Studying Bacterial Pathogenesis and Therapy - Term Paper Example

How useful are invertebrate infection models for studying bacterial pathogenesis and therapy Infectious diseases are responsible for over a quarter of human deaths annually according to the World Health Organization report. Also causing economic losses and its consequent suffering are pathogens causing crop and livestock damage. To identify new microbial targets and effective combat mechanisms it is important to understand the underlying cellular and molecular mechanism of pathogenesis. Pathogenesis is a complex pathogen-host interaction in which the pathogen deploys multiple factors leading to infection and host adopts diverse resistant strategies. The infection cycle involves the disease causing agent managing entry into the host either through adhesion or penetration, assimilation of nutrients to generate more copies of itself and subvert the defence systems of the host and eventually exit from the host to start another cycle in a different target. Numerous in vitro and in vivo infection models have been developed over the years to identify virulence factors and understand its regulation. The fact that some of the host-pathogen interactions have been evolutionarily conserved has led to the establishment of model systems to understand pathogenesis from both the hosts’ and pathogens’ side. Much remains to be understood about the host-pathogen interaction at the molecular level and model systems that are most informative of this could be systems in which the pathogen and host are both amenable to genetic analysis (Pradel and Ewbank, 2004). A number of non-vertebrate model organisms have been developed in order to study host-pathogen interactions which facilitates not only a better understanding of virulence mechanisms but also permit direct genetic techniques to study host defences while reducing cost and ethical constraints associated with mammalian model systems. Non-vertebrate models have also been more popular in bacterial pathogenesis studies because of the following factors: 1. The presence of an innate immune response in these simpler organisms which is important in the host response in bacterial pathogenesis 2. The Toll receptors’ discovery in Drosophila melanogaster 3. The understanding that the virulence mechanisms of microbes are conserved among different host types 4. Well developed genetic techniques for the manipulation of simpler hosts 5. Suggestions that origin of mammalian virulence in certain microbes is from the nonmammalian host interactions in the environment Some of the more common invertebrate model organisms are listed below: Species Taxonomic information Types of study Pathogen used Drosophila melanogaster Arthropoda, Insecta Virulence Pseudomonas aeruginosa Caenorhabditis elegans Nematoda, Secementea Virulence Pseudomonas aeruginosa, Burkholderia sp, Bacillus thuringiensis Galleria mellonella (Greater wax moth) Arthropoda, Insecta, lepidoptera Virulence, antibiotic testing Acinetobacter baumannii, Pseudomonas aeruginosa Dictyostelium discoideum Amoebozoa, Mycetozoa, Dictyostelia Virulence Pseudomonas aeruginosa, Legionella pneumophila, Mycobacterium spp. To understand the complexity of virulence-defence interactions a number of pathogen-host systems are required. Simple easy to handle organism such as D. discoideum, C. elegans, D. melanogaster and G. mellonella helps in identifying virulence factors and understanding their functions. Comparative studies in host models also contribute to the identification of novel elements involved in host susceptibility and resistance. Some of these elements conserved over species may also contribute to our understanding of pathogenesis in vertebrates. The genetic tractability of the simple host models will also make possible refined approaches such as gene profiling and genomic techniques to identify and elucidate specific virulence factors or pathogens with restricted host range. Cell fractioning techniques have made genome-wide transcription profiling of pathogen obtained from biopsy materials of infected patients. The widest range of model hosts amenable to maximum analytical methods makes understanding of host-pathogen interaction studies possible. Drosophila melanogaster Drosophila melanogaster emerged as an invertebrate model system to study innate immunity after parallels were drawn between the immune systems of the fly and mammals in response to microbial infection (Lemaitre, 1995). Following that, studies by Lemaitre et al demonstrated the influence of Toll and imd genes in the control of antimicrobial defence (Lemaitre, 1995). One of the important features that makes D. melanogaster an excellent model to study innate immune responses during pathogenesis is the lack of adaptive components in its immune system besides its genetic tractability. Both cellular and humeral components are however present in the innate immune system. Cellular response includes plasmocytes or phagocytes