Bartonella henselae (Bh) is an emerging zoonotic pathogen that has been associated with a variety of human diseases, including bacillary angiomatosis that is characterized by vasoproliferative tumor-like lesions on the skin of some immunosuppressed individuals. The study of Bh pathogenesis has been limited to in vitro cell culture systems due to the lack of an animal model. Therefore, we wanted to investigate whether the zebrafish embryo could be used to model human infection with Bh. Our data showed that Tg(fli1:egfp)y1 zebrafish embryos supported a sustained Bh infection for 7 days with >10-fold bacterial replication when inoculated in the yolk sac. We showed that Bh recruited phagocytes to the site of infection in the Tg(mpx:GFP)uwm1 embryos. Infected embryos showed evidence of a Bh-induced angiogenic phenotype and an increase in the expression of genes encoding pro-inflammatory factors and pro-angiogenic markers. However, infection of zebrafish embryos with a deletion mutant in the major adhesin (BadA) resulted in little or no bacterial replication and a diminished host response, providing the first evidence that BadA is critical for in vivo infection. Thus, the zebrafish embryo provides the first practical model of Bh infection that will facilitate efforts to identify virulence factors and define molecular mechanisms of Bh pathogenesis.
Get full access to this article
View all access options for this article.
References
1.
AndersonBE, NeumanMA. Bartonella spp. as emerging human pathogens. Clin Microbiol Rev, 1997; 10:203–219.
2.
BattermanHJ, PeekJA, LoutitJS, FalkowS, TompkinsLS. Bartonella henselae and Bartonella quintana adherence to and entry into cultured human epithelial cells. Infect Immun, 1995; 63:4553–4556.
3.
VigilA, OrtegaR, JainA, Nakajima-SasakiR, TanX, ChomelBB, et al.Identification of the feline humoral immune response to Bartonella henselae infection by protein microarray. PLoS One, 2010; 5:e11447.
4.
JamesonP, GreeneC, RegneryR, DrydenM, MarksA, BrownJ, et al.Prevalence of Bartonella henselae antibodies in pet cats throughout regions of North America. J Infect Dis, 1995; 172:1145–1149.
5.
RelmanDA, LoutitJS, SchmidtTM, FalkowS, TompkinsLS. The agent of bacillary angiomatosis. An approach to the identification of uncultured pathogens. N Engl J Med, 1990; 323:1573–1580.
AlsmarkCM, FrankAC, KarlbergEO, LegaultBA, ArdellDH, CanbackB, et al.The louse-borne human pathogen Bartonella quintana is a genomic derivative of the zoonotic agent Bartonella henselae. Proc Natl Acad Sci U S A, 2004; 101:9716–9721.
8.
HoiczykE, RoggenkampA, ReichenbecherM, LupasA, HeesemannJ. Structure and sequence analysis of Yersinia YadA and Moraxella UspAs reveal a novel class of adhesins. EMBO J, 2000; 19:5989–5999.
RiessT, AnderssonSG, LupasA, SchallerM, SchaferA, KymeP, et al.Bartonella adhesin a mediates a proangiogenic host cell response. J Exp Med, 2004; 200:1267–1278.
11.
KempfVA, VolkmannB, SchallerM, SanderCA, AlitaloK, RiessT, et al.Evidence of a leading role for VEGF in Bartonella henselae-induced endothelial cell proliferations. Cell Microbiol, 2001; 3:623–632.
12.
KempfVA, SchairerA, NeumannD, GrasslGA, LauberK, LebiedziejewskiM, et al.Bartonella henselae inhibits apoptosis in Mono Mac 6 cells. Cell Microbiol, 2005; 7:91–104.
13.
KempfVA, LebiedziejewskiM, AlitaloK, WalzleinJH, EhehaltU, FiebigJ, et al.Activation of hypoxia-inducible factor-1 in bacillary angiomatosis: evidence for a role of hypoxia-inducible factor-1 in bacterial infections. Circulation, 2005; 111:1054–1062.
14.
BuchmannAU, KempfVAJ, KershawO, GruberAD. Peliosis hepatis in cats is not associated with Bartonella henselae infections. Vet Pathol, 2010; 47:163–166.
15.
ZhangP, ChomelBB, SchauMK, GooJS, DrozS, KelminsonKL, et al.A family of variably expressed outer-membrane proteins (Vomp) mediates adhesion and autoaggregation in Bartonella quintana. Proc Natl Acad Sci U S A, 2004; 101:13630–13635.
