Reference

Exploration of host-pathogen interactions using Listeria monocytogenes and Drosophila melanogaster.

Mansfield BE Dionne MS Schneider DS Freitag NE
Cellular microbiology. 2003.

Abstract

The facultative intracellular bacterial pathogen Listeria monocytogenes is capable of replicating within a broad range of host cell types and host species. We report here the establishment of the fruit fly Drosophila melanogaster as a new model host for the exploration of L. monocytogenes pathogenesis and host response to infection. Listeria monocytogenes was capable of establishing lethal infections in adult fruit flies and larvae with extensive bacterial replication occurring before host death. Bacteria were found in the cytosol of insect phagocytic cells, and were capable of directing host cell actin polymerization. Bacterial gene products necessary for intracellular replication and cell-to-cell spread within mammalian cells were similarly found to be required within insect cells, and although previous work has suggested that L. monocytogenes virulence gene expression requires temperatures above 30 degrees C, bacteria within insect cells were found to express virulence determinants at 25 degrees C. Mutant strains of Drosophila that were compromised for innate immune responses demonstrated increased susceptibility to L. monocytogenes infection. These data indicate L. monocytogenes infection of fruit flies shares numerous features of mammalian infection, and thus that Drosophila has the potential to serve as a genetically tractable host system that will facilitate the analysis of host cellular responses to L. monocytogenes infection.

Main claims

  1. Listeria monocytogenes infection of Drosophila shares numerous features with mammalian infection, making Drosophila a genetically tractable host for analysis of host cellular responses to L. monocytogenes infection [v1]
    Supported by subsequent studies of L. monocytogenes infection in Drosophila (Ayres et al., 2008; Buchon et al., 2009; Chambers et al., 2012; Cheng and Portnoy, 2003; Parks et al., 2021; Yano et al., 2008)

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Major claims

  1. Listeria monocytogenes is capable of establishing lethal infections in adult fruit flies and larvae [v1]
    Supported by subsequent studies (Ayres et al., 2008; Buchon et al., 2009)

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    1. Thoracic pricking of adult flies with a log phase culture of L. monoctyogenes followed by incubation at 29C established an infection that was lethal to wild-type flies in 6-8 days [v1]

      To be assessed

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    2. CFUs showed that L. monocytogenes exhibited exponential growth in infected flies, reaching numbers as high as 1010 before death. Inoculation with 10-20 CFU was sufficient for lethality in 80% of flies. [v1]

      To be assessed

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  2. Listeria monocytogenes is taken up by insect phagocytes and directs host cell actin polymerization. [v1]
    (Yano et al., 2008) confirm that L. monocytogenes is taken up by Drosophila phagocytes and replicates inside of them, and upon escape to the cytoplasm causes distinctive rearrangements of the actin cytoskeleton. Also supported in S2 cells by (Cheng and Portnoy, 2003).

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    1. Inoculation of larvae with L. monocytogenes expressing GFP showed that bacteria were present at the wound site and contained within phagocytes [v1]

      To be assessed

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    2. At 20hpi, microscopy showed that larval phagocytes were often completely filled with bacteria, which were present in the cytosol as well as within vacuoles, as evidenced by bacteria-filled protrusions of the hemocyte cell walls. Staining for filamentous actin showed that L. monocytogenes were closely associated with host actin and actin comet tails, indicating that the bacteria is directing host actin polymerization to facilitate bacterial movement as it does in mammalian cells. [v1]

      To be assessed

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  3. Both Toll and Imd pathways contribute to defense against Listeria monocytogenes infection [v1]
    Supported by (Ayres et al., 2008) (same group) who show that mutations in imd and kenny increase susceptibility of flies to L. monocytogenes; supported by (Buchon et al., 2009)

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    1. Dif mutant flies had a strong increase in susceptibility to L. monocytogenes, whereas imd mutant flies had a moderate increase in susceptibility. [v1]

      To be assessed

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Minor claims

  1. Infection of larvae with Listeria monocytogenes causes extensive melanization [v1]
    Supported in a metabolic study by the same group showing that melanin precursors are consumed following infection with L. monocytogenes (Chambers et al., 2012). Supported by independent group in preprint (Parks et al., 2021)

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    1. Melanization in infected larvae was present at the wound site, but single cells floating within the hemolymph were also melanized. As infection progressed, large areas of tissue including the fat body and epithelium became melanized. This was greater than the degree of melanization observed with E. coli, S. typhimurium, M. luteus or M. marinum infections [v1]

      To be assessed

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  2. Although previous work has suggested that Listeria monocytogenes virulence gene expression requires temperatures above 30C, bacteria within insect cells were found to express virulence genes at 25C [v1]
    This was also found to be the case in a Drosophila S2 cell system (Cheng and Portnoy, 2003). Several studies show that virulence genes are expressed in L. monocytogenes at low temperatures in response to various types of stress (Manso et al., 2020; Neuhaus et al., 2013). As Drosophila is unlikely to be a preferred/natural host for L. monocytogenes, it would be logical if stress-related responses such as these were activated in bacteria infecting flies. Indeed, an alternate temperature independent isoform of the prfA gene that controls virulence factors has been suggested to mediate expression of virulence genes at lower temperatures in systems such as Drosophila (Lemon et al., 2010).

