Spirochetes of the genus Leptospira are highly motile gram-negative bacteria that cause leptospirosis, a zoonotic bacterial disease that occurs in diverse epidemiological settings but lays its greatest burden on resource-poor populations [1–6]. The disease has a broad geographical distribution due to the large spectrum of its mammalian hosts that harbor and excrete the spirochete agent from renal tubules [1,3,7]. Leptospirosis affects risk groups that are exposed to animal reservoirs or contaminated environments, such as abattoir and sewage workers, military personnel, and individuals partaking in water sports and recreation [8–12].
Moreover, leptospirosis has a broader health impact on tropical regions impoverished subsistence farmers [13–15], cash croppers, and pastoralists [16]. Other wildlife and domestic animal species are also important in the epidemiology of disease. Organisms contaminate soil and water and can remain viable in the environment for weeks to months when conditions are optimal [64].Cats may be an underrecognized source of transmission in some regions and should be considered in One Health investigations that employ sequence typing methods to advance knowledge of disease epidemiology. All dogs are at risk of leptospirosis, regardless of their signalment, geographic location, lifestyle, and the time of year. Those dogs at risk of leptospirosis include small breed dogs from urban areas, puppies as young as 11 weeks of age, geriatric dogs, dogs in rural areas, and dogs that have been inadequately vaccinated for leptospirosis (like dogs vaccinated with 2-serovar Leptospira vaccines in some regions). [65].
Furthermore, leptospirosis has emerged as a health threat in new settings as a result of globalization and climate change. Disasters and extreme weather events can precipitate epidemics [6]. The emergence of leptospirosis in Thailand [17] and Sri Lanka [18] highlight the disease's potential for the disease to rapidly spread and cause large unexplainable nationwide outbreaks. Finally, the global expansion of urban slums worldwide has created better conditions for rat-borne transmission of the disease[19–24]. Epidemics are reported in cities throughout the developing world [6,19,25] and will likely intensify as the world’s slum population doubles to two billion by 2030 [26].
Leptospirosis has life-threatening manifestations. The symptoms from this bacterial infection are usually grouped into 2 forms, Anicteric and icteric leptospirosis [49,50]. Anicterie leptospirosis causes a common symptom of Acute febrile bacteremia, which patients get flu-like symptoms like headaches and fevers [51]. Another hallmark characteristic of anicteric leptospirosis is that it causes conjunctival suffusion, which is the dilation of blood vessels in your eyes[51]. Anicteric leptospira is almost always misunderstood as a regular flu and lacks proper treatment, worsening the patient's situation.
Icteric leptospirosis, also known as Weil’s disease, On the other hand, is a more severe form of leptospirosis [50]. Patients infected with Icteric leptospirosis are jaundiced, meaning they would have yellowish sclera and skin [52]. Moreover, icteric leptospirosis [31] has emerges as a major cause of diseases such as pulmonary hemorrhage syndrome [27–30], acute kidney injury [53] and multi-organ failure [54]. Case fatalities for pulmonary hemorrhage syndrome and Weil’s disease are more than 10% and 70% respectively [14]. The majority of leptospirosis patients are misdiagnosed as undifferentiated fever [16,32–38], malaria [16], dengue [39–41] and other types of acute febrile illnesses. This misdiagnosis contributes to under-reporting of cases [44,45], as well as unnecessary deaths [39].
[46]The complement system is composed of more than 50 plasma proteins and receptors. Traditionally considered as one of the first lines of defense against invading microorganisms due to its opsonic, inflammatory, and lytic activities, the complement roles extend far beyond pathogen killing. Complement effector functions result from the activation of three different pathways: classical, alternative, and/or lectin (CP, AP, and LP, respectively). While the AP and LP participate in the innate immunity, the CP is generally activated by the presence of IgG or IgM that are specifically bound to antigens.
The AP is initiated by the spontaneous hydrolysis of an intra-chain thioester bond located in the C3 molecule. While the LP is being activated with lectins such as mannose-binding lectin or ficolins, its binding to carbohydrates is commonly found on microorganisms’ surfaces. During activation, fragments C3b and C4b are generated and they bind covalently to acceptor surfacessuch as immune complexes, foreign, and host cells that are located in the vicinity of the activation site. On these surfaces, C3 and C5 convertases are formed which further lead the formation of the membrane attack complex to culminate with microorgan-ism lysis. As a consequence of the activation, particles opsonized with iC3b, C3b, and C4b are more efficiently internalized by neutrophils, monocytes, and macrophages once bound to complement receptors present on these cells’ membranes. CR2 promotes activation and proliferation of B lymphocytes in the presence of C3d/C3dg fragments covalently bound to antigens inducing the production of antibodies. In addition, C3a and C5a fragments are important anaphylatoxins and also chemoattractant factors for inflammatory cells [47]. In order to protect the host against self-damage, complement activation is tightly controlled at all stages of the cascade by several soluble and cell surface regulators. C1 inhibitor, Factor I (FI), Factor H (FH), and C4b-binding protein (C4BP) are all soluble complement regulators. Complement receptor type 1 (CR1 or CD35), membrane cofactor protein (MCP or CD46), decay accelerator factor (DAF or CD55), and CD59 are all cell-anchored regulatory receptors.[47,48]
However, there are some proteins in Leptospira which evade the complement system through binding their outer membrane proteins with certain proteins of our very own complement system, inhibiting the formation of MAC. The best characterized complement evasion molecules from Leptospira include (i) leptospiral endostatin-like proteins A and B [LenA and LenB (56, 57)], (ii) Leptospira immunoglobulin-like (Lig) proteins A and B [LigA and LigB(55, 58)], and (iii) Leptospiral complement regulator-acquiring protein A [LcpA (59)]. All of these proteins have been shown to bind to more than one complement regulator, and they seem to be involved in not only immune evasion but also in adhesion and invasion by them interacting with ECM and plasma proteins such as plasminogen (PLG) [60].
After intensive literature review and data mining, we have found potential biomarkers (miRNAs) which are present at scale towards acute leptospirosis. This includes the hsa-miR-144-3p, hsa-miR-21-5p [61], and hsa-miR-601 [62] that target toll-like receptors; and miR-3646, miR-4427, miR-3352, miR-4430, miR-148b-5p, miR-6874-3p, and miR-6787-3p that target complement factor H. With significant target score [63] such hosts miRNA down regulate the mRNAs which produce complement proteins in our system.
We are looking for miRNA and target sequence interaction to propose to design our synthetic miRNA mimics/ inhibitors based on these biomarkers. Our aim is to upregulate our immune system, specifically immune system response such as TLR2 and complement factor H to enhance our complement system. Our project aims to last for 2 years.
We believe our project is a useful application of synthetic biology as our therapeutics are a better alternative, in terms of specificity, effectiveness and economical efficiency, to the current ineffective antibiotics. In terms of specificity, for example, in response to leptospirosis, the liver and kidneys are more affected. miRNA based drug therapy is more targeted, more effective and more cost efficient. Unlike conventional therapy that has many side effects, our miRNA based drugs not have this problem.
We began with a problem. A student interested in watersports found an article that shed light on an infection of leptospirosis after a watersport contest that was held in Malaysian Borneo. Out of the 189 contest participants contacted by the Centers for Disease Control in Malaysia, 80 (42 %) of them met the case definition for leptospirosis. The contraction factors extented exposure for in the rain-swollen Segama river [66]. Upon further research we found that even after 24 years of the incident, humankind still lacks early diagnosis and cure tools. Besides, Leptospira infects pets which can transmit this disease to humans. Both pets and sports are integral parts of our lives and we shall look out for solutions and spread awareness through this competition.