinants of fungal diseases of humans, animals, and plants. Secondary metabolites exhibit a staggering variation in chemical structures and biological activities, yet their biosynthetic pathways share a number of key characteristics. produced, and how their biosynthetic genes are distributed across the Fungi.  Mice models with human tissue developed to date have already enhanced our knowledge of human parasites, and are useful tools for assessing anti-parasitic interventions.

Although these systems are imperfect, their continued refinement will likely broaden their utility. Some of the malaria parasite’s interactions with human hepatocytes and human erythrocytes can already be modeled with available humanized mice systems. Malaria is a vector-borne disease that involves multiple parasite species in a variety of ecological settings.

However, the parasite species causing the disease, the prevalence of subclinical infections, the emergence of drug resistance, the scale-up of interventions, and the ecological factors affecting malaria transmission, among others, are aspects that vary across endemic areas.

Such complexities have propelled the study of parasite genetic diversity patterns in the context of epidemiologic investigations. Importantly, molecular studies indicate that the time and spatial distribution of malaria cases reflect epidemiological processes that cannot be fully understood without characterizing the evolutionary forces shaping parasite population genetic patterns

CHAPTER ONE

INTRODUCTION AND LITERATURE REVIEW

1.1 Introduction

Malaria is a vector-borne parasitic disease endemic in tropical and sub-tropical regions worldwide. Despite progress on reducing its burden, nearly 40% of the world’s population remains at risk of infection (World Health Organization (WHO). 2018).

Malaria is caused by protozoa of the genus Plasmodium (Apicomplexa: Plasmodiidae), a diverse group that infects a variety of vertebrate hosts including primates (Muehlenbein et al., 2015). Such diversity has led to a disease involving multiple parasites and vector species across various ecosystems worldwide (World Health Organization (WHO) 2018).

There are four species of Plasmodium that commonly infect humans: Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax. Of those, P. falciparum and P. vivax cause most of malaria morbidity and mortality. These parasites emerged independently as human pathogens during the radiation of a Plasmodium clade associated with non-human primates and rodents hosts ((Muehlenbein et al., 2015) and that appears to be older than the origin of hominids (Loy et al., 2018).

Such complex evolutionary history may explain the biological differences between parasite species causing human malaria. There are definite similarities in the life cycles among all Plasmodium. In all species, a fraction of the circulating parasites in the blood (merozoite) differentiates into sexual stages (gametocytes) that are then taken up by the mosquito vector.

However, there are also marked differences between these parasite species in terms of their life histories. For example, when compared to P. vivax, a P. falciparum infection produces gametocytes after a longer period of time of being inoculated, but their gametocytes are infectious longer than those in P. vivax (Bousema  et al., 2011). Such differences may affect their transmission and how interventions affect their fitness (Schneider KA et al., 2013).

Plasmodium vivax and P. ovale develop a dormant liver-stage (hypnozoite) that reactivates causing relapse (infection of the red blood cells) after several weeks (to months or years) of the primary infection. Thus, a radical cure of a malaria patient with any of these two parasites requires eliminating those dormant stages not found in P. falciparum or P. malariae (World Health Organization(WHO). 2018).

On the other hand, P. falciparum-infected erythrocytes can adhere to the endothelium of capillaries and venules, a process mediated by a gene family without clear orthologs in the other human malarias (Wahlgren et al ., 2017).

Malaria illnesses are generally associated with periodic fever, chills, shivering, headache, nausea, vomiting, and may other clinical conditions, However, in the case of P. falciparum, severe diseases such as severe anemia, respiratory distress, cerebral malaria and other organ dysfunction are also common (Trampuz et al., 2003)10.

It has long been believed that P. vivax infections are relatively benign and cause mild clinical symptoms, and parasites do not sequester in the deep capillaries of organs (Price et al., 2007). However, recent studies have suggested the possibility of parasite sequestration in organs as evidenced by the P. vivax infection-associated severe illnesses and deaths (Kochar et al., 2009). Clinical symptoms of malarial infections are initiated soon after the initial liver stag

The distributions of these malaria parasites also vary worldwide (World Health Organization (WHO) 2018); there could be only one species in some endemic areas while all four Plasmodium can coexist in others. Plasmodium vivax can be present in temperate zones, whereas the other human parasites are generally restricted to tropical and sub-tropical regions.

Plasmodium falciparum and P. vivax may have overlapping distributions in many endemic areas outside sub-Saharan Africa but could differ in their temporal and spatial occurrence at a local scale (Arevalo et al., 2012). In addition to these four Plasmodium species, zoonotic infections and potential animal reservoirs further complicate malaria epidemiology.

The most recognizable zoonotic malaria is Plasmodium knowlesi, a parasite found in nonhuman primates from Southeast Asia (Yusof et al., 2016). Although human-to-human transmission has not been demonstrated, the incidence of this zoonosis is increasing in some areas when compared to the more common human malarias (William et al., 2014).

