The orange spotted cockroach (Blaptica dubia, Serville 1839) is a permissive experimental host for Francisella tularensis

Bridget E. Eklund, Osama Mahdi, Jason F. Huntley, Elliot Collins, Caleb Martin, Joseph Horzempa, Nathan A. Fisher

Abstract


Francisella tularensis is a zoonotic bacterial pathogen that causes severe disease in a wide range of host animals, including humans. Well-developed murine models of F. tularensis pathogenesis are available, but they do not meet the needs of all investigators. However, researchers are increasingly turning to insect host systems as a cost-effective alternative that allows greater increased experimental throughput without the regulatory requirements associated with the use of mammals in biomedical research. Unfortunately, the utility of previously-described insect hosts is limited because of temperature restriction, short lifespans, and concerns about the immunological status of insects mass-produced for other purposes. Here, we present a novel host species, the orange spotted (OS) cockroach (Blaptica dubia), that overcomes these limitations and is readily infected by F. tularensis. Intrahemocoel inoculation was accomplished using standard laboratory equipment and lethality was directly proportional to the number of bacteria injected. Progression of infection differed in insects housed at low and high temperatures and F. tularensis mutants lacking key virulence components were attenuated in OS cockroaches. Finally, antibiotics were delivered to infected OS cockroaches by systemic injection and controlled feeding; in the latter case, protection correlated with oral bioavailability in mammals. Collectively, these results demonstrate that this new host system provides investigators with a new tool capable of interrogating F. tularensis virulence and immune evasion in situations where mammalian models are not available or appropriate, such as undirected screens of large mutant libraries.

Keywords


Francisella; cockroach; infection model; tularemia

Full Text:

PDF

References


Abd, H., Johansson, T., Golovliov, I., Sandström, G., and Forsman, M. (2003). Survival and growth of Francisella tularensis in Acanthamoeba castellanii. Appl Environ Microbiol, 69:600–6.

Ahlund, M.K., Rydén, P., Sjöstedt, A., and Stöven, S. (2010). Directed screen of Francisella novicida virulence determinants using Drosophila melanogaster. Infect Immun, 78:3118–28.

Ahmad, S., Hunter, L., Qin, A., Mann, B.J., and Hoek M.L. van. (2010). Azithromycin effectiveness against intracellular infections of Francisella. BMC Microbiol, 10:123.

Aperis, G., Fuchs, B.B., Anderson, C.A., Warner, J.E., Calderwood, S.B. and Mylonakis, E. (2007). Galleria mellonella as a model host to study infection by the Francisella tularensis live vaccine strain. Microbes Infect, 9:729–34.

Arvanitis, M., Glavis-Bloom, J., Mylonakis, E. (2013). Invertebrate models of fungal infection. Biochim Biophys Acta, 1832:1378–83.

Bangi, E. (2013). Drosophila at the intersection of infection, inflammation, and cancer. Front Cell Infect Microbiol, 3:103.

Beetz, S., Holthusen, T.K., Koolman, J., and Trenczek, T. (2008). Correlation of hemocyte counts with different developmental parameters during the last larval instar of the tobacco hornworm, Manduca sexta. Arch Insect Biochem Physiol, 67:63–75.

Booth, K., Cambron, L., Fisher, N., and Greenlee, K.J. (2015). Immune Defense Varies within an Instar in the Tobacco Hornworm, Manduca sexta. Physiol Biochem Zool, 88:226–36.

Bradburne, C.E., Verhoeven, A.B., Manyam, G.C. Chaudhry, S.A., Chang, E.L., Thach, D.C., Bailey, C.L., and Hoek van, M.L. (2013). Temporal transcriptional response during infection of type II alveolar epithelial cells with Francisella tularensis live vaccine strain (LVS) supports a general host suppression and bacterial uptake by macropinocytosis. J Biol Chem, 288:10780–91.

Bröms, J.E., Meyer, L., Sun, K., Lavander, M., and Sjöstedt, A. (2012). Unique substrates secreted by the type VI secretion system of Francisella tularensis during intramacrophage infection. PLoS One, 7:e50473.