16.
RegnathT, MielkeME, ArvandM, HahnH. Murine model of Bartonella henselae infection in the immunocompetent host. Infect Immun, 1998; 66:5534–5536.
17.
KunzS, OberleK, SanderA, BogdanC, SchleicherU. Lymphadenopathy in a novel mouse model of Bartonella-induced cat scratch disease results from lymphocyte immigration and proliferation and is regulated by interferon-alpha/beta. Am J Pathol, 2008; 172:1005–1018.
18.
FouquetB, WeinsteinBM, SerlucaFC, FishmanMC. Vessel patterning in the embryo of the zebrafish: guidance by notochord. Dev Biol, 1997; 183:37–48.
19.
WillettCE, CherryJJ, SteinerLA. Characterization and expression of the recombination activating genes (rag1 and rag2) of zebrafish. Immunogenetics, 1997; 45:394–404.
20.
van der SarAM, MustersRJ, van EedenFJ, AppelmelkBJ, Vandenbroucke-GraulsCM, BitterW. Zebrafish embryos as a model host for the real time analysis of Salmonella typhimurium infections. Cell Microbiol, 2003; 5:601–611.
21.
NicoliS, RibattiD, CotelliF, PrestaM. Mammalian tumor xenografts induce neovascularization in zebrafish embryos. Cancer Res, 2007; 67:2927–2931.
22.
NicoliS, De SenaG, PrestaM. Fibroblast growth factor 2-induced angiogenesis in zebrafish: the zebrafish yolk membrane (ZFYM) angiogenesis assay. J Cell Mol Med, 2009; 13:2061–2068.
23.
TraverD, HerbomelP, PattonEE, MurpheyRD, YoderJA, LitmanGW, et al.The zebrafish as a model organism to study development of the immune system. Adv Immunol, 2003; 81:253–330.
24.
TredeNS, LangenauDM, TraverD, LookAT, ZonLI. The use of zebrafish to understand immunity. Immunity, 2004; 20:367–379.
25.
Lugo-VillarinoG, BallaKM, StachuraDL, BanuelosK, WerneckMB, TraverD. Identification of dendritic antigen-presenting cells in the zebrafish. Proc Natl Acad Sci U S A, 2010; 107:15850–15855.
26.
MackichanJK, GernsHL, ChenYT, ZhangP, KoehlerJE. A SacB mutagenesis strategy reveals that the Bartonella quintana variably-expressed outer membrane proteins (Vomp) are required for bloodstream infection of the host. Infect Immun, 2008; 76:788–795.
27.
GillaspieD, PerkinsI, LarsenK, McCordA, PangonisS, SwegerD, et al.Plasmid-based system for high-level gene expression and antisense gene knockdown in Bartonella henselae. Appl Environ Microbiol, 2009; 75:5434–5436.
28.
RiessT, DietrichF, SchmidtKV, KaiserPO, SchwarzH, SchaferA, et al.Analysis of a novel insect cell culture medium-based growth medium for Bartonella species. Appl Environ Microbiol, 2008; 74:5224–5227.
29.
BrannonMK, DavisJM, MathiasJR, HallCJ, EmersonJC, CrosierPS, et al.Pseudomonas aeruginosa type III secretion system interacts with phagocytes to modulate systemic infection of zebrafish embryos. Cell Microbiol, 2009; 11:755–768.
30.
MathiasJR, PerrinBJ, LiuTX, KankiJ, LookAT, HuttenlocherA. Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish. J Leukoc Biol, 2006; 80:1281–1288.
31.
MathiasJR, DoddME, WaltersKB, YooSK, RanheimEA, HuttenlocherA. Characterization of zebrafish larval inflammatory macrophages. Dev Comp Immunol, 2009; 33:1212–1217.
32.
WesterfieldM. The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 5th ed. Eugene, OR: University of Oregon Press, 2007.
33.
ColbornJM, KosoyMY, MotinVL, TelepnevMV, ValbuenaG, MyintKS, et al.Improved detection of Bartonella DNA in mammalian hosts and arthropod vectors by real-time PCR using the NADH dehydrogenase gamma subunit (nuoG). J Clin Microbiol, 2010; 48:4630–4633.
34.
LeungYF, MaP, DowlingJE. Gene expression profiling of zebrafish embryonic retinal pigment epithelium in vivo. Invest Ophthalmol Vis Sci, 2007; 48:881–890.
35.
SchmittgenTD, LivakKJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc, 2008; 3:1101–1108.
