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Infection in a dish: high-throughput analyses of bacterialpathogenesisC Le´opold Kurz1,2,3 and Jonathan J Ewbank1,2,3 Diverse aspects of host–pathogen interactions have been in the infection process regardless of the host. These can studied using non-mammalian hosts such as Dictyostelium be identified and characterised using genetically tractable discoideum, Caenorhabditis elegans, Drosophila melanogaster and inexpensive non-mammalian models. In addition, the and Danio rerio for more than 20 years. Over the past two years, molecular and genetic tools that have been developed for the use of these model hosts to dissect bacterial virulence use with these simple organisms, combined with their mechanisms has been expanded to include the important well-studied cellular biology and/or immunology, enable human pathogens Vibrio cholerae and Yersinia pestis.
one to decipher the complex interactions between host Innovative approaches using these alternative hosts have also and pathogen.
been developed, enabling the isolation of new antimicrobialsthrough screening large libraries of compounds in a C. elegans The four organisms listed above have many factors in Enterococcus faecalis infection model. Host proteins required common that make them very useful as model hosts, such by Mycobacterium and Listeria during their invasion and as the availability of their fully sequenced genomes intracellular growth have been uncovered using high- and their ease of culture [1]. These alternative hosts throughput dsRNA screens in a Drosophila cell culture system, are being used for approaches as diverse as testing the and immune evasion mechanisms deployed by Pseudomonas virulence of chosen pathogen mutants [6,7], screening aeruginosa during its infection of flies have been identified.
large banks of pathogen mutants for those with attenu- Together, these reports further illustrate the potential and ated virulence [8,9,10] or dissecting the host mechan- relevance of these non-mammalian hosts for modelling many isms involved in pathogen invasion and intracellular facets of bacterial infection in mammals.
Addresses1 Centre d'Immunologie de Marseille-Luminy, Universite´ de la In addition, they have unique features that are relevant to Me´diterrane´e, Case 906, 13288 Marseille Cedex 9, France the study of specific aspects of host–pathogen interac- 2 Institut national de la sante´ et de la recherche me´dicale (INSERM), tions. The amoeba D. discoideum is a professional phago- U631, 13288 Marseille, France3 Centre National de la Recherche Scientifique (CNRS), UMR6102, cyte that can be used to decipher the molecular basis of 13288 Marseille, France phagocytosis and phagosome maturation [4]. Addition-ally, it can give insights into how certain intracellular Corresponding author: Ewbank, Jonathan J ([email protected]) bacterial pathogens survive in the phagolysosome [14].
The fly D. melanogaster possesses a very well-studied Current Opinion in Microbiology 2007, 10:10–16 innate immunity [15] that has contributed to the under-standing of immune mechanisms in mammals. More This review comes from a themed issue on recently, it has been used to analyze the mechanisms Host–microbe interactions: bacteria used by pathogens to evade the host immune system Edited by Pamela Small and Gisou van der Goot [16,17,18]. Finally, genetic screens for bacterial viru- Available online 18th December 2006 lence genes in a vertebrate with a fully developedimmune system [19] are possible with the fish D. rerio.
1369-5274/$ – see front matter This review focuses on recent work with the alternative # 2006 Elsevier Ltd. All rights reserved.
model hosts D. discoideum, C. elegans, D. melanogaster and D. rerio in these new investigative paradigms.
New infections modelled with alternative The use of mamma Author's personal copy
lian models to identify and understand An increasing number of human bacterial pathogens are the virulence factors of human pathogens is indispensa- being tested in non-mammalian hosts in order to con- ble. Alternative models such as the amoeba Dictyostelium veniently study their virulence. In addition to established discoideum, the nematode Caenorhabditis elegans, the insect models such as Pseudomonas aeruginosa [20,21], Salmonella Drosophila melanogaster and the fish Danio rerio can be typhimurium [22–24] or Serratia marcescens [25,26], several complementary systems for such studies [1–5]. This is pathogens including Listeria monocytogenes [27,28], Yersi- possible because many human pathogens have a low nia pestis (see below) and Vibrio cholerae, the causal agent species-specificity and can infect hosts ranging from of cholera, have recently been added to the list of micro- insects and nematodes to fish, as well as other mammals.
organisms that are capable of causing lethal infection of They rely on universal virulence factors that are involved the nematode and the fly.
