Zebrafish as a Genetic Model to Study Tuberculosis

Tuberkuloosi on yhä maailmanlaajuinen terveysongelma, sillä joka vuosi siihen sairastuu 10 miljoonaa ihmistä ja yli miljoona kuolee. Tuberkuloosin aiheuttaa Mycobacterium tuberculosis, joka leviää ilmateiden kautta. Tartunta voi johtaa erilaisiin tuloksiin oireettomasta latentista tilasta aktiiviseen tautiin. Koska tämänhetkiset hoitomuodot ovat riittämättömiä, taistelussa tuberkuloosia vastaan tarvitaan uusia lähestymistapoja. On näytetty, että isännän geneettinen tausta vaikuttaa M. tuberculosis-tartunnan lopputulokseen. Siksi seulomalla isännän geneettisten vaihteluiden vaikutusta mykobakteeri-infektion taudinkulkuun saadaan uutta tietoa, joka on hyödyllistä uusia hoitomuotoja kehitettäessä. Tuberkuloositutkimusta on hidastanut hyvien eläinmallien puute. Hyvä malli jäljittelisi tuberkuloosin taudinkulkua, mutta olisi silti sujuva käyttää ja huomioisi myös eettiset näkökohdat. Seeprakala on kasvattanut suosiotaan immunologisena tutkimusmallina. On myös olemassa monenlaisia menetelmiä tuottaa seeprakalalle sekä satunnaisia että kohdennettuja mutaatioita. Seeprakalan luontainen taudinaiheuttaja, Mycobacterium marinum, on M. tuberculosis -bakteerin läheinen sukulainen. Se aiheuttaa seeprakalassa koko elimistöön vaikuttavan taudin. Erityisesti M. marinum -infektio seeprakalan poikasessa on vakiintunut tuberkuloosimalli. Väitöskirjatutkimuksessa selvitettiin aikuisen seeprakalan M. marinum -infektion käytettävyyttä tuberkuloosimallina. Tämän väitöskirjan ensimmäinen tavoite oli tutkia kyseisen infektion kulkua aikuisessa seeprakalassa histologisilla menetelmillä ja transkriptiotason tutkimuksella. Toinen tavoite oli tutkia interleukiini 10 (il10) -geenin tehtäviä M. marinum -infektion aikana. Lopullinen tavoite oli tehdä geneettinen seulonta ja tunnistaa isännän geenejä, joilla on tehtävä seeprakalan puolustautuessa mykobakteeri-infektiota vastaan. Päästäksemme ensimmäiseen tavoitteeseemme värjäsimme M. marinum -bakteerin pitkittäisistä kudosleikkeistä, jotka oli otettu aikuisesta seeprakalasta infektion eri vaiheissa. Tutkimus osoitti, että infektoitaessa pienellä M. marinum -annoksella granuloomien ja infektoituneiden elinten lukumäärä vakiintui neljännestä viikosta eteenpäin. Suurella annoksella infektoitaessa infektoituneiden elinten lukumäärä oli korkeampi ja granuloomien lukumäärä kasvoi infektion edetessä. Selvittääksemme M. marinum -bakteerin aiheuttamaa immuunivastetta tarkemmin teimme koko genomin kattavan transkriptomianalyysin munuaisnäytteistä seeprakalan pääasiallinen verta muodostava elin). Tutkimus osoitti, että 14 päivää infektoinnin jälkeen synnynnäiseen immuunivasteeseen liittyvät prosessit olivat rikastuneet positiivisesti säädeltyjen geenien joukossa, kun taas rasva-aineenvaihduntaan liittyvät prosessit olivat rikastuneet negatiivisesti säädeltyjen geenien joukossa. Käytimme il10e46 -mutanttiseeprakalalinjaa tutkiaksemme il10-geenin tehtäviä M. marinum infektion aikana seeprakalassa. Kyseisessä linjassa on mutaatio, joka aiheuttaa toimimattoman proteiinin syntymisen. Näytimme, että kyseisen mutaation suhteen homotsygootit kalat selvisivät villityypin kontrollikaloja paremmin M. marinum -infektion aikana ja kantoivat vähemmän bakteeria 8/9 viikon kuluttua pienellä bakteeriannoksella infektoimisesta. Nämä tulokset viittaavat parantuneeseen vastustuskykyyn M. marinum -bakteeria vastaan. il10e46/e46-mutanteilla tnfb ja il1b tulehdussytokiinien ilmentyminen oli lisäksi vähentynyt mahdollisesti kuvastaen infektion hitaampaa etenemistä. interferoni gamma 1 -geenin ilmentyminen oli lisääntynyt ja T-auttajasolu 1 (Th1) -vaste vahvistunut il10e46/e46-mutanteilla mahdollisesti selittäen parantuneen vastustuskyvyn. Suoritimme geneettisen seulonnan tunnistaaksemme geenejä, joilla on merkitystä aikuisen seeprakalan puolustautuessa mykobakteeri-infektiota vastaan. Tunnistimme 10 mutanttiseeprakalalinjaa, jotka selviytyivät huonommin ja yhden linjan, joka selviytyi paremmin pienen M. marinum -bakteeriannoksen aiheuttaman infektion aikana. Herkin linja, jonka tunnistimme, mutant463, kantoi 14 päivää