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The major emerging infectious diseases that cause human epidemics for example HIV, Influenza and Elbola have evolved in other animal hosts, but it is still lacking in the knowledge about pathogenesis, rate of evolution, strategies of vaccination and the behaviour of vaccines in human hosts. It has now been found that the rate at which pathogens spread through populations appears to be due to the rate of spread through individual hosts. (1) Pathogenesis results from several parts of the host-pathogen interactions. (1) Our immune system is well adapted to compacting these infectious organisms. This is through the relationship of multiple pathogenic substances that have shaped the immune system to become what it is now. The continual interaction of microbes with cells of the immune system has allowed many different kinds of microbes to develop approaches directed at avoiding the host immune defense mechanism. Throughout the years, there have been great advances in the understanding of immunity and the exact role of the different cell types as well as the numerous molecules involved in making up an immune response. In the recent years, it is becoming strong that the most determined and infectious microbes are finding their own way to succeed within the host, despite the occurrence of a well-functioning immune system. (2-3)
Studying the pathogenesis of many animals has been achievable and has been very useful with the use of invertebrate hosts as infection models. Using these models has allowed many studies to occur from the perspectives of both the pathogen and the host.(3) This essay intends to explain the four of the most used invertebrate models in determining host pathogen interactions. These models include: Amoebae, the nematode C.elegans, Zebrafish, and drosophila melanogaster. Invertebrate models have numerously been used because of their size and can be used to scan pathogen genomes for virulence related genes. (3) Also, they have been greatly used in combination with a pathogen to study host innate immunity. Different host models have different advantages and disadvantages, for example, to determine the right model host the following questions should be made: 1) Is the host to be used in drug discovery, 2) What host-related tools are available and are they any advantages to the study, 3). Will host tissue need to be removed and studied, 4) Will host phagocytoisis of the pathogen be studied and 5) what temperature conditions are best for studying a particular gene. (3,5,7)
The nematode worm Caenorhabditis elegans is classified as one of the simplest and widely used invertebrate model. Its characteristics make this organism a suitable invertebrate model to study. The characteristics of this organism include it being a small hermaphroditic animal which can grow up to 1mm in length and it being transparent makes the manipulation and observation of the worm easier. (4) C.elegans are also free-living organisms, which live at an optimum temperature of 20°C. Its habitat is in the soil and it feeds on bacteria such as E.coli. The soil they live in suggests these metazoans must of evolved protective responses against pathogens. This relationship of the C.elegans and microorganisms makes the worm an excellent host-pathogen model. C.elegans has been used in many studies and has been extensively studied for over 20 years. The model has many advantages, which include a fast generation time, developed genetic and molecular equipment that is used for manipulation, and the growth conditions of this model are very simple. The birth rate of these organisms is very high compared to vertebrate hosts, C.elegans can give birth to around 300 progenies by self-fertilization making it an ideal host to study. The C.elegans has been the first multicellular animal in which the genome sequence was successful for completion. The discoveries made with this model and the many cellular processes such as DNA replication, transcription are very similar to the studies of higher organisms and because of this it has been shown that C.elegans are a vital tool to understanding path finding, vertebrate neuronal growth and intra- and intercellular signalling pathways. Recent studies using this model and interacting it with enterobacterium Pseudomonas aeruginosa has shown and represents an important model for the study of pathogen and host defences. (6) The disadvantages of C.elegans are it cannot survive at 37°C. The size of the organism is also a disadvantage as well as an advantage because it is difficult to get biochemical and pathological information.