Current Opinion in Microbiology 2007, 10:10–16 Infection in a dish: high-throughput analyses of bacterial pathogenesis Kurz and Ewbank In humans, expression of cholera toxin (CT) by V. cholerae to identify in vivo new antibacterial molecules. A similar provokes a rise in cAMP in the intestinal epithelium, the system involving flies to be used to identify antifungal opening of ion channels and consequently, loss of water drugs is also being developed [31,32].
into the intestinal lumen. In mice, this secretory diarrhoeacan be successfully treated with the channel-blocker Random screens for the identification of clotrimazole. It has now been reported that oral infection bacterial virulence genes of the fruit fly by V. cholerae leads to the death of the Three recent reports [8,9,10] using D. discoideum, C.
animals in a manner somewhat similar to that observed in elegans and D. rerio as hosts to screen bacterial mutant humans, including rapid weight-loss [7]. CT is required libraries of Klebsiella pneumoniae, Y. pestis and Streptococcus for full virulence in the fly model and, remarkably, flies iniae, respectively, have further strengthened the rele- with loss-of-function mutations in genes encoding homo- vance of these simple hosts.
logues of the known targets of CT resist infection.
Furthermore, clotrimazole can help cure flies infected K. pneumoniae is an important human pathogen that, as its with V. cholerae [7].
name suggests, causes pneumonia. Its interaction withalveolar macrophages can be modelled using D. discoideum During the lethal colonization of the C. elegans intestine as a surrogate phagocyte. D. discoideum is normally able to by V. cholerae, however, CT does not appear to play an feed on wild type Klebsiella. Cosson and colleagues important role [6]. But, using a reverse genetic approach, [10,33] elegantly combined the genetics of D. discoideum Vaitkevicius et al. [6] demonstrated that the quorum and the genetics of K. pneumoniae. They first identified a sensing regulated protease PrtV is essential for this kill- new gene ( phg1) that, when mutated, rendered the ing. Moreover, they obtained data suggesting that this amoeba especially susceptible to infection and unable protease is important to V. cholerae in its natural niche [29] to grow on Klebsiella. They then isolated Klebsiella for its resistance to the marine plankton that graze on the mutants that supported the growth of the phg1 mutant bacterium. Finally, they measured an increased interleu- amoeba: among the mutated bacterial genes were several kin-8 (IL–8) secretion in human epithelial intestinal cells that were required for biosynthesis of lipopolysaccharides exposed to a V. cholerae prtV deletion mutant, compared to and amino acids. They tested several of the isolated that of the parental strain, suggesting a role for this bacterial mutants in a mouse pneumonia model and found protease in modulating (directly or indirectly) the host an attenuation of virulence [10].
response in vertebrates [6].
The genetic manipulation of both host and pathogen Together, these reports illustrate to what extent nema- enabled the authors to create a 2D virulence array show- tode and fly can be relevant for the study of the causative ing that distinct groups of host genes are necessary to agent of cholera. Importantly, the work by Blow et al.
resist infection by various bacterial pathogens and [7] are compatible with the idea of using Drosophila to mutants (Figure 2). They were also able to demonstrate screen for chemicals that inhibit CT in vivo, following conservation of both virulence factors and defence genes a precedent set by the Ausubel laboratory [30], using because Drosophila phg1 mutants are more susceptible to K. pneumoniae infection [10].
In vivo screens for new antimicrobials Y. pestis, the causative agent of plague, can form a biofilm The massive use of antibiotics, combined with the high that is important for dissemination by its vector, the adaptation capacity of bacteria has created a huge public flea. A Y. pestis biofilm can also accumulate on the head health problem with many human pathogens becoming of C. elegans, and this is clearly a more accessible model resistant to multiple antibiotics. Therefore, there is a real for studying biofilm function than is looking in the gut need for new antibiotic molecules. Moy et al. [30] of the flea [34]. As biofilm formation is only one aspect of cleverly used an infection system involving a C. elegans Y. pestis pathogenicity, Styer et al. [9] developed a immunocompromised mutant and Enterococcus faecalis to nematode-based infection system to identify Y. pestis screen thousands of synthetic and natural molecules to virulence genes not related to biofilm formation. They Author's personal copy
promoted host survival (Figure 1). This in showed that a biofilm-deficient mutant of Y. pestis colo- vivo screen not only permitted the identification of eight nises the intestine of C. elegans and provokes an early molecules that affect bacterial growth in vitro (minimum death of the host. They used this infection model to inhibitory concentration [MIC] <35 mg ml 1) but also of screen a bank of Y. pestis mutants for those with attenu- eight other products that either impair pathogen viru- ated virulence in the nematode. Remarkably, despite the lence or boost host innate immunity in the absence of differences between nematodes and mammals, they significant in vitro activity (MIC > 125 mg ml 1) [30].
identified two genes necessary for full virulence in an Even though the efficiency and toxicity of the identified intranasal mouse model of Y. pestis pathogenesis, genes molecules does need to be tested in mammals, this that had previously not been implicated in Y. pestis system represents a very promising screening platform pathogenicity [9].