In addition, there is growing evidence indicating that another non-human primate parasite from Southeast Asia, Plasmodium cynomolgi, could naturally infect humans, even as an asymptomatic infection (Imwong et al., 2019).

1.2. Literature Review

Fungi, plants, and bacteria are the major kingdoms of life with well-developed secondary metabolism. About 500,000 secondary metabolites (also referred to as natural products) have been described to date. About 100,000 of these are derived from animals, 350,000 are from plants, and 70,000 are from microbes (Nett et al., 2009). Roughly 33,500 bioactive microbial metabolites have been described.

Of these 33,500 microbial metabolites, about 12.5% (4,200) are metabolites of unicellular bacteria and cyanobacteria, 41% (13,700) are products of Actinomycete fermentations, and about 47% (15,600) are of fungal origin (Berdy ;2012). Furthermore, the rate of discovery of new fungal metabolites has accelerated significantly in the past two decades relative to the rate of discovery in the actinomycetes, filamentous bacteria that traditionally have been the richest source of microbial natural products (Nett et al., 2009).

This complex and rich secondary metabolism is highly developed in the filamentous Ascomycota and Basidiomycota, while it is underdeveloped in the unicellular forms of the Ascomycota and Basidiomycota and in the Zygomycota, Blastocladiomycota, and Chytridiomycota (Fig. 1). The diversity of fungal species, particularly in the Ascomycota and Basidiomycota, and the accompanying diversification of biosynthetic genes and gene clusters points to an almost limitless potential for metabolic variation. In fact, one can argue that much of the ecological success of the filamentous fungi in colonizing virtually all habitats on the planet is owed to their ability to deploy arrays of secondary metabolites in concert with their penetrative and absorptive life forms.

This dependence of the fungi on secondary metabolites to conquer diverse habitats and sustain their existence within them is evidenced by the facts that most specie make multiple types of secondary metabolites, their expression is orchestrated with the life cycle and environment, and significant portions of their genomes are devoted to encoding and regulating the production of these products.

Secondary metabolites display an incredibly broad range of biological activities. Some have afforded society changing benefits, while others are associated with serious, almost intractable problems. This dichotomy is indicative of the diversity of natural products that fungi can produce. Fungal metabolites with the greatest negative impact include mammalian toxins commonly known as mycotoxins.

As many as 1,000 fungal compounds have been afforded this label, with the most widely known including aflatoxins, trichothecenes, fumonisins, ochratoxin, cytochalasins, and various indole-terpene tremogenic compounds (Brase et al., 2009). Organisms in all domains of life produce toxins, including many that are more toxic than those from fungi, but mycotoxins tend to be more problematic because of their widespread occurrence as contaminants of food for humans and livestock, as well as mold-contaminated indoor environments (Miller et al., 2014).

Understanding mycotoxin chemistry is essential to improving the ability to monitor and reduce the levels of exposure to such compounds. Fungi that produce mycotoxins and phytotoxins associated with crop diseases can cause considerable economic losses and require significant investment in their control. Metabolites that function as phytotoxins and small-molecule virulence factors produced by plant pathogenic species are responsible for many of the damaging effects of plant diseases (Miyamoto et al., 2014)

On the other hand, many important pharmaceuticals have been discovered through studies of fungal chemistry . Natural products continue to be among the most important therapeutic agents and lead compounds in medicine and have been particularly important in the development of effective therapies for cancer, malaria, bacterial and fungal infections, neurological and cardiovascular diseases, and autoimmune disorders (Newman et al., 2016).

Many agricultural chemicals are also natural productderived (Asolkar et al., 2013; Rimando et al., 2006). Fungi are particularly prolific sources of bioactive secondary metabolites  and have contributed in spectacular and indispensable ways to improvements in human and animal health .

The best known examples are the β-lactam antibiotics, which include penicillins and cephalosporins . It is difficult to overstate the tremendous effect these compound classes have had on global health because of their effectiveness against bacterial infections.

Moreover, the success of penicillins effectively spawned the development of major technological advances in microbiology, chemistry, biochemistry, and engineering and contributed in many ways to the establishment of the modern pharmaceutical industry.

Many other fungal metabolites with valuable activities are known and further examples are incorporated into the discussion below. One ongoing question regarding the dimensions of fungal biodiversity relates to judging the potential for discovery of even more novel and useful secondary metabolites, as well as the likelihood of further applications for those that are already known.

Often one reads provocative statements such as “To date, fewer than 7% of the more than 1.5 million species of fungi thought to exist have been investigated for bioactive components” (Miyamoto et al., 2014). Such statements are based on the number of metabolites described in the literature and the number of species from which those metabolites have been reported.