Pseudomonas aeruginosa is a human pathogen that kills C.elegans through different mechanisms, depending on the P.aeruginosa strain and medium on which this organism is grown on (the nematode growth medium NG). C.elegans are grown in laboratories and are fed with Escherichia coli and spread through the organism. On the other hand when C.elegans are fed with p. aeruginosa strain PA14 and grown on NG medium, PA14 strain spreads within the lumen of the C.elegans intestine killing the worms (4). P.aeruginosa have many strains, PA14 strain has been found to kill C.elegans in two ways: "Fast Killing" where a toxic produced by bacteria' diffusible phenazine' toxin can give rise to a killer oxidative stress. Another way C.elegans can be killed through this stain is by exposing the worm on minimal medium, this process is called "Slow Killing". Many virulence factors important in the fast and slow killing of C.elegans have been found in P. aeruginosa showing the relationship of a common virulence mechanism. (4,6) Overall, it has been established that the C.elegans model for studying pathogenicity is only limited to pathogens that can infect the nematode worm.(8)
C.elegans have also been found to act as a host for specialized pathogens. An example of this is Salmonella enterica. Salmonella enterica uses host specific approaches to establish a pathogenic relationship. It has been found that when salmonella are within the intestinal lumen they can respond to a lot of environmental conditions, through the use of protein effectors that are translocated into host cells by the type III secretory system and change signal transduction pathways within host cells as seen in figure 1. (19) It is still being studied to whether effector proteins and their targets in the host are conserved. Many S.enterica for example salmonella typhimurium have been found to kill C.elegans. Both the P.aeruginosa and S.typhimurium models have one feature in common, the establishment of a persistent infection by S.typhimurium in the C.elegan intestine, that cannot be moved by transferring the worms from S.typhimurium lawn to an Ecoli lawn. (4) In comparison to worms fed with the P.aeruginosa the normal rhythmic defecation process rapidly removed the P. aeruginosa that was left in the intestine and worms were shown to live normal life spans. (8)
Figure 1. Illustration of how salmonella enterica can enter the host cell through a type III secretion sysytem. SopE turns on Rac while SptP turns off Rac. The virulence factors that enter the host cell are degraded through ubiqutin-mediated proteasomal degradation(red). It has been shown that SopE is more senstitve to UPS-mediated degration than SptP and their changes in the half lifes favours the incactivation of Rac by SptP. The mechnaism of SptP is therefore a method of controlling the host inflammatory responses triggered by infection following salmonella internalization.(19)
Gram-positive bacteria have been used in studies and have shown to kill C. elegans. Recent works on streptococcus pneumonia, Enterococcus faecalis and streptococcus aureus have all shown to kill adult C.elegans through bacterial lawns grown on brain-heart infusion medium compared to streptococcus pyogenes and enterococcus faecium. (4) Also gram-positive bacteria showed they do not kill. Both E.faecalis and E.faecium were chosen amongst gram-positive human pathogens for characterization because both showed to kill C.elegan eggs. This shortens the model of pathogenesis because S. typhimurium do not kill worms faster to stop the generation of brood. E. faecium do not kill C.elegans but have shown to accumulate in the intestinal lumen and E.faecalis have shown to produce an infection in the C.elegans intestine. (4)(6)(8)
Zebrafish as an infection model compared to drosophila and C.elegans, have a fully developed immune system a great advantage for using as a model. A developed immune system allows the interaction of a pathogen with innate and adaptive system to be studied further in detail. The disadvantages of a Zebrafish model include that the genetics of a nematode are more advanced, the removal of nematode mutants and suppression of gene expression by RNAi is a useful tool. Genetics on Zebrafish rely on the use of injections of antisense non-DNA oligos inserted into fertilized eggs to knock out the expression of particular genes because RNAi is not a viable technique for gene splicing. Compared to vertebrate models e.g. mice the Zebrafish have the advantage of genetic screens. Zebrafish are a high hope for drug screening models. In these recent years, Zebrafish models are being used in studying human pathogens through the use of adult fish and embryos or larvae with a fully developed adaptive system or innate immunity. The Zebrafish and the human immune system have been known be very similar. Zebrafish have been very useful in studying infectious disease because of invivo imaging of host pathogen interactions with the use of many tools. Zebrafish have many features that make it a useful model to study, some of which are: they can produce hundreds of offspring every week; the embryos remain transparent during many days of the larval development. Many methods are now being targeted at using embryos under germ free conditions to study host-microbe interactions in a controlled environment. The embryos of Zebrafish from day 1 already start to produce functional macrophages that allow them to respond to microbial infections. (9)(10) The extent to which embryos developing immune systems quicker make this model possible to study the different immune cells types to host pathogen interactions. The size, high population density, and high reproductive capability make Zebrafish an excellent model for forward genetic screens. The genome sequence of Zebrafish and the many tools for reverse genetics make Zebrafish a useful model to study. Another advantage of using Zebrafish is the use of microarray and sequencing data sets that provides understandings into the Zebrafish transcriptome. Another feature that makes this model an advantage is its size of the embryo and larvae, which makes them suited for screening chemical libraries. A main disadvantage of using Zebrafish is that even though they have a fully developed immune system the tools for analysis such as marker antibodies are still lacking. (9-10)