Current Opinion in Microbiology 2007, 10:10–16

Host–microbe interactions: bacteria Protocol used by Moy et al. [30] to screen in vivo for new antimicrobial compounds using an established C. elegans–E. faecalis infection system.
After culture and amplification of nematode numbers on growth plates (seeded with the Escherichia coli strain OP50) synchronised populationsof worms are transferred to infection plates, seeded with E. faecalis strain MMH594. After 8 h, worms are washed off the plates and approximately25 individuals added to each well of a 96-well microtitre plate and then assayed for their survival. Compounds or extracts that extended wormsurvival by twofold to threefold after 6–8 day's culture were selected for further analyses. Whereas this screen was carried out manually, automationof different steps is possible with tools such as Union Biometrica Biosort ( It is important to notethat similar screens for antimicrobial compounds can be designed using C. elegans and other pathogens. The time when worm survival is scoredwill vary depending on the pathogen used.
S. iniae is a bacterial pathogen that is able to infect fish involved in invasion and survival in human macrophages and humans. To analyze the interaction between strep- tococcal pathogens and their natural hosts, Miller et al.
[8] created a bank of bacterial mutants and screened it These three studies further validate the use of non-mam- using zebrafish. They wished to identify bacterial malian hosts for large-scale screens to identify bacterial mutants specifically deficient in their capacity to disse- virulence genes relevant to infection in mammals. More- minate in the brain. To facilitate the screening process, over, the genetic manipulation of the host, as exemplified they used a signature-tagged mutagenesis strategy by the work of Benghezal et al. [10], expands the range of Author's personal copy
rmitted the analysis of fish co-infected by models available for this kind of screening approach, in a a pool of 12 distinct mutant strains. Doing so, they manner reminiscent of the directed modification of mice, screened 1128 signature-tagged transposon bacterial through trangenesis [36] or the creation of human-mouse mutants and determined which bacterial mutants were chimeras [37], but without any of the ethical concerns.
not present in brain extracts from infected fish. Interest-ingly, 7 out of the 41 bacterial mutants isolated had Identification of host molecules required for transposon insertions in genes required for the production pathogenesis and how the pathogen evades of capsular polysaccharides. Finally, using the bacterial the immune system mutants they isolated, they showed in a human whole The host factors involved in the infection processes are blood assay for phagocytosis that the capsule of S. iniae is not restricted to ‘immunity genes' such as those coding Current Opinion in Microbiology 2007, 10:10–16

Infection in a dish: high-throughput analyses of bacterial pathogenesis Kurz and Ewbank Hypothetical host–pathogen 2D array inspired by data from Benghezal et al. [10]. The ability of host mutants to resist (blue) or their susceptibilityto (red) different bacterial strains and bacterial mutants is indicated. Gene names are arbitrary with hrg and pvf for ‘host resistance gene' and‘pathogen virulence factor', respectively. Based on this matrix, it can be speculated that hrg-1 is specifically involved in a mechanism necessaryfor host resistance to bacterial virulence factors encoded by pvfB and pvfC. The hrg-3–pvfD interaction would correspond to the case describedby Liehl et al. [18] with hrg-3 and pvfD being the Drosophila Imd and Pseudomonas aprA genes, respectively. Finally, hrg-2 and hrg-4 couldencode host proteins necessary for bacterial invasion by pathogen C and pathogens B and C, respectively, corresponding to the observationsdescribed in the reports by Philips et al. [12] and Agaisse et al. [13].
for interleukins or Toll-like receptors (TLRs). This is family. This work also highlighted a role for autophagy in especially the case for intracellular bacterial pathogens the control of L. monocytogenes infection [12].
that have to enter the cell and avoid being degraded inphagolysosomes. Therefore, intracellular bacteria have In contrast to these two studies, which used automated developed many ways to hijack the endocytic or phago- microscopy, a third study was performed manually [11].