infektoinnin jälkeen merkittävästi enemmän bakteeria verrattuna villityypin kaloihin. Tämä viittaa ennemmin heikentyneeseen vastustuskykyyn kuin heikentyneeseen sietokykyyn M. marinum -bakteeria kohtaan. Teimme myös mutant463-linjalle transkriptomianalyysin. Tulokset osoittivat, että mutant463-seeprakaloilla oli 27 geenin ilmentyminen heikentynyt munuaissoluissa villityypin kaloihin verrattuna 14 päivää infektoinnin jälkeen. Näiden geenien joukossa on seitsemän geeniä, joilla on tunnettu tai ennustettu immunologinen tehtävä. Ensimmäisessä osatyössä näytimme, että seeprakalan M. marinum -infektion histologiset piirteet muistuttavat tuberkuloosin piirteitä. Toisen osatyön tulokset il10- geenin tehtävistä sopivat yhteen aiemman kirjallisuuden kanssa, sillä ne korostavat Th1-vasteen osuutta vastustuskyvyssä mykobakteeria kohtaan. Nämä tulokset vahvistavat käyttämäämme tuberkuloosimallia. Lisäksi kolmannessa osatyössä tunnistimme geenejä, joilla on mahdollisesti tehtävä immuunipuolustuksessa mykobakteeri-infektiota vastaan. Näiden geenien tehtävien varmentamiseksi ja tarkentamiseksi on tarpeen tehdä lisätutkimuksia.Tuberculosis is still a major global health issue, with 10 million new cases and over one million casualties each year. Tuberculosis is caused by M. tuberculosis, which spreads via airways. This infection may result in a wide spectrum of outcomes, from an asymptomatic latent state to active disease. Due to the insufficiency of current treatments, new approaches to combat tuberculosis are needed. Host genetics has been shown to affect the outcome of M. tuberculosis infection. Therefore, screening the effect of different genetic variations of the host on the progression of mycobacterial infection provides information useful for developing new treatments. Moreover, the research on tuberculosis has been impeded by the lack of good animal models that mimic the progression of tuberculosis and are still feasible to use whilst meeting ethical challenges. The zebrafish has become more and more popular as an immunological research model. There are various tools available to carry out both forward and reverse genetic modifications in the zebrafish. Moreover, Mycobacterium marinum is a close relative of M. tuberculosis and a natural zebrafish pathogen. It causes a systemic infection in zebrafish. The M. marinum infection in zebrafish larvae is an especially well-established tuberculosis model. Thus our group considered the M. marinum infection in adult zebrafish as an advantageous tuberculosis model. The first aim of this thesis was to study the progression of M. marinum infection in adult zebrafish with histological methods and transcriptional analysis. The second aim was to study the role of interleukin 10 (il10) during this infection. The final aim was to perform a forward genetic screen to identify host genes having a role in the defense against mycobacterial infection in zebrafish. To reach the first aim, we used Ziehl-Neelsen staining to identify M. marinum in longitudinal tissue sections taken from adult zebrafish at different time-points of the infection. Importantly, analysis showed that in low-dose M. marinum infection, the number of granulomas and affected organs remained stable from four weeks post infection onwards, whereas in a high-dose infection the number of affected organs was higher and granuloma numbers kept rising along the course of infection. To further characterize the immune response to M. marinum, we did a whole genome- level transcriptome analysis from the kidney (the main hematopoietic organ of fish) samples. Analysis revealed that at 14 days post infection (dpi), innate immune response related processes were enriched among the upregulated genes and many lipid metabolism-related processes were enriched among the downregulated genes. To study the role of il10 during M. marinum infection in zebrafish, we used a mutant zebrafish line, il10e46, having a non-sense mutation resulting in a truncated protein product. We showed that these homozygous mutated fish had improved survival during M. marinum infection compared to wild type (WT) control