36.
PressleyME, PhelanPE, 3rd, WittenPE, MellonMT, KimCH. Pathogenesis and inflammatory response to Edwardsiella tarda infection in the zebrafish. Dev Comp Immunol, 2005; 29:501–513.
37.
van der SarAM, AppelmelkBJ, Vandenbroucke-GraulsCM, BitterW. A star with stripes: zebrafish as an infection model. Trends Microbiol, 2004; 12:451–457.
38.
NeelyMN, PfeiferJD, CaparonM. Streptococcus-zebrafish model of bacterial pathogenesis. Infect Immun, 2002; 70:3904–3914.
39.
PrajsnarTK, CunliffeVT, FosterSJ, RenshawSA. A novel vertebrate model of Staphylococcus aureus infection reveals phagocyte-dependent resistance of zebrafish to nonhost specialized pathogens. Cell Microbiol, 2008; 10:2312–2325.
40.
HerbomelP, ThisseB, ThisseC. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development, 1999; 126:3735–3745.
41.
DavisJM, ClayH, LewisJL, GhoriN, HerbomelP, RamakrishnanL. Real-time visualization of mycobacterium-macrophage interactions leading to initiation of granuloma formation in zebrafish embryos. Immunity, 2002; 17:693–702.
42.
LeeYS, ChoiI, NingY, KimNY, KhatchadourianV, YangD, et al.Interleukin-8 and its receptor CXCR2 in the tumour microenvironment promote colon cancer growth, progression and metastasis. Br J Cancer, 2012; 106:1833–1841.
43.
VajkoczyP, FarhadiM, GaumannA, HeidenreichR, ErberR, WunderA, et al.Microtumor growth initiates angiogenic sprouting with simultaneous expression of VEGF, VEGF receptor-2, and angiopoietin-2. J Clin Invest, 2002; 109:777–785.
44.
ZhaoC, WangX, ZhaoY, LiZ, LinS, WeiY, et al.A novel xenograft model in zebrafish for high-resolution investigating dynamics of neovascularization in tumors. PLoS One, 2011; 6:e21768.
45.
SchmidMC, SchuleinR, DehioM, DeneckerG, CarenaI, DehioC. The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells. Mol Microbiol, 2004; 52:81–92.
46.
Resto-RuizSI, SchmiedererM, SwegerD, NewtonC, KleinTW, FriedmanH, et al.Induction of a potential paracrine angiogenic loop between human THP-1 macrophages and human microvascular endothelial cells during Bartonella henselae infection. Infect Immun, 2002; 70:4564–4570.
47.
McCordAM, Resto-RuizSI, AndersonBE. An autocrine role for IL-8 in Bartonella henselae-induced angiogenesis. Infect Immun, 2006; 74:5185–5190.
DehioM, QuebatteM, FoserS, CertaU. The transcriptional response of human endothelial cells to infection with Bartonella henselae is dominated by genes controlling innate immune responses, cell cycle, and vascular remodelling. Thromb Haemostasis, 2005; 94:347–361.
50.
MussoT, BadolatoR, RavarinoD, StornelloS, PanzanelliP, MerlinoC, et al.Interaction of Bartonella henselae with the murine macrophage cell line J774: infection and proinflammatory response. Infect Immun, 2001; 69:5974–5980.
51.
LiangD, XuX, ChinAJ, BalasubramaniyanNV, TeoMA, LamTJ, et al.Cloning and characterization of vascular endothelial growth factor (VEGF) from zebrafish, Danio rerio. Biochim Biophys Acta, 1998; 1397:14–20.
52.
DiazMH, BaiY, MalaniaL, WinchellJM, KosoyMY. Development of a novel genus-specific real-time PCR assay for detection and differentiation of Bartonella species and genotypes. J Clin Microbiol, 2012; 50:1645–1649.
53.
SchuleinR, GuyeP, RhombergTA, SchmidMC, SchroderG, VergunstAC, et al.A bipartite signal mediates the transfer of type IV secretion substrates of Bartonella henselae into human cells. Proc Natl Acad Sci U S A, 2005; 102:856–861.
54.
MonteilRA, MichielsJF, HofmanP, Saint-PaulMC, HitzigC, PerrinC, et al.Histological and ultrastructural study of one case of oral bacillary angiomatosis in HIV disease and review of the literature. Eur J Cancer B Oral Oncol, 1994; 30B:65–71.
55.