cytic routes [38,39]. Macrophages are often confronted by In this painstaking project, interest was focused espe- intracellular pathogens because they are professional cially on the interaction between the L. monocytogenes phagocytes. The Drosophila S2 macrophage-like cell line toxin listeriolysin O (LLO) and host factors that enable has now been used in three large-scale RNA interference the bacteria to escape from the phagosome. The authors (RNAi) screens in order to identify host factors required used RNAi to inactivate host genes and combined this for entry and survival of intracellular bacterial pathogens with bacteria mutated in LLO. In a first set of experi- [11,12,13]. The first two analyses combined auto- ments, they used an LLO-deficient bacterial strain mated microscopy with the use of green fluorescent unable to leave the phagosomes of normal cells and protein (GFP)-tagged Mycobacterium fortuitum [13] or screened for dsRNAs that restored the capacity of these Listeria monocytogenes [12] to screen a bank containing mutants to escape into the cytoplasm. The corresponding 21 300 dsRNAs (targeting >95% of annotated Drosophila genes would be expected to be elements of the host genes in a redundant fashion). They showed that factors pathways targeted by LLO. In a second set of experi- involved in vesi Author's personal copy
cle trafficking and actin cytoskeleton ments, they used a bacterial mutant producing a LLO organization are necessary for internalization and intra- toxin that lacks the PEST sequence which normally cellular survival of these two pathogens. Moreover, they makes the protein relatively unstable. They screened identified Peste (French for ‘plague'), a Drosophila homo- for dsRNAs that rendered S2 cells more susceptible to logue of the scavenger receptor CD36, as being crucial for this stable toxin in order to determine which host entry of L. monocytogenes and M. fortuitum into the S2 cells, enzymes control LLO toxicity. On the basis of their whilst being dispensable for phagocytosis in general results, they proposed a model in which the pore-forming [12,13]. On the basis of these observations, the study LLO inserts into the membrane of the L. monocytogenes- was extended to mammalian cells and new roles in uptake containing phagosome, thus impairing its acidification of bacteria were described for members of the CD36 and maturation. Concerning the host's control of LLO Current Opinion in Microbiology 2007, 10:10–16 Host–microbe interactions: bacteria toxicity, their screen identified serine palmitoyl-CoA bacterial pathogens for their mammalian host. For transferase (SPT), which is an enzyme necessary for instance, some virulence genes involved in mammalian sphingolipid metabolism, as a key factor for host resis- pathogenesis are only expressed at 37 8C, whereas not all tance [11].
the model animals described in this review can be grownat this temperature [5]. Moreover, C. elegans does not The experimental systems described in these three reports possess macrophage-like cells [42] and some receptors can thus be used to shed light on the complex interactions necessary for the engulfment of intracellular bacterial between the host and an intracellular pathogen that are pathogens in mammalian cells are absent from the surface both fighting for their survival. But just as is the case for any of non-mammalian cells, thus limiting the utility of sim- model system, the results come with several caveats. It is ple organisms for the study of intracellular pathogenesis.
well known that a dsRNA can interfere with off-target The same is true concerning mammalian signalling path- genes and so generate false positive results [40]. Conver- ways specifically targeted and hijacked by some patho- sely, important genes can be missed if they are not gens (e.g. although it possesses one TLR, NF-kB expressed in or on S2 cells, as is indeed the case for some transcription factors, crucial for mammalian immunity, receptors involved in phagocytosis (Istvan Ando and Dan are not present in C. elegans [43]).
Hultmark, personal communication). Nevertheless, in thelong term, by combining large-scale screens in the host and the pathogen, it will be possible to define a host–pathogen Although evolutionary divergence from mammals can interactome (Figure 2) [41].
limit the pertinence of simple model animals, the papersdescribed in this review demonstrate that there is a Extracellular bacterial pathogens are usually not able to wide-spread conservation of host–pathogen interactions survive phagocytosis. Many, however, have developed at the molecular and physiological levels. In the light of strategies to counteract the humoral arm of the host this, the phylogenetic distance between a model system immune system. A handful of recent articles have demon- and mammals can even be considered a boon because strated that infection of D. melanogaster with Pseudomonas conserved interactions are frequently the most impor- is a most suitable system to study the host immune tant. Therefore, given the practical advantages asso- response and to uncover the strategies used by the ciated with their use, non-mammalian models are pathogen to elude defence mechanisms. In one article increasingly being recognized as attractive alternatives [17], the role of the Pseudomonas exotoxin ExoS was to more traditional models [5]. Moreover, it is probable directly addressed by expressing this toxin either ectopi- that many of the virulence mechanisms that pathogens cally in the eye or ubiquitously throughout the fly. The use during their infection of humans in fact evolved authors showed with these transgenic systems that ExoS because they confer a survival advantage in the natural inhibits the activity of a host Rho GTPase in vivo and that ecological niche, and so are best studied using their ubiquitous ExoS expression impairs the phagocytic capa- natural predators, such as D. discoideum and C. elegans.