which eliminate invading micro organisms by engulfment, lamellocytes differentiates and encapsulates parasites and crystal cells eliminate the invading parasite with the help of encapsulation and melanisation (Meister and Lagueux, 2003). Antimicrobial peptides synthesized by fat body and secreted into haemolymph comprise the humoural response. Mutant D. melanogaster were used to demonstrate the involvement of Toll and IMD pathways in the humoural immune response before genetic screens were performed to identify the particular gene (Lemaitre, 1995). Ensuing studies drew parallels between these immune pathways and their vertebrate counterparts (Hoffmann and Reichhart, 2002). Toll pathway is a key response against gram positive bacteria and the Imd pathway against gram negative bacteria in D. melanogaster. The toll pathway is activated following infection by a serine protease that results in a Spaetzle processing which along with Toll initiates a cascade within the cell involving adaptor proteins dMyD88 and Tube and Pelle the threonine-serine kinase. This results in Cactus degradation and nuclear translocation of Dorsal and Dif, NF-ĸB like transcription factors which control the antimicrobial peptide expression (Janssens and Beyaert, 2003). Imd pathway regulate the antimicrobial peptide expression through transcription factor Relish which involves the mitogen activated protein kinase signalling pathway and caspase Dredd pathway (Brennan and Anderson, 2004). A reporter strain which expresses β-galactosidase under diptericin gene promoter encoding an antimicrobial peptide was used by Wu and Anderson (1998) in one of the first genetic screens. Toll signalling pathways were not involved in diptericin activation only imd genes were required for its expression. Ethyl methane sulfonate the commonly used chemical mutagen was used to randomly induce point mutations on chromosome 3 of homozygous males carrying dipt::lac Z transgene and assays performed for immune response (Corbo and Levine, 1996). Another method used to study defence responses in D. Melanogaster was expression profiling analyses using microarray studies. This helps in assessing the innate immune response following microbial infection as well as to dissect pathways involved. RNA interference has also been employed to study phagocytosis. Extending this technology to conduct genome-wide screens could elucidate similarities and differences in vertebrate and non-vertebrate immune systems. Though several human pathogens are not able to penetrate D. Melanogaster and represent a limitation for it to be used in genetic analyses, P.aeruginosa virulence factors have been successfully identified using this model system (D’Argenio, 2001). Caenorhabditis elegans Caenorhabditis elegans is a nematode which has gained considerable popularity as a model organism owing to its defined development patterns which involve specific number of cells. Being an organism with its complete cell lineage determined C. elegans is particularly useful in cellular differentiation studies. C. elegans is bilaterally symmetrical, unsegmented, has a cuticle integument, four main epidermal cords and a pseudocoelomate cavity which is fluid filled. There are considerable research advantages in using it as a model organism as it is a multicellular eukaryote which is simple enough to study in detail. C. elegans is one of the simplest organisms with nervous system and also the first multicellular organism to have a completely sequenced genome. Also the organism is easy to propagate with a short life cycle of three days at 25˚C and easy to store make it one of the most popular invertebrate model organisms. Double stranded RNA interference techniques make it possible to obtain C. elegans knock out or knock down mutants. C. elegans as a bacterial virulence factor model Known bacterial pathogens of C.elegans Gram positive Gram negative Bacillus megaterium Burkholderia pseudomallei Bacillus thuringiensis B. thailandensis Enterococcus faecalis Salmonella typhimurium Microbacterium nematophilum Pseudomonas aeruginosa Staphylococcus aureus P. fluorescens Streptococcus pneumoniae Serratia marcescens Table Ref: (Ewbank, 2002) An approach to identifying the virulence factors of bacterial pathogens is to perform screens for mutants with attenuated virulence in C. elegans. Pseudomonas aeruginosa mutants screen carried out in Ausbel lab was based on this principle (Tan, 1999). A disease model of C. elegans and P.aeruginosa was the first pathogen to be characterized (Tan, 2000). Type PA14 pathogen used by the Ausbel group was seen to produce toxins of low-molecular weight under high osmolarity conditions to kill the worms in a process called fast killing within a span of 4-24 hrs(Rahme, 1995). Mutant C. elegans resistant to oxidative stress were seen resistant to the killing. Exposing the nematode to mild heat shock or low sodium hypochlorite concentration prevented them from the fast killing process whereas nematodes that