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23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis

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23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis, Page 1 of 2

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Abstract:

This chapter describes an alternative method for identifying pathogen virulence factors that is based on the finding that many mammalian pathogens are also capable of causing disease in simple nonvertebrate hosts. The ability of to infect plants as well as animals was subsequently extended to show that the same clinical isolate of that can infect , strain PA14, was also a pathogen of the nematode worm as well as the insects and . Toxin-mediated killing is characterized by rapid worm killing, usually within one day, and by the ability of the conditioned media to kill in the absence of live bacteria. Infection-associated killing usually occurs over the course of several days and thus far has been characterized by the accumulation and, in some cases, proliferation of bacteria within the worm digestive tract. is an ideal insect host for genetic analysis of the host innate immune response. However, as is relatively small, it is difficult to inject bacteria directly into the body cavity. An alternative approach for investigating the function of microbial products that manipulate host cells is to express these bacterial toxins within a genetically tractable host system. Interactive genetic analysis was also used to define the role of P-glycoproteins (PGP) in protecting from the toxic effects of phenazines.

Citation: Sifri C, Ausubel F. 2004. 23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis, p 543-564. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch23

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Figures

Image of Figure 23.1
Figure 23.1

Identification of bacterial virulence factors by screening for avirulent mutants in model nonvertebrate hosts. A human bacterial pathogen, in this case strain PA14, which is also infectious in a nonvertebrate host (or hosts), is subjected to random transposon mutagenesis. Individual transposants are picked to 96- or 384-well microtiter plates and then tested individually in a killing assay or in an leaf infiltration assay. Mutants that are less pathogenic in or are characterized by determining the gene into which the transposon has inserted and verifying that the mutant phenotype corresponds to the transposon insertion. Finally, validated mutants are tested in a mouse pathogenesis model.

Citation: Sifri C, Ausubel F. 2004. 23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis, p 543-564. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch23
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Image of Figure 23.2
Figure 23.2

Isolation of mutants by direct screening in plants and nematodes and their phenotypes in plants, nematodes, and mice. Refer to Figure 23.1 for the overall experimental strategy. In the nematode assay, one gene was hit twice and one gene was identified in both the plant and nematode screens. Thus, the total number of genes identified is less than the number of mutants.

Citation: Sifri C, Ausubel F. 2004. 23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis, p 543-564. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch23
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Image of Figure 23.3
Figure 23.3

Conservation of innate immunity signaling pathways in mammals, insects, nematodes, and plants. In mammals and insects, pathogen-associated molecular patterns (PAMPs), macromolecules that are synthesized by microbes but not host cells, are detected by a family of transmembrane leucine-rich repeat (LRR) Toll and Toll-like receptors (TLRs). The signal is then transduced via conserved adapter proteins including MyD88, leading to the activation of Rel/NF-кB transcription factors as well as a mitogen-activated protein kinase (MAPK) signaling cassette that includes the highly conserved p38 MAPK. Analogous pathways function in , although the components of the signaling pathways do not appear to be direct homologues of the proteins in the mammalian and insect pathways. For example, the transmembrane LRR receptor kinase FLS2 is involved in the recognition of a 22-amino-acid component of the eubacterial flagellar protein. Mammals have a corresponding TLR that also responds to eubacterial flagellar protein. In comparison to mammals, insects, and plants, relatively little is known about the innate immune response in . The genome encodes a single Toll-like protein but does not encode transcription factors such as mammalian NF-кB or DIF. also has homologues of the mammalian TLR pathway components IRAK (interleukin-1 receptor-associated kinase; PIK-1 in worms) and TRAF6 (TNF receptor-associated factor; TRF-1 in worms). However, , or mutants do not exhibit an immunocompromised phenotype. Recent genetic analysis has led to the identification of a p38 MAPK signaling cascade that is directly homologous to the mammalian MAPK cascade that plays a central role in mediating mammalian immunity. Moreover, a Toll interleukin-1 resistance (TIR) domain- containing protein, TIR-1, functions upstream of p38 in , and it may couple a yet-to-be-identified PAMP receptor to the innate immune response.

Citation: Sifri C, Ausubel F. 2004. 23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis, p 543-564. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch23
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Image of Figure 23.4
Figure 23.4

-mediated killing of under so-called slow-killing conditions that involve an infection-like process. strains die when they are transferred from a petri plate where they are feeding on their normal food source, strain OP50, to a petri plate containing a lawn of . The rate of killing depends on the strain of and on the medium on which the lawn was grown. When grown on minimal media and then fed to worms, accumulates in the intestine and kills the worms by an infectious-like process over the course of a couple of days that requires live bacteria. In contrast, when grown in rich medium, appears to produce a variety of low-molecular- weight toxins, including phenazines, which kill the nematodes much more quickly. Live bacteria are not required for this latter type of toxin-mediated killing. Other human pathogens also kill . The rate of killing varies from 24 h to several days, depending on the pathogen and the medium on which it is grown.

Citation: Sifri C, Ausubel F. 2004. 23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis, p 543-564. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch23
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Image of Figure 23.6
Figure 23.6

proliferates in plant leaves similarly to the well characterized plant pathogen , and attaches perpendicularly to and perforates plant cell walls. As seen in the panel on the left, when infiltrated directly into an leaf, proliferates in the intercellular spaces in the leaf similarly to the well-studied plant pathogen . Once inside the leaf, attaches perpendicularly to plant cell walls and in some mesophyll cells gains entry via small holes that it makes in plant cell walls.

