Font Size: a A A

Listeria Monocytogenes:Germ-free Zebrafish Infection Model And The Antibacterial Role Of Mmp-9 During Infection

Posted on:2017-03-05Degree:DoctorType:Dissertation
Country:ChinaCandidate:Y DanFull Text:PDF
GTID:1223330488983718Subject:Prevention of Veterinary Medicine
Abstract/Summary:PDF Full Text Request
Listeria monocytogenes (Lm) is a one of the major food-borne pathogens that can ubiquitously present in the environments. The bacterium, once ingested via contaminated food, can cross the intestinal barrier and disseminate to the brain and placenta via blood stream. It may lead to severe diseases in humans and ruminant animals with clinical features that vary from septicemias, meningitis, and abortions. L. monocytogenes has evolved with an elaborate arsenal of virulence factors. In its intracellular life cycle during infection, each step from adhesion to host cells, intracellular invasion, proliferation within cells to cell-to-cell spread is mediated by specific virulence factors. Internalin A (InlA), one of the adhesion factors, can covalently bind with E-cadherin on the host cell surface. L. monocytogenes can invade different eukaryotic hosts, but the efficiency of infection is species-dependent. A single amino acid difference at position 16 of mouse E-cadherin impairs its interaction with InlA, leading to less efficient oral infection than in humans. Zebrafish E-cadherin is proline at ths site, same as in humans.Zebrafish are highly fecund and fertile with ex vivo development and short sexual maturation period. The embryos are optically accessible and genetically tractable for large-scale mutagenesis and genetic screening. The immune cells in zebrafish are similar to mammals and the innate immune system is independent of the acquired immune system during the embryonic period. Therefore, using zebrafish as an alternative pathogen model is of great use for understanding the pathogenesis of infectious diseases and mechanism of host innate immunity. However, the microbes in their living environments and gut may compromise the research results with pathogens of interests when conventional fish are used. Thus, germ-free zebrafish have to be used for studies on the host immune responses to infections.In this study, we attempt to:(1) establish a germ-free zebrafish embryo culture system; (2) develop germ-free zebrafish models of L. monocytogenes infection through different routes of inoculation; (3) dissect the transcriptomic characteristics of Listeria immersion-infected germ-free zebrafish embryos; and (4) explore the role of matrix metalloproteinase 9 (Mmp-9) in macrophage migration during L. monocytogenes infection. I. Establishement and evaluation of germ-free zebrafish embryos.Zebrafish larva has an independent innate immune system. The adaptive immune system is immature until 4-6 weeks post fertilization. This developing stage during the first 4 weeks can be employed to study innate immunity to infections by pathogenic organisms. Microorganisms proliferation in the living environment and gut microbiota may activate the innate immunity and compromise the investigation on the host innate immune responses to infection. We established a germ-free zebrafish embryos model as shown by absence of microbial growth in the artificial media. We also tested the transcriptional level of Toll-like receptors (TLRs), the mark genes indicating activation of the innate immune system. The transcription levels of TLRs were barely detectable in zebrafish raised in the germ-free system, but highly induced in conventionally raised zebrafish. This germ-free zebrafish embryos model could serve as a useful tool for studying the function of innate immune system and the interaction between specific pathogen and host intestinal mucosa.Ⅱ. Pathogenecity of Listeria infection in zebrafish varied with routes of inoculation and species/strainsWe established germ-free zebrafish infection models of L. monocytogenes through different routes of inoculation:oral immersion and injection via yolk sac, brain ventricle and blood island. Of the three injection routes, infection with virulent Lm strain EDGe by yolk sac led to rapid death with only 18% hatchability in early stage of infection, while hatchability in Lm M7 strain, L. innocua (Ln) and mock control remained high (80%,88% and 90% respectively). Injection into zebrafish embryos via brain ventricle or blood island led to progressive lethal infection. Strain EGDe caused 100% death in one week when infected intravenously at a dose of 100 CFU/fish, and showed steady replication in fish embryos and was far more pathogenic than strain M7 and Ln which were readily cleared within certain time after infection, consistent with findings in the murine model. Immersion of zebrafish larva even with 1010 CFU/mL Lm EGDe strain in egg