lacked P glycoprotein efflux pumps were easily susceptible to fast killing (Mahajan-Miklos, 1999). However under low osmolarity conditions several days were taken for the pathogen to colonise C. elegans and kill the animal. In this type of slow killing model, eight bacterial mutants out of the 2400 clones tested were found to be greatly attenuated for their virulence (Tan, 1999) which remarkably coincided with a murine model of P. aeruginosa pathogenesis (Tan 2000). C. elegans has also been used as a model for investigating the mechanism of action of Burkholderia species in which killing appeared to be via a toxin which acts by disrupting calcium signalling. Mutations in C. elegans that affect the calcium channels were seen to provoke increased susceptibility to paralysis. However a particular C. elegans mutant egl-9 which is resistant to lethal paralysis by P. aeruginosa strain PA01 was seen to be more resistant than wild type worms indicating a possible similarity in the mechanism of action against both the toxins (O’Quinn, 2001). Infection models to study the action of Bt toxin by Bacillus thuringiensis have been undertaken in C. elegans. A specific toxin Cry5B that provokes damage in the worm intestine has been used in studies recently to understand the action. Genetic screens of worm resistant to the toxin isolated five Bacillus-toxin resistant mutants which significantly were no more resistant than non-mutant worms to a structurally different Cry6A nematicidal toxin. Cloning of one of the mutants showed a protein homologous to mammalian β1, 3-alactosyltransferases. Characterisation of the remaining mutants could give greater insights into the cellular targets and mechanism of action of the toxin (Marroquin, 2000). Salmonella typhimurium, a pathogen well adapted to murine hosts is also capable of infecting C. elegans. However the bacteria remain extracellular throughout the course of infection unlike in the mice (Aballay, 2000). Studies have shown that infected C. elegans produce very few progeny probably due to apoptosis of germ cells which is also a strategy to protect the worm from lethal consequences of the infection (Aballay, 2001). Inducible C. elegans defences C. elegans do not possess the Rel/NF-ĸB family member in its immune pathway as in D. melanogaster. However genes homologous to Toll, pelle and cactus as well dTraf1 which also function in signalling of Rel/NF-ĸB in the pathway are present in the nematode suggesting a partial conservation of the Toll pathway in the nematode (Fallon, 2001). However studies have shown apparent functional divergence in the innate immunity pathways of the nematode and insect pathways (Pujol, 2001). Microarray techniques have been used to identify inducible genes in C. elegans. Comparative analyses of infected and non-infected populations upon Serratia marcescens infection have shown upregulation of host genes which includes genes with high sequence similarities to lectins (Drickamer and Dodd, 1999). Certain lectins are believed to have key functions in both vertebrate and invertebrate innate immunity. Certain lysozyme homologous to Entamoaeba histolytica was also seen to be induced upon infection (Nickel, 1998). These findings suggest infection-induced defence responses in C. elegans which might lead to evolutionarily conserved innate immunity mechanisms. Recent studies have shown that C. elegans require the evolutionarily conserved pathways such as the insulin signalling pathway, p38 and MAPK pathways and TGF-β pathway are required for pathogen resistance. Microarray expression profiling in recent studies have identified candidate genes which are involved in the recognition and elimination of microbial pathogens as well as those gene classes induced in response to pathogens. PMK-1 p38 MAPK pathway was observed to regulate genes induced by pathogens where as DAF-16 the Forkhead family transcription factor imparts resistance to pathogens by regulation of genes that are non-overlapping with genes induced by pathogen (Shivers, 2008). C. elegans has proved its excellence as an in vivo experimental model for universal virulent factor identification through attenuated mutant isolation. The nematode being a genetically tractable model has great potential to understand host pathogen interactions and eventually contribute to identifying new strategies to fight infectious diseases. However a limitation of C. elegans as a model is its inability to survive at 37˚C as in D. melanogaster and the administration of exact infection inocula or antimicrobial substances are technically exerting. Galleria mellonella Galleria mellonella belongs to the greater wax moth family and has been increasingly utilized to study host-pathogen interactions. It has been used to model pathogenesis in a number of organisms including Pseudomonas aeruginosa (Miyata et al., 2003), Burkholderia mallei (Schell et al., 2008), Burkholderia cepaciai (Seed and Dennis, 2008),  Proteus mirabilis (Morton et al., 