Zebrafish have been used to study infections that have a number of pathogens such as Mycobacterium marinum, Salmonella enterica Typhimurium, and Straphylococcus aureus. A pathogen found in Zebrafish P.aeruginosa uses a number of virulence mechanisms to fight host defense, these include a type III secretion system and the production of many exotoxins. A number of host species, including Dictyostelium discoideum, Arabidopsis thaliana, C.elegans, and D. melanogaster have been used to identify the roles of these virulence causes in P. aeruginosa pathogenesis. Because of the similarities seen in Zebrafish and mammalian immune responses and the many advantages of Zebrafish as a model organism, it was investigated whether P.aeruginosa could be recognized in Zebrafish embryos. It was found that the insertion of P.aeruginosa into the circulation of Zebrafish embryos produced a lethal infection and involved quorum sensing and type III secretion system for complete virulence in late-stage embryos. An advantage to P.aeruginosa infection model in Zebrafish is the simplicity to which pathogen mechanisms can be analysed while changing host components like looking at different P.aeriginsoma mutants infected at different stages of development. Another reason is small molecules to the embryo medium can control the result of infection and therefore the model can be used to review the P.aeruginosa pathogenesis in the whole host by using chemical genetics.(11-13)
The advantage of using fish pathogens is the association between the pathogen and its host, which results in an intricate relationship. Mammalian pathogens are rarely used because of the incubation temperature. Zebrafish maintain temperatures between 26°C and 29°C and show a decreased viability at higher temperatures. In comparison to mammalian pathogens that are adapted to the temperature of their host at 37°C, streptococcus pyogenes has been the only mammalian pathogen to be studied in adult Zebrafish. In Zebrafish embryos, Salmonella arizonae and S.typhimurium have been tested and have been shown to cause an infection. S.typhimurium in small amounts is lethal and is able to replicate within and outside of the fish. S.typhimurium-infected macrophages compared to M.marinum infected macrophages are not forced out of bloodstream, which results in infection mainly in the bloodstream. It has been shown that mammalian pathogens are able to express the important virulence factors to engulf the innate immune system of Zebrafish embryos. (10)
Mycobacterium marinum is a fast growing human and animal pathogen that is used to study tuberculosis. M.marium causes an all round granulomatous infection and disease in its natural host for example in Zebrafish. A Zebrafish embryo model has been used because it can describe the events following mycobacterial infections where the macrophage accumulates producing tuberculosis granulomas. The Zebrafish model through genetic manipulation of the host and pathogen shows a relationship between pathogenic mycobacteria and host phagocytes. Reducing the macrophages in innate immunity has shown a role in limiting mycobacterial numbers while enabling the pathogens to create a systemic infection by distributing them to deeper tissues. Recent evidence of M.marinum infection suggests the contribution of innate and adaptive immunity in controlling mycobacterial infection is the same in fish and mammals. Furthermore, it has also been seen that the increase in mycobacterial numbers in both adult and embryonic Zebrafish has been seen in mammalilan models of tuberculosis.(11-12)
Drosophila melanogaster also known as the fruit fly spend their entire life cycle in the decaying organic matter. (4) Drosophila are used as models for the innate immune response to pathogens and in identifying toll like receptors as important mediators of protection against pathogen attack. The immune response against microorganisms in D.melangaster lacks the adaptive parts and relies fully on innate defences. The relying of innate defences together with the genetic tractability makes drosophila an excellent animal model to study innate responses. Before the identification of innate immunity-related genes, D.melangaster mutants were used to express the Toll and IMD pathways for antimicrobial defence in flies. Studies showed similarities between these pathways, which regulate the expression of many of the defence related genes compared to fungal and bacterial infection with the use of NF-kB like transcription factors. The toll pathway in D.melanogaster as illustrated in figure 2 is an important defence response against gram- positive bacteria. Another pathway that looks at antimicrobial peptides is the Imd pathway, which is important against gram-negative bacteria. (14-15)
Figure 2. A diagram showing two of the main signalling pathways in Drosophila. (21). Toll (in Blue) is known as a transmembrane receptor made up of leucine rich repeat proteins that contain Toll-IL-IR (TIR) domains. The toll pathway can be activated after an infection through a series of serine protease cascades that lead to the processing of spatzle(in purple). The interaction of spatzle and toll causes a intracellular cascade with the use of intracellular proteins: dMyD88 (red) and Tube (red) and pelle( a threonine-serine kinase) causing a degradation of NF-kB like transcription factors Dorsal and Dif, which then turn on the expression of antimicrobial peptides. Imd pathway regulates the expression of antimicrobial peptides with the use of the rel family transcription factor called Relish. Two cascades have been involved in the turning on of Relish these include the mitogen-activated protein kinase (MAPK) signalling pathway and a pathway involving the caspase Dredd. Recently it has also been found that the MAPK Jun N- terminal protein kinase can be regulated by Relish and be involved in transient expression of innate immunity- related genes The toll and imd pathways control the expression of a subsection of Drosophila genes that are stimulated upon septic injury.(19)