Browne, N. Surlis, C., and Kavanagh, K. (2014). Thermal and physical stresses induce a short-term immune priming effect in Galleria mellonella larvae. J Insect Physiol, 63:21–6.

Burke, D.S. (1977). Immunization against tularemia: analysis of the effectiveness of live Francisella tularensis vaccine in prevention of laboratory-acquired tularemia. J Infect Dis, 135:55–60.

Chong, A. and Celli, J. (2010). The Francisella intracellular life cycle: toward molecular mechanisms of intracellular survival and proliferation. Front Microbiol, 1:138

Chong, A., Child, R. Wehrly, T.D., Rockx-Brouwer, D., Qin, A., Mann, B.J., and Celli, J. (2013). Structure-Function Analysis of DipA, a Francisella tularensis Virulence Factor Required for Intracellular Replication. PLoS One, 8:e67965, 2013.

Dean, S.N., and Hoek van, M.L. (2015). Screen of FDA-approved drug library identifies maprotiline, an antibiofilm and antivirulence compound with QseC sensor-kinase dependent activity in Francisella novicida. Virulence, 6:487–503.

Dennis, D.T., Inglesby, T.V., Henderson, D.A., Bartlett, J.G., Ascher, M.S., Eitzen, E., Fine, A.D., Friedlander, A.M., Hauer, J., Layton, M., Lillibridge, S.R., McDade, J.E., Osterholm, M.T., O’Toole, T., Parker, G.,

Perl, T.M., Russell, P.K., and Tonat, K. (2001). Tularemia as a biological weapon: medical and public health management. JAMA, 285:2763–73.

El-Etr, S.H., Margolis, J.J., Monack, D., Robison, R.A., Cohen, M., Moore, E., and Rasley. A. (2009) Francisella tularensis type A strains cause the rapid encystment of Acanthamoeba castellanii and survive in amoebal cysts for three weeks postinfection. Appl Environ Microbiol, 75:7488–500.

Eleftherianos, I., Baldwin, H., ffrench-Constant, R.H., and Reynolds, S.E. (2008). Developmental modulation of immunity: changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus. J Insect Physiol, 54:309–18.

Ellis, J., Oyston, P.C., Green, M., and Titball, R.W. (2002). Tularemia. Clin Microbiol Rev, 15:631–46.

Fisher, N.A., Ribot, W.J., Applefeld, W., and DeShazer, D. (2012). The Madagascar hissing cockroach as a novel surrogate host for Burkholderia pseudomallei, B. mallei and B. thailandensis. BMC Microbiol, 12:117, Jun 2012.

Forestral, C.A., Malik, M., Catlett, S.V., Savitt, A.G., Benach, J.L., Sellati, T.J., and Furie M.B. (2007). Francisella tularensis has a significant extracellular phase in infected mice. J infect Dis. 196: 134-7. Doi:10.1086/518611

Golovliov, I., Sjöstedt, A., Mokrievich, A., and Pavlov, V. (2003). A method for allelic replacement in Francisella tularensis. FEMS Microbiol Lett, 222:273–80.

Hernández, Y., López, D., Yero, J.M., Pinos-Rodríguez, and Gibert. I. (2015). Animals devoid of pulmonary system as infection models in the study of lung bacterial pathogens. Front Microbiol, 6:38.

Horn, L., Leips, J., and Starz-Gaiano, M. (2014). Phagocytic ability declines with age in adult Drosophila hemocytes. Aging Cell, 13:719–28, Aug 2014.

Horzempa, J., Carlson, P.E. Jr., O’Dee, D.M., Shanks, R.M., and Nau, G.J. (2008). Global transcriptional response to mammalian temperature provides new insight into Francisella tularensis pathogenesis. BMC Microbiol, 8:172.

Horzempa, J., O’Dee, D.M., Shanks, R.M., and Nau, G.J. (2010). Francisella tularensis ∆pyrF mutants show that replication in nonmacrophages is sufficient for pathogenesis in vivo. Infect Immun, 78:2607–19.