city of fly macrophages without affecting induction of After a period when these model systems were used in antimicrobial peptide genes [17]. In a complementary essentially one-sided approaches (e.g. screening banks of study, Liehl et al. [18] used host and pathogen mutants bacterial mutants for virulence genes or identifying the to demonstrate that the Pseudomonas AprA metallopro- host targets of bacterial virulence factors), more and tease directly degrades fly antimicrobial peptides. This more studies are now exploiting a combination of bac- protease thereby acts as a virulence factor by enhancing terial and host genetics to address the molecular basis of bacterial survival within the host body fluid. In addition to pathogenicity and defence. The future promises to these reports, Apidianakis et al. [16] compared microarray reveal details of the intimate but deadly dance between results from flies infected by virulent or avirulent P.
pathogen and host that has been going on since the birth aeruginosa strains. Strikingly, this analysis revealed an of eukaryotes.
as yet uncharacterised mechanism used by P. aeruginosain the early phases of the infection to limit expression of Drosophila antimicrobial genes at the transcriptional level.
It has recently been shown using C. elegans, the P. aeru-ginosa strain PA14 and a PA14 gacA mutant (that is highly Author's personal copy
studies illustrate the potential use of attenuated) that the intrinsic virulence of PA14 is a major genetically tractable non-mammalian hosts, with charac- elicitor of the host's innate immune response [44]. In terized immune systems, to decipher the mechanisms addition, by using the same animal model and by com- pathogens employ to evade host defenses. As exemplified paring the genome of the P. aeruginosa strain PA14 with above, it is possible to have a global approach and/or to that of PA01, it has been demonstrated that Pseudomonas precisely address the role of a specific bacterial protein.
virulence is multifactorial and necessitates the combina-torial action of multiple virulence factors that interact The principal drawbacks with these models are associated in a distinct manner, depending on the bacterial genetic with bacterial physiology and the specificity of certain background [45].
Current Opinion in Microbiology 2007, 10:10–16 Infection in a dish: high-throughput analyses of bacterial pathogenesis Kurz and Ewbank 12. Agaisse H, Burrack LS, Philips JA, Rubin EJ, Perrimon N, Higgins DE: Genome-wide RNAi screen for host factors We thank Pierre Golstein for helpful criticism. Work in the authors' required for intracellular bacterial infection. Science 2005, laboratory is supported by the Fondation Recherche Me´dicale, INSERM, the CNRS, the French Ministry of Research, Marseille-Nice Ge´nopole, See annotation for [13].
the Re´seau Nationale des Ge´nopoles, the European Union and theFrench National Research Agency (ANR).
13. Philips JA, Rubin EJ, Perrimon N: Drosophila RNAi screen reveals CD36 family member required for mycobacterialinfection. Science 2005, 309:1251-1253.
References and recommended reading To uncover host factors necessary for intracellular bacterial infection, Papers of particular interest, published within the period of review, these two papers used a genome-wide RNAi bank, a Drosophila macro- have been highlighted as: phage-like cell line, automated microscopy and GFP-tagged L. mono-cytogenes [12] or M. fortuitum [13]. Several hundred dsRNA that altered  of special interest the progression of infection were identified. The results obtained in the  of outstanding interest two screens were directly compared [12]. Peste, a member of the CD36family was found to be crucial for the entry of both bacteria into cells.
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The antimalarial and people suffering from the disease per year. Africa accounts forover 90% of reported cases, with an annual 20% increase of cytotoxic drug cryptolepine malaria-related illness and death. Malaria is responsible for as many deaths per annum as AIDS for all of the last 15 years. Drug intercalates into DNA at resistance to malaria has become one of the most significantthreats to human health and the search for new effective drugs is

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FULL PAPER International Journal of Recent Trends in Engineering, Vol 2, No. 1, November 2009 EVISTA – Interactive Visual Clustering System K. Thangavel1, P. Alagambigai2 1 Department of Computer Science, Periyar University, Salem, Tamilnadu, India Email: [email protected] 2 Department of Computer Applications, Easwari Engineering College, Chennai, Tamilnadu, India