fish and decreased bacterial burden at 8/9 weeks post low-dose infection, indicating improved resistance against M. marinum. In addition, il10e46/e46 mutants had decreased expression of the pro-inflammatory cytokines tnfb and il1b possibly reflecting a slower progression of infection. Most importantly however, il10e46/e46 mutants showed improved interferon gamma 1 (ifng1) expression and a shift towards a T helper 1 (Th1) cell response, which provides a potential explanation for the improved resistance. In order to identify genes participating in the immune defense against mycobacterial infection in adult zebrafish, we performed a forward genetic screen. We identified ten mutant zebrafish lines with impaired survival and one line with improved survival following low-dose infection with M. marinum. The most susceptible line, mutant463, showed a significantly higher bacterial burden compared to WT control fish at 14 dpi, indicating impaired resistance rather than tolerance against M. marinum. We also included mutant463 in the transcriptome analysis we performed. The results showed impaired expression of 27 genes in zebrafish kidney cells at 14 dpi compared to WT. These included seven genes with known or predicted immunological roles. In the first original publication, we showed that the histological features of M. marinum infection in adult zebrafish are similar to tuberculosis. Results from the experiments with il10 mutants in the second original publication are in line with earlier literature highlighting the role of the Th1 response in resistance against mycobacterial infection. This further validates the feasibility of our model in studying tuberculosis infection. Moreover, in the third original communication we found genes potentially required for defense against mycobacterial infection. Further studies are warranted to confirm and characterize the roles of these genes in more detail

Zebrafish as a Genetic Model to Study Tuberculosis

Tuberkuloosi on yhä maailmanlaajuinen terveysongelma, sillä joka vuosi siihen sairastuu 10 miljoonaa ihmistä ja yli miljoona kuolee. Tuberkuloosin aiheuttaa Mycobacterium tuberculosis, joka leviää ilmateiden kautta. Tartunta voi johtaa erilaisiin tuloksiin oireettomasta latentista tilasta aktiiviseen tautiin. Koska tämänhetkiset hoitomuodot ovat riittämättömiä, taistelussa tuberkuloosia vastaan tarvitaan uusia lähestymistapoja. On näytetty, että isännän geneettinen tausta vaikuttaa M. tuberculosis-tartunnan lopputulokseen. Siksi seulomalla isännän geneettisten vaihteluiden vaikutusta mykobakteeri-infektion taudinkulkuun saadaan uutta tietoa, joka on hyödyllistä uusia hoitomuotoja kehitettäessä. Tuberkuloositutkimusta on hidastanut hyvien eläinmallien puute. Hyvä malli jäljittelisi tuberkuloosin taudinkulkua, mutta olisi silti sujuva käyttää ja huomioisi myös eettiset näkökohdat. Seeprakala on kasvattanut suosiotaan immunologisena tutkimusmallina. On myös olemassa monenlaisia menetelmiä tuottaa seeprakalalle sekä satunnaisia että kohdennettuja mutaatioita. Seeprakalan luontainen taudinaiheuttaja, Mycobacterium marinum, on M. tuberculosis -bakteerin läheinen sukulainen. Se aiheuttaa seeprakalassa koko elimistöön vaikuttavan taudin. Erityisesti M. marinum -infektio seeprakalan poikasessa on vakiintunut tuberkuloosimalli. Väitöskirjatutkimuksessa selvitettiin aikuisen seeprakalan M. marinum -infektion käytettävyyttä tuberkuloosimallina. Tämän väitöskirjan ensimmäinen tavoite oli tutkia kyseisen infektion kulkua aikuisessa seeprakalassa histologisilla menetelmillä ja transkriptiotason tutkimuksella. Toinen tavoite oli tutkia interleukiini 10 (il10) -geenin tehtäviä M. marinum -infektion aikana. Lopullinen tavoite oli tehdä geneettinen seulonta ja tunnistaa isännän geenejä, joilla on tehtävä seeprakalan puolustautuessa mykobakteeri-infektiota vastaan. Päästäksemme ensimmäiseen tavoitteeseemme värjäsimme M. marinum -bakteerin pitkittäisistä kudosleikkeistä, jotka oli otettu aikuisesta seeprakalasta infektion eri vaiheissa. Tutkimus osoitti, että infektoitaessa pienellä M. marinum -annoksella granuloomien ja infektoituneiden