Horzempa, J., Shanks, R.M., Brown, M.J., Russo, B.C., O’Dee, D.M., and Nau, G.J. (2010). Utilization of an unstable plasmid and the I-SceI endonuclease to generate routine markerless deletion mutants in Francisella tularensis. J Microbiol Methods, 80:106–8.

Kaunisto, S., Härkönen, L., Rantala, M.J., and Kortet, R. (2015). Early-life temperature modifies adult encapsulation response in an invasive ectoparasite. Parasitology, 142:1290–6.

Klein S.L., Flanagan K.L. (2016). Sex differences in immune responses. Nat Rev Immunol. 16(10):626-38. doi: 10.1038/nri.2016.90.

Lai, X.H., Golovliov, I., and Sjöstedt, A. (2004). Expression of IglC is necessary for intracellular growth and induction of apoptosis in murine macrophages by Francisella tularensis. Microb Pathog, 37:225–30.

Lauriano, C.M. Barker, J.R. Yoon, S.S., Nano, F.E., Arulanandam, B.P., Hassett, D.J., and Klose, K.E. (2004). MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival. Proc Natl Acad Sci USA, 101:4246–9.

Law, H.T., Sriram, A., Fevang, C., Nix, E.B., Nano, F.E., and Guttman, J.A. (2014). IglC and PdpA are important for promoting Francisella invasion and intracellular growth in epithelial cells. PLoS One, 9:e104881.

Lazzaro, B.P., Flores, H.A., Lorigan, J.G., and Yourth, C.P. (2008). Genotype-by-environment interactions and adaptation to local temperature affect immunity and fecundity in Drosophila melanogaster. PLOS Pathog 4(3); e1000025. doi:10.1371/journal.ppat.1000025

Meylaers, K., Freitak, D., and Schoofs, L. (2007). Immunocompetence of Galleria mellonella: sex- and stage-specific differences and the physiological cost of mounting an immune response during metamorphosis. J Insect Physiol, 53:146–56.

Morner, T., and Addison, E. (2001). Tularemia. In ES Williams and IK Barker, editors, Infectious Diseases of Wild Mammals, chapter 18. Iowa State University Press, Ames, Iowa, USA, 3 edition. doi: 10.1002/9780470344880.ch18.

Moule, M.G., DM Monack, D.M. and DS Schneider, D.S. (2010). Reciprocal analysis of Francisella novicida infections of a Drosophila melanogaster model reveal host-pathogen conflicts mediated by reactive oxygen and imd-regulated innate immune response. PLoS Pathog, 6:e1001065.

Plotly Technologies Inc. (2015) Collaborative data science.

R Core Team. (2013). R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.

R Studio Team. (2015). RStudio: Integrated Development Environment for R. RStudio, Inc., Boston, MA.

Ramarao, N., Nielsen-Leroux, C., Lereclus, D. (2012). The insect Galleria mellonella as a powerful infection model to investigate bacterial pathogenesis. J Vis Exp, page e4392.

Ren, G., Champion, M.M., Huntley, J.F. (2014). Identification of disulfide bond isomerase substrates reveals bacterial virulence factors. Mol Microbiol, 94:926–44.

Steele, S., Brunton, B., Ziehr, B., Taft-Benz, S., Moorman, N., and Kawula, T. (2013). Francisella tularensis harvests nutrients derived via ATG5-independent autophagy to support intracellular growth. PLoS Pathog, 9:e1003562.

Santic, M. and Kwaik Y. A. (2013). Nutritional virulence of Francisella tularensis. Front Cell Infect Microbiol, 3:112.

Schmidt, M., Klimentova, J., Rehulka, P, Straskova, A., Spidlova, P., Szotakova, B., Stulik, J., and Pavkova, I. (2013). Francisella tularensis subsp. holarctica DsbA homologue: a thioredoxin-like protein with chaperone function. Microbiology, 159:2364–74.