1984), Bacillus cereus (Fedhila et al, 2006), Francisella tularensis (Aperis et al, 2007), and a number of pathogenic fungi (Cotter et al, 200). Also a positive correlation in mammalian models and G.mellonella has been established in the case of P. aeruginosa virulence (Jander et al, 2000). A number of factors make G.mellonella an excellent host model to study human pathogens when compared to other non-vertebrate model systems. They can be maintained at 37˚C temperatures and the infection inoculums can be injected precisely into the caterpillar’s body due to its relatively larger size. The presence of both humoural and cellular immune response pathways in G. mellonella mediated respectively by antimicrobial peptides and phagocytic cells enable host response analyses and assessments (Kavanagh and Reeves, 2004). Also to its advantage as a model organism is the fact that G. mellonella is amenable to antibiotic treatment making assessment of antimicrobial agents on it possible (Aperis et al., 2007). Studies have shown G. mellonella larvae to be quite sensitive to P.aeruginosa with a lethal dose of 50% of less than 10 bacteria injected into the haemolymph (Lysenko, 1963). The results showed bacterial inhibition as a defence response by the insect (Jarosz, 1997). A P. aeruginosa study showed a mutant variety of lipopolysaccharide deficient isolates reduced virulence in the insect whereas mutants with defects in proteases, pili or flagella did not (Jerrell and Kropinski, 1982). G. mellonella as a host model in P. aeruginosa pathogenesis G. mellonella was successfully used to study the role of Type III secretion systems or TTSS in Pseudomonas aeruginosa pathogenesis. TTSS is a highly conserved pathogenesis feature in animal and plant pathogens required for virulence factor translocation into the cytosol of target cells. The translocated virulence factors or effector proteins alter the host cytoskeleton and innate immune response pathway. The G. mellonella model was used to examine the role of known effector proteins based on the observation that TTSS pscD gene mutation in strain PA14 of P. aeruginosa resulted in attenuated virulence phenotype in the moth. A positive correlation between the results obtained in the wax moth and cytopathology assay results performed in mammalian tissue culture validated the role of G. mellonella in the study indicating it to be a successful nonmammalian host to study P. aeruginosa virulence (Miyata et al., 2003). G. mellonella model system in therapeutics A host-pathogen interaction mode of G. mellonella with gram-negative bacteria Acinetobacter baumannii to study antibacterial efficacy showed that it is an efficient system to conduct in-vivo therapeutic studies as well. Infecting the wax moth with the pathogen strain showed the rate of killing was dependent on the inoculums injected as well as the post infection incubation temperature with a greater killing at 37˚C than at 30. Antibiotics that had activity against the pathogen in vitro prolonged the survival of G.mellonella during treatment of a lethal A. baumannii infection when treated with them as against treatment with antibiotics to which the pathogen was resistant. A correlation in pathogenicity between the wax moth and humans was observed making it an ideal tool and infection model to study novel therapeutic compounds for in vivo activity against A. baumannii without the ethical, economical and logistical constraints associated with mammalian models (Peleg et al., 2009). Dictyostelium discoideum Dictyostelim discoideum is a soil-living amoeba which is particularly useful as a model organism in studying chemotaxis and cell-cell communications. Adverse conditions such as starvation result in the independent D. discoideum cells to interact with each other to form multicellular structures. The capacity of D. discoideum to function both as individual cells and a collection of cells during its lifecycle provide great insights into the fundamental principles of cell behaviour. The genome of the organism has been sequenced and it is also extremely experiment friendly from a research perspective. The simple lifecycle makes mutant selection easy by plating cells clonally on bacterial lawns. The amoeba form a plaque by ingesting the bacteria s they grow and the cells within the plaque enter the multicellular development program due to starvation. Studies using D. discoideum have established the haploid social soil amoeba as a host model for pathogens including Pseudomonas aeruginosa, Legionella pneumophila, Mycobacterium spp. and Cryptococcus neoformans. The genetic tractability of the organism makes it ideal for the study of cellular and molecular mechanisms. D. discoideum is amenable to diverse molecular genetic techniques including conventional mutagenesis, gene silencing using RNA interference, targeted gene disruption, gene replacement and antisense technique, green fluorescence protein-fusions and restriction enzyme mediated integration. The haploid