Citation: Sifri C, Ausubel F. 2004. 23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis, p 543-564. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch23
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Image of Figure 23.7
Figure 23.7

Wax moth caterpillars () are large enough to be directly injected by hand with pathogenic bacteria. This allows very accurate determination of LD50s. In the strain PA15, the LD50 varies from about 1 to 10 cells, depending on the particular experimental conditions.

Citation: Sifri C, Ausubel F. 2004. 23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis, p 543-564. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch23
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Image of Figure 23.8
Figure 23.8

Relative importance of PA14 virulence factors in wax moths and mice. Each data point represents an individual PA14 mutant that was identified by screening in plants or nematodes (see Figures 23.1 and 23.2 ) and was subsequently tested for virulence in wax moth caterpillars and in mice. The black square represents wild-type PA14.

Citation: Sifri C, Ausubel F. 2004. 23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis, p 543-564. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch23
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References

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1. Aballay, A.,, P. Yorgey,, and F. M. Ausubel. 2000. Salmonella typhimurium proliferates and establishes a persistent infection in the intestine of Caenorhabditis elegans. Curr. Biol. 10:15391542.
2. Asai, T.,, G. Tena,, J. Plotnikova,, M. R. Willmann,, W.-L. Chiu,, L. Gomez-Gomez,, T. Boller,, F. M. Ausubel,, and J. Sheen. 2002. MAP kinase signaling cascade in Arabidopsis innate immunity. Nature 415:977983. This work describes the identification and characterization of a MAP kinase cascade that induces the expression of Arabidopsis early-defense genes in response to bacterial flagellin.
3. Darby, C.,, C. L. Cosma,, J. H. Thomas,, and C. Manoil. 1999. Lethal paralysis of Caenorhabditis elegans by Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 96:1520215207.
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5. Fauvarque, M. O.,, E. Bergeret,, J. Chabert,, D. Dacheux,, M. Satre,, and I. Attree. 2002. Role and activation of type III secretion system genes in Pseudomonas aeruginosa-induced Drosophila killing. Microb. Pathog. 32:287295.
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8. Lemaitre, B.,, J. Reichhard,, and J. Hoffmann. 1997. Drosophila host defense: differential induction of antimicrobial peptide genes after infection by various classes of microorganisms. Proc. Natl. Acad. Sci. USA 94:1461414619.
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10. Mahajan-Miklos, S.,, M.-W. Tan,, L. G. Rahme,, and F. M. Ausubel. 1999. Molecular mechanisms of bacterial virulence: Caenorhabditis elegans pathogenesis model. Cell 96:4756. This paper describes the C. elegans-P. aeruginosa fast-killing model and shows that the system could be used to identify virulence factors important for both C. elegans and mammalian pathogenesis. In addition, the work shows that the host and pathogen could be simultaneously examined genetically to determine the mechanisms of virulence of the P. aeruginosa phenazine toxins.
11. Mallo, G.,, C. Kurz,, C. Couillault,, N. Pujol,, S. Granjeaud,, Y. Kohara,, and J. Ewbank. 2002. Inducible Antibacterial Defense System in C. elegans. Curr. Biol. 12:12091214. In this paper, a cDNA microarray analysis of C. elegans demonstrated induction of several nematode lysozymes and lectins in response to Serratia marcescens infection. Previous re- search had shown a similar pattern of expression under the control of the C. elegans TGF-β- related signaling molecule DBL-1. A dbl-1 mutant was shown to be hypersusceptible to S. marcescens infection.
12. Mylonakis, E.,, F. M. Ausubel,, J. R. Perfect,, J. Heitman,, and S. B. Calderwood. 2002. Killing of Caenorhabditis elegans by Cryptococcus neoformans as a model of yeast pathogenesis. Proc. Natl. Acad. Sci. USA 99:1567515680.
13. Neely, M. N.,, J. D. Pfeifer,, and M. Caparon. 2002. Streptococcus-zebrafish model of bacterial pathogenesis. Infect. Immun. 70:39043914.
14. Rahme, L.,, E. Stevens,, S. Wolfort,, J. Shao,, R. Tompkins,, and F. M. Ausubel. 1995. Common virulence factors for bacterial pathogenicity in plants and animals. Science 268:18991902. In this landmark paper, clinical Pseudomonas aeruginosa isolates were shown to cause softrot disease in Arabidopsis thaliana and severe disease in a murine infection model. Three P. aeruginosa pathogenicity-related genes (toxA, plcS, and gacA) were shown to be essential for infectivity in both plants and animals.
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Tables

Generic image for table
Table 23.1

PA14 virulence-related genes identified in nonvertebrate hosts

Citation: Sifri C, Ausubel F. 2004. 23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis, p 543-564. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch23
Generic image for table
Table 23.2

Model hosts and the human pathogens that infect them

Citation: Sifri C, Ausubel F. 2004. 23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis, p 543-564. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch23
Generic image for table
Table 23.3

General characteristics of nonvertebrate host-pathogen model systems

Citation: Sifri C, Ausubel F. 2004. 23 Use of Simple Nonvertebrate Hosts To Model Mammalian Pathogenesis, p 543-564. In Cossart P, Boquet P, Normark S, Rappuoli R (ed), Cellular Microbiology, Second Edition. ASM Press, Washington, DC. doi: 10.1128/9781555817633.ch23

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