Schmitt, D.M., Barnes, R, Rogerson, T., Haught, A., Mazzella, L.K., Ford, M., Gilson, T., Birch, J.W.-M., Sjöstedt A., Reed, D.S., Franks, J.M., Stolz, D.B., Denvir, J., Fan, J., Rekulapally, S., Primerano, D.A., Horzempa, J. (2017). The role and mechanism of erythrocyte invasion by Francisella tularensis. Frontiers in Cellular and Infection Microbiology, 7:1-173. doi:10.3389/fcimb.2017.00173

Schmitt, D.M., Connolly, K.L, Jerse, A.E., Detrick, M.S, Horzempa J. (2016). Antibacterial activity of resazurin-based compounds against Neisseria gonorrhoeae in vitro and in vivo. Int J Antimicrob Agents. 48(4):367-72. doi: 10.1016/j.ijantimicag.2016.06.009

Schmitt, D.M. O’Dee, D.M., Cowan, B.N., Birch, J.W. Mazzella, L.K., Nau, G.J., and Horzempa, J (2013). The use of resazurin as a novel antimicrobial agent against Francisella tularensis. Front Cell Infect Microbiol, 3:93.

Shaik, H.A., and Sehnal, F. (2009). Hemolin expression in the silk glands of Galleria mellonella in response to bacterial challenge and prior to cell disintegration. J Insect Physiol, 55:781–7.

Silva, M.T., and Pestana, N.T. (2013). The in vivo extracellular life of facultative intracellular bacterial parasites: role in pathogenesis. Immunobiology, 218:325–37.

Sprynski, N., Valade, E., and Neulat-Ripoll, F. (2014). Galleria mellonella as an infection model for select agents. Methods Mol Biol, 1197:3–9.

Therneau, T.M. and Grambsch, P.M. (2000). Modeling Survival Data: Extending the Cox Model. Springer, New York.

Therneau, T.M. (2015). A Package for Survival Analysis in S. version 2.38.

Tian, L., Guo, E., Diao, Y., Zhou, S., Peng, Q., Cao, Y., Ling, E., and Li, S. (2010). Genome-wide regulation of innate immunity by juvenile hormone and 20-hydroxyecdysone in the Bombyx fat body. BMC Genomics, 11:549.

Torson, A.S., Yocum, G.D., Rinehart, J.P., Kemp, W.P., and Bowsher, J.H. (2015). Transcriptional responses to fluctuating thermal regimes underpinning differences in survival in the solitary bee Megachile rotundata. J Exp Biol, 218:1060–8.

Verma, P. and Tapadia, M.G. (2012). Immune response and anti-microbial peptides expression in Malpighian tubules of Drosophila melanogaster is under developmental regulation. PLoS One, 7:e40714.

Weiss, D.S., Brotcke, A., Henry, T., Margolis, J.J., Chan, K., and Monack, D.M. (2007). In vivo negative selection screen identifies genes required for Francisella virulence. Proc Natl Acad Sci U S A, 104:6037–42.

Woltedji, D., Fang, Y., Han, B., Feng, M., Li, R., Lu, X., and Li, J. (2013). Proteome analysis of hemolymph changes during the larval to pupal development stages of honeybee workers (Apis mellifera ligustica). J Proteome Res, 12:5189–98.

Wu, X., Ren, G., and Huntley, J.F. (2015). Generating Isogenic Deletions (Knockouts) in Francisella tularensis, a Highly-infectious and Fastidious Gram-negative Bacterium. Bio Protoc, 5:e1500.

Xu, Q., Lu, A., Xiao, G., Yang, B., Zhang, J., Li, X., Guan, J., Shao, Q., Beerntsen, B.T., Zhang, P., Wang, C., and Ling, E. (2012). Transcriptional profiling of midgut immunity response and degeneration in the wandering silkworm, Bombyx mori. PLoS One, 7:e43769.




Copyright (c) 2017 Proceedings of the West Virginia Academy of Science

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.