nature of D. discoideum genome allows a rich variety of mutant generation (Noegel and Schleicher, 2000; Martens et al, 2002). The efficient phagocytosis shown by D. discoideum makes it particularly interesting in the study of pathogenesis. D. discoideum shares a number of traits with mammalian cells even though the evolutionary position is represented before metazoan and fungi branching (Baldauf et al, 2000). The non-specific and lectin receptors involved in phagocytosis can both recognize bacteria and following surface recognition, signal transduction and cytoskeleton mobilization shows areas of active research. The chemotactic capacity of D. discoideum is similar to leukocyte and particle uptake in macrophage pahgocytosis (Noegel and Schleicher, 2000). These features have made D. discoideum a powerful tool for bacterial virulence screening systems and models for infection analysis on the host side. D. discoideum as host model in the analysis of virulence factors of pathogens Pathogen: Pseudomonas aeruginosa Virulence trait Effect Quorum sensing system (las, rhl) Host cell growth inhibition Type III secretion (PscJ) Host cell lysis Rhamnolipids Host cell lysis Antibiotic reistance efflux pump Better survival of host cell Cytotoxin Host cell killing Pathogen: Mycobacterium avium Virulence trait Effect Intracellular growth Host cell lysis Pathogen: Mycobacterium marinum Virulence trait Effect PE-PGRS family protein Intracellular growth contribution Pathogen: Legionella pneumophila Viulence trait Effect Type IV secretion system Host cell uptake Alternative σ28 factor Intracellular growth Flagellin Intracellular growth Macrophage infectivity potentiator Intracellular growth LigA gene Intracellular growth Components homologous to SNARE system Non-lytic exocytosis Table Ref: (Steinert and Heuner, 2005) Wild type D. discoideum in Pseudomonas mutant analysis Pseudomonas aeruginosa is an environmental bacterium implicated in infections in cystic fibrosis patients and burn victims and has a wide spectrum of hosts including plants, nematodes and insects suggesting functionally universal virulence factors. Simple plating assays showed conserved virulence pathways of the pathogen in the infection of D. discoideum. D. discoideum when plated on nutrient agar plates with wild type and mutant strains of P. aeruginosa gets embedded in the bacterial lawn formed. Virulent bacteria were seen to kill the amoeba resulting in an intact bacterial lawn whereas certain avirulant mutant strains were removed by the amoeba resulting in the formation of plaques (Pukatzki et al., 2002). Mutant Dictyostelim in the analysis of Mycobacterium species Mycobacterium species are involved in opportunistic infections in patients with impaired immune systems such as trauma, AIDS and immunosuppressive therapy. They grow within human macrophages upon infection. Invasion assays with D. discoideum demonstrated Mycobacterium avium surviving within the amoeba and the intracellular life cycle which results in lysis of the host cell appeared to be similar to that observed in human macrophages (Skriwan et al., 2002). Mutant D. discoideum which was achieved by knocking out Nramp1 which is D. discoideum natural resistance-associated macrophage protein was seen to be more permissive than the wild types. Dictyostelium-Legionella infection model The dictyostelim-legionella infection model was established by analyzing the intracellular growth and activities and sub cellular localization of different strains and mutants of the pathogen. Analysis of L.pneumophila demonstrated its intracellular growth in the single cell stage of D. discoideum within its membrane bound vacuoles making it a suitable model for the analysis of Legionella infection (Solomon et al, 2000). D. discoideum cells in phagocytosis assays using cellular inhibitors demonstrated the uptake of Legionella by conventional phagocytosis involving heterotrimeric G-protein and phospholipase C pathway. The experiments also revealed the involvement of calcium levels in the cytoplasm, cytoskeleton associated proteins and calreticulin and calrexin, the calcium binding proteins of the endoplasmic reticulum in Legionella uptake (Fajardo et al., 2004). D. discoideum is an excellent model for studying pathogenesis and the availability of high throughput techniques for transcriptional studies and cryoelectron tomography for infected cell image studies make future research exciting and more refined. However inherent limitations of D. discoideum such as its inability to survive in temperatures above 27 degree C and cellular traits redundancy due to multiple proteins involved in overlapping functions should be taken into consideration. CONCLUSION Each non-vertebrate model system used has both advantage and disadvantage which itself states the need to use as many model systems required to understand the mechanisms by which infectious agents manipulate innate immune system. 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