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  3. Bacterial gene products necessary for intracellular replication and cell-to-cell spread of Listeria monocytogenes within mammalian cells are similarly required in insect cells. [v1]
    Supported in a follow-up study by the same group (Ayres et al., 2008), supported in S2 cells by (Cheng and Portnoy, 2003)

    Last change on 2022-08-16 09:22 by Anonymous

    1. Establishment of an L. monocytogenes infection in S2 cells followed by treatment with gentimicin to eliminate extracellular bacteria and application of antibodies to detect bacteria showed that L. monocytogenes effectively spread in monolayers of S2 cells, and that the bacteria replicated intracellularly. [v1]

      To be assessed

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    2. As in mammalian cells, L. monocytogenes with a mutation of actA were unable to spread cell-to-cell, although intracellular replication was unaffected. [v1]

      To be assessed

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    3. As in mammalian cells, L. monocytogenes with a mutation of the virulence-controlling transcriptional activator prfA failed to replicate, but small numbers persisted intracellularly, suggesting that unlike wild-type bacteria they were unable to escape host vacuoles. [v1]

      To be assessed

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    4. L. monocytogenes with these mutations had reduced virulence in flies, with delayed lethality. [v1]

      To be assessed

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    5. L. monocytogenes with these mutations had reduced virulence in flies, with delayed lethality. [v1]

      To be assessed

      Last change on 2022-08-14 15:33 by Anonymous

Methods

  1. Septic infection of larvae and adult flies, survivals. Infection with GFP-expressing bacteria, immunohistochemistry, microscopy. Infection of S2 cell cultures. Infection with mutated strains of Listeria monocytogenes. [v1]

    Last change on 2022-08-16 09:22 by Anonymous

Additional context

Not annotated yet

Additional files

Not annotated yet

References

  1. Ayres JS, Freitag N, Schneider DS. 2008. Identification of Drosophila mutants altering defense of and endurance to Listeria monocytogenes infection. Genetics 178:1807–1815. [DOI link]

    Buchon N, Poidevin M, Kwon H-M, Guillou A, Sottas V, Lee B-L, Lemaitre B. 2009. A single modular serine protease integrates signals from pattern-recognition receptors upstream of the Drosophila Toll pathway. Proc Natl Acad Sci U S A 106:12442–12447. [DOI link]

    Chambers MC, Song KH, Schneider DS. 2012. Listeria monocytogenes Infection Causes Metabolic Shifts in Drosophila melanogaster. PLOS ONE 7:e50679. [DOI link]

    Cheng LW, Portnoy DA. 2003. Drosophila S2 cells: an alternative infection model for Listeria monocytogenes. Cell Microbiol 5:875–885. [DOI link]

    Lemon KP, Freitag NE, Kolter R. 2010. The Virulence Regulator PrfA Promotes Biofilm Formation by Listeria monocytogenes. J Bacteriol 192:3969–3976. [DOI link]

    Manso B, Melero B, Stessl B, Jaime I, Wagner M, Rovira J, Rodríguez-Lázaro D. 2020. The Response to Oxidative Stress in Listeria monocytogenes Is Temperature Dependent. Microorganisms 8:521. [DOI link]

    Neuhaus K, Satorhelyi P, Schauer K, Scherer S, Fuchs TM. 2013. Acid shock of Listeria monocytogenes at low environmental temperatures induces prfA, epithelial cell invasion, and lethality towards Caenorhabditis elegans. BMC Genomics 14:285. [DOI link]

    Parks SC, Nguyen C, Nasrolahi S, Juncaj D, Lu D, Ramaswamy R, Dhillon H, Buchman A, Akbari OS, Yamanaka N, Boulanger MJ, Dillman AR. 2021. Parasitic nematode fatty acid- and retinol-binding proteins compromise host immunity by interfering with host lipid signaling pathways. bioRxiv 2021.03.25.436866. [DOI link]

    Yano T, Mita S, Ohmori H, Oshima Y, Fujimoto Y, Ueda R, Takada H, Goldman WE, Fukase K, Silverman N, Yoshimori T, Kurata S. 2008. Autophagic control of listeria through intracellular innate immune recognition in drosophila. Nat Immunol 9:908–916. [DOI link]

     

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