water was unable to cause mortality, but GFP-expressing bacteria in the gut lumen can be observed in frozen sections at 24 and 48 hours post infection (hpi), but cleared at 72 hpi. Several selected maker genes of the innate immune system, including cypla, irgll, illb, and mmp9, were significantly induced by oral immersion not only with strain EGDe, but also with strain M7 and Ln, though to a lesser degree (P< 0.01). Such induction appears to be transient with peak at 48 hpi, but returned to basal level at 72 hpi.III. Comparative transcriptomics of germ-free zebrafish larvae immersion-infected by pathogenic and non-pathogenic ListeriaWe used an Affymetrix gene chip to examine the expression profiles of 14,900 genes of zebrafish immersion-infected with Lm EGDe and Ln. A total of 239 genes were up-regulated (fold> 2) and 56 genes down-regulated (fold< 0.5) compared with uninfected fish. There were only 25 genes up-regulated and 4 genes down-regulated in Ln infected fish. L. monocytogene infection led to up-regulation of 102 genes, as compared with Ln. Highest expression (> 20-fold) was seen with the mmp-9 gene encoding the matrix metalloproteinase-9 (Mmp-9) known to degrade the extracellular matrix proteins. The expression levels of some genes by the microarray method were verified by qPCR. Gene ontology analysis revealed that most of the 239 and 102 up-regulated genes in Lm infected fish relative to mock-infected fish or Ln infected fish were enriched in GO:0005576 and GO:0050896, involved in maintaining a steady state of extracellular matrix and responding to stress respectively. Genes in immunity-related GO categories were performed with cluster analysis. Of the nine clusters, up-regulation was seen with genes related to tight junction, signaling, immune cells, interferons, receptors, heat shock proteins, fibrinogen, complement and actin. These findings suggest that zebrafish might employ these molecules to maintain homeostasis of the extracellular matrix to resist Listeria infection where Mmp-9 might paly a role.IV. The role of Mmp-9 in zebrafish against Listeria infectionMmp-9, a member of the matrix metallopreteinases, is a zinc-dependent collagenase. It is expressed in various cells and involved in many physiological and pathological events such as tissue repair, cell movement, inflammation. Early studies have revealed that Mmp-9 could create a path by degrading extracellular matrix (ECM) for macrophages and neutrophils crawling in the ECM. Recent reports indicate that Mmp-9 functions in cancer cell migration by inducing signaling pathways including motility-related signaling. We used morpholino knockdown method to silence Mmp-9 expression from one-cell stage. The morphants were infected with Lm via intravenous, intraventricular and immersion routes. Rapid death was seen within 3 days with much high bacterial load after intravenous or intraventricular (brain ventricle) infection. Macrophages in mmp-9-knockdown morphants had significant defect in migrating to the brain cavity upon intraventricular infection. Immersion infection did not cause any lethality, but there were hemorrhagic spots in mmp-9-silenced fish,To verify the role of Mmp-9 in macrophage migration and the possible signaling, we used RNAi and specific inhibitors in murine macrophage cell line RAW264.7 cultured in transwell inserts with 8-μm pore polycarbonate membrane. Decreased migration of murine macrophages with knockdown of mmp-9 and cd44 were seen, as compared with the scrambled RNA. However, silence of mmp-9 did not impair the actin tail formation around the bacterial cells, neither did it affect the formation of cell pseudopodia. By using the RhoA pathway inhibitor, we found that inhibition of FAK and ROCK counteracted Mmp-9 induced migration of macrophages. Mmp-9 induced migration was abolished after treating with cytochalasin D. These results suggest that Mmp-9 played a protective role against infection by enhancing migration of zebrafish macrophages to the site of infection via a non-proteolytic role.In conclusion, this study established a simple method to generate germ-free zebrafish embryos without activation of the innate immune system useful for host responses of innate immunity to infections. Multi-route infection models were tested using L. monocytogenes as a model pathogen to compare pathogenicity of different bacterial species or strains. Immersion infection with L. monocytogenes significantly induced several innate immune responsive genes. Among them, mmp-9 was the most induced gene. Mmp-9 is protective against Listeria infection by enhancing migration of zebrafish macrophages to the site of infection via a non-proteolytic pathway.
Keywords/Search Tags:Listeria monocytogenes, Germ-free zebrafish, Transcriptomics, Mmp-9, Macrophage migration
PDF Full Text Request
Related items