Many studies of D. melanogaster are showing that selected microorgainsms are able to get through the exoskeleton or through the intestinal epithelium to cause infection. This is either through physiological or natural route of infection. The physiological way involves feeding the D.melagaster larvae or adult flies with the microorganism of interest in the food or through spraying the fungal spores. Many different types of microorganisms, however cannot break the first line of defence and therefore require to be inoculated. This is either through pricking the dorsal part of the fly throax body cavity of the fly with a needle dipped into a microbial suspension or through microinjecting a reasonable dose of microbes directly into the body cavity. There are disadvantages with these methods, which include: the mechanical manipulation can infect the host defence response, which can cause a significant difference in the drosophila defence response depending on the route of inoculation.(14-16)
Straphyloccus aureus is an important pathogen of humans, which can cause life-threatening diseases ranging from wound infections to serious conditions such as endocarditis. The fruit fly has a short generation time and contains an innate immune system similar to humans. Infections through gram-positive bacteria induce the toll-signalling cascade, similar to the toll-like receptor cascade in vertebrates. In insects this cascade leads to the expression of many antimicrobial peptides. S. aurea has previously been seen in D. melanogaster. Recent studies have shown two pattern recognition receptors (PRRs) for detecting gram- negative bacteria in D. melanogaster. PGRP-SA (a peptiglycan recognition protein) and GNBP1, a gram-positive binding protein has recently been found to be important for toll activation following infection by the gram-negative bacteria S. aurea.(16)
D. discoideum are used as a model because of their biological features and experimental tools. Compared to other protozoa D. discoideum are open to genetic manipulation and have mostly been used for the study of signal transductions and phagocytosis. A remarkable point of this model is that it can be infected by many different pathogens because of the lower level of difficulty compared to mammalian models. A disadvantage of using this model is that they cannot survive above 27°C. D. discoideum can be used as infection models for L. pneumophila, M. marinum, Mycobacterium avium, Mycobacterium Tuberculosis, Salmonella typhimurium, Pseudomonas aeruginosa, and Burkholderia cenocepacia. These protozoa have essential features for an infection model and are very similar to human macrophages. Also, they are easy to cultivate making it a suitable host for many pathogenic bacteria and in identifying host susceptibility.(17)(20)
L.pneumophila is a gram-negative bacterium that upon spread to humans causes life-threatening pneumonia. Infection of L.pnemophila in humans happens through the inhalation of aerosolized bacteria from contaminated water sources, which then results to L. pneumophila being phagoscytosed by macrophages in the lungs and results to them being replicated. The ability to replicate is important to L.pneumophila pathogenesis because mutants defective in this process are avirulent. Experiments have shown the growth of L.pneumophila and infection in D. discoideum happens by the same process as macrophages and fresh-water amoebae.(20-21)
Pseudomonas aeruginosa is another pathogen that causes life-threatening infections in people with cooperated immune systems. Treating this pathogen involves different strategies for example one approach is to interfere with the interaction between P.aeruginosa and its host. Many models involving the nematode Caenorhabditis elegans, the fruit fly Drosphila melanogaster, and mice have been used to study host pathogen interactions. Dictyostelium discoideum is a eukaryote that lives most of its life cycle as a unicellular amoeba. The food source for amoebae is bacteria through phagocytic mechanism, which is similar to mammalian macrophages. The unicellular feature is the reason to studying the interaction between a host and a pathogen and makes D. discoideum suited for studying the host biology of pathogens because it contains different genetic manipulations. A plating assay was a technique used to study the interaction between P. aeruginosa and D. discoideum. Results showed the pathogen formed lawns on the plates with amoebae inserted in them. Virulent P. aeruginosa strains have shown to kill amoebae but if they become avirulent towards the host, the amoebae feed on the bacteria and form plaques in bacterial lawns. Results identified many P. aeruginosa mutants suggesting this pathogen uses two major virulence pathways, quorum-sensing mediated virulence and type III secretion of virulence factors to infect amoebae. Overall, it has shown that D. discoideum is a system that can be used to study host pathogen interaction of P.aeruginsoma.(17)(22)
The models discussed in this essay for host pathogen interactions have been found to be dependent on genetics and have been found to be similar to mammals. The immune system of these models is a result of an established living with pathogenic microbes. Most pathogens rely on a host for growth and replication and if not controlled can lead to the damage of the host and death of the pathogen. Therefore, a pathogen-host relationship has been discovered that confirms a balance between microbial growth and immune control by the host. The successes of this relationship has led to many approaches to fighting pathogens that been known to kill millions worldwide.