LeBoitPE, BergerTG, EgbertBM, BecksteadJH, YenTS, StolerMH. Bacillary angiomatosis. The histopathology and differential diagnosis of a pseudoneoplastic infection in patients with human immunodeficiency virus disease. Am J Surg Pathol, 1989; 13:909–920.
56.
LiX, WangS, QiJ, EchtenkampSF, ChatterjeeR, WangM, et al.Zebrafish peptidoglycan recognition proteins are bactericidal amidases essential for defense against bacterial infections. Immunity, 2007; 27:518–529.
57.
WangZ, ZhangS, WangG, AnY. Complement activity in the egg cytosol of zebrafish Danio rerio: evidence for the defense role of maternal complement components. PLoS One, 2008; 3:e1463.
58.
TobiaC, De SenaG, PrestaM. Zebrafish embryo, a tool to study tumor angiogenesis. Int J Dev Biol, 2011; 55:505–509.
59.
ZahringerU, LindnerB, KnirelYA, van den AkkerWM, HiestandR, HeineH, et al.Structure and biological activity of the short-chain lipopolysaccharide from Bartonella henselae ATCC 49882T. J Biol Chem, 2004; 279:21046–21054.
60.
DehioC, MeyerM, BergerJ, SchwarzH, LanzC. Interaction of Bartonella henselae with endothelial cells results in bacterial aggregation on the cell surface and the subsequent engulfment and internalisation of the bacterial aggregate by a unique structure, the invasome. J Cell Sci, 1997; 110(Pt 18):2141–2154.
61.
MengaudJ, OhayonH, GounonP, MegeRM, CossartP. E-cadherin is the receptor for internalin, a surface protein required for entry of L. monocytogenes into epithelial cells. Cell, 1996; 84:923–932.
62.
BabbSG, MarrsJA. E-cadherin regulates cell movements and tissue formation in early zebrafish embryos. Dev Dyn, 2004; 230:263–277.
63.
TruttmannMC, GuyeP, DehioC. BID-F1 and BID-F2 domains of Bartonella henselae effector protein BepF trigger together with BepC the formation of invasome structures. PLoS One, 2011; 6:e25106.
64.
van Lookeren CampagneM, WiesmannC, BrownEJ. Macrophage complement receptors and pathogen clearance. Cell Microbiol, 2007; 9:2095–2102.
65.
LambrisJD, Muller-EberhardHJ. The multifunctional role of C3: structural analysis of its interactions with physiological ligands. Mol Immunol, 1986; 23:1237–1242.
66.
EhlenbergerAG, NussenzweigV. The role of membrane receptors for C3b and C3d in phagocytosis. J Exp Med, 1977; 145:357–371.
67.
LuYY, FranzB, TruttmannMC, RiessT, Gay-FraretJ, FaustmannM, et al.Bartonella henselae trimeric autotransporter adhesin BadA expression interferes with effector translocation by the VirB/D4 type IV secretion system. Cell Microbiol, 2012; 16:12070.
68.
OehlersSH, FloresMV, HallCJ, O'TooleR, SwiftS, CrosierKE, et al.Expression of zebrafish cxcl8 (interleukin-8) and its receptors during development and in response to immune stimulation. Dev Comp Immunol, 2010; 34:352–359.
69.
LemmensR, Van HoeckeA, HersmusN, GeelenV, D'HollanderI, ThijsV, et al.Overexpression of mutant superoxide dismutase 1 causes a motor axonopathy in the zebrafish. Hum Mol Genet, 2007; 16:2359–2365.
70.
HooperAT, ShmelkovSV, GuptaS, MildeT, BambinoK, GillenK, et al.Angiomodulin is a specific marker of vasculature and regulates vascular endothelial growth factor-A-dependent neoangiogenesis. Circ Res, 2009; 105:201–208.
71.
KubotaY, OikeY, SatohS, TabataY, NiikuraY, MorisadaT, et al.Isolation and expression patterns of genes for three angiopoietin-like proteins, Angptl1, 2 and 6 in zebrafish. Gene Expr Patterns, 2005; 5:679–685.
72.
ClatworthyAE, LeeJS, LeibmanM, KostunZ, DavidsonAJ, HungDT. Pseudomonas aeruginosa infection of zebrafish involves both host and pathogen determinants. Infect Immun, 2009; 77:1293–1303.
73.
TangR, DoddA, LaiD, McNabbWC, LoveDR. Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization. Acta Biochim Biophys Sin (Shanghai), 2007; 39:384–390.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