Ultimately, no one really knows what percentage of the total number of species has been tested for biologically active compounds, but it is in all probability much higher. Our experience and that of our colleagues who have worked in the pharmaceutical and agrochemical industries tells us that far more species have been surveyed for secondary metabolites of interest over the decades in discovery screening programs than the literature would indicate, because inactive, strongly toxic, redundant, or unproductive fermentation extracts and products are rarely pursued, let alone published or otherwise documented (Katz et al., 2016).

Recognition of already known compounds in fungal fermentations—a process referred to as dereplication (Nielsa et al.,2015; Gaudencio et al., 2015)—is a vital step in natural product discovery processes. The high frequency of encounters with known compounds in screening programs represents a substantial resource cost that generally affords little value. Although improvements in the efficiency of dereplication processes continue to develop with advances in relevant analytical technologies (Gaudencio et al., 2015), the costs in time and effort associated with “rediscovery” of known metabolites pose a significant obstacle to natural product discovery efforts. Such findings generally result in disinterest, and certainly not in publication.

Other phenomena, such as compound decomposition or loss during separation efforts or the possibility that mixtures may exert effects not detected in purified/ individual metabolites, may ultimately prevent identification of secondary metabolites that may be present. Fermentations of most strains in major service collections have probably been examined for biologically active compounds at some level (Bills et al., 2009), yet most of this work likely is undocumented.

For example, in the past, pharmaceutical companies would routinely buy strains from major service collections when the collection staff made routine transfers of their stock collections. It is impossible to know how many fungi have really been screened or how many compounds have been produced, detected, purified, and partially or fully characterized but then discarded for lack of potency and target specificity in the assays being employed for screening.

To complicate such speculation even further, the window of desired biological activity in a given screening program often may be quite narrow, e.g., inhibition of bacterial type II topoisomerases, and negative data from these efforts are seldom considered worthy of publication. What is clear is that the numbers of possible applications for fungal metabolites in medicine, agriculture, cell biology, and consumer products are limitless and constantly evolving.

Even well-known metabolites, previously discarded for one application, e.g., an antibiotic can suddenly become recognized for new and exciting biology. In our opinion, exploration of fungal chemistry with the aim of finding new applications is likely to be far more productive than surveying what one believes are new fungi using dated screening methods, e.g., growth inhibition of typical model strains of human pathogens, and certainly more productive than simple chemical screening with no biological detection method at all.

No example illustrates the unforeseen value of “ordinary” fungal metabolites better than mycophenolic acid (MPA) (Bently;2005). MPA was discovered by Bartolomeo Gosio from Penicillium brevicompactum and it has the distinction of being the first antibiotic to be purified in crystalline form (Bently; 2005).

It inhibited the growth of Bacillus anthracis, but quantities were insufficient for further investigation. Its medical application as an antibiotic was later investigated by C. L. Alsberg and O. M. Black   and by Howard Florey’s team at Oxford   but it was discarded as a potential antibiotic due to its toxicity.

This review will briefly summarize general aspects of fungal secondary metabolism and recent developments in our understanding of how and why fungi make secondary metabolites, how these molecules are encoded, and how their encoding genes are distributed across the Fungi. Readers seeking in-depth and detailed works on fungal metabolites and their biosynthesis should consult information from more comprehensive works  and a new database on functionally characterized secondary metabolite gene clusters (Liz et al., 2016).

In addition, we will illustrate the breadth of fungal secondary metabolite diversity by highlighting recent information on the biosynthesis of some of the most important fungus-derived metabolites that have contributed to human health and agriculture, as well as those that have negatively impacted crop production, food distribution, and human-built environments

1.3. Rational

Malaria is a vector-borne disease that involves multiple parasite species in a variety of ecological settings. However, the parasite species causing the disease, the prevalence of subclinical infections, the emergence of drug resistance, the scale-up of interventions, and the ecological factors affecting malaria transmission, among others, are aspects that vary across endemic areas. Such complexities have propelled the study of parasite genetic diversity patterns in the context of epidemiologic investigations.

This study therefore seeks to investigate the characteristics and properties of antimalarial of secondary metabolites from fungi. It is hope that the finding of this study will therefore aid to reduce malaria in Cameroon and the world at large.

1.4. Research Objectives

This study seeks to fulfil the following research objectives

1.4.1. Main Research objective

The aim to evaluate the antimalarial properties and characteristics of secondary metabolites from fungi

1.4.2. Specific research objectives

1. To isolate fungi.
2. To culture fungi and allow to grow producing secondary metabolites.
3. To use the secondary metabolites to prevent (Profilactive activity) and cure malaria (Curative activity)

1.5. Research questions

1. What have the government of Cameroon and other partners done to address malaria problems among under-fives in Cameroon?
2. How do the government and other key actors use communication as a tool to fight malaria?

1.6. Hypothesis

H0: The prevalence of malaria is a communication problem.

H1: The prevalence of malaria is not a communication problem.