elinten lukumäärä vakiintui neljännestä viikosta eteenpäin. Suurella annoksella infektoitaessa infektoituneiden elinten lukumäärä oli korkeampi ja granuloomien lukumäärä kasvoi infektion edetessä. Selvittääksemme M. marinum -bakteerin aiheuttamaa immuunivastetta tarkemmin teimme koko genomin kattavan transkriptomianalyysin munuaisnäytteistä seeprakalan pääasiallinen verta muodostava elin). Tutkimus osoitti, että 14 päivää infektoinnin jälkeen synnynnäiseen immuunivasteeseen liittyvät prosessit olivat rikastuneet positiivisesti säädeltyjen geenien joukossa, kun taas rasva-aineenvaihduntaan liittyvät prosessit olivat rikastuneet negatiivisesti säädeltyjen geenien joukossa. Käytimme il10e46 -mutanttiseeprakalalinjaa tutkiaksemme il10-geenin tehtäviä M. marinum infektion aikana seeprakalassa. Kyseisessä linjassa on mutaatio, joka aiheuttaa toimimattoman proteiinin syntymisen. Näytimme, että kyseisen mutaation suhteen homotsygootit kalat selvisivät villityypin kontrollikaloja paremmin M. marinum -infektion aikana ja kantoivat vähemmän bakteeria 8/9 viikon kuluttua pienellä bakteeriannoksella infektoimisesta. Nämä tulokset viittaavat parantuneeseen vastustuskykyyn M. marinum -bakteeria vastaan. il10e46/e46-mutanteilla tnfb ja il1b tulehdussytokiinien ilmentyminen oli lisäksi vähentynyt mahdollisesti kuvastaen infektion hitaampaa etenemistä. interferoni gamma 1 -geenin ilmentyminen oli lisääntynyt ja T-auttajasolu 1 (Th1) -vaste vahvistunut il10e46/e46-mutanteilla mahdollisesti selittäen parantuneen vastustuskyvyn. Suoritimme geneettisen seulonnan tunnistaaksemme geenejä, joilla on merkitystä aikuisen seeprakalan puolustautuessa mykobakteeri-infektiota vastaan. Tunnistimme 10 mutanttiseeprakalalinjaa, jotka selviytyivät huonommin ja yhden linjan, joka selviytyi paremmin pienen M. marinum -bakteeriannoksen aiheuttaman infektion aikana. Herkin linja, jonka tunnistimme, mutant463, kantoi 14 päivää infektoinnin jälkeen merkittävästi enemmän bakteeria verrattuna villityypin kaloihin. Tämä viittaa ennemmin heikentyneeseen vastustuskykyyn kuin heikentyneeseen sietokykyyn M. marinum -bakteeria kohtaan. Teimme myös mutant463-linjalle transkriptomianalyysin. Tulokset osoittivat, että mutant463-seeprakaloilla oli 27 geenin ilmentyminen heikentynyt munuaissoluissa villityypin kaloihin verrattuna 14 päivää infektoinnin jälkeen. Näiden geenien joukossa on seitsemän geeniä, joilla on tunnettu tai ennustettu immunologinen tehtävä. Ensimmäisessä osatyössä näytimme, että seeprakalan M. marinum -infektion histologiset piirteet muistuttavat tuberkuloosin piirteitä. Toisen osatyön tulokset il10- geenin tehtävistä sopivat yhteen aiemman kirjallisuuden kanssa, sillä ne korostavat Th1-vasteen osuutta vastustuskyvyssä mykobakteeria kohtaan. Nämä tulokset vahvistavat käyttämäämme tuberkuloosimallia. Lisäksi kolmannessa osatyössä tunnistimme geenejä, joilla on mahdollisesti tehtävä immuunipuolustuksessa mykobakteeri-infektiota vastaan. Näiden geenien tehtävien varmentamiseksi ja tarkentamiseksi on tarpeen tehdä lisätutkimuksia.Tuberculosis is still a major global health issue, with 10 million new cases and over one million casualties each year. Tuberculosis is caused by M. tuberculosis, which spreads via airways. This infection may result in a wide spectrum of outcomes, from an asymptomatic latent state to active disease. Due to the insufficiency of current treatments, new approaches to combat tuberculosis are needed. Host genetics has been shown to affect the outcome of M. tuberculosis infection. Therefore, screening the effect of different genetic variations of the host on the progression of mycobacterial infection provides information useful for developing new treatments. Moreover, the research on tuberculosis has been impeded by the lack of good animal models that mimic the progression of tuberculosis and are still feasible to use whilst meeting ethical challenges. The zebrafish has become more and more popular as an immunological research model. There are various tools available to carry out both forward and reverse genetic modifications in the zebrafish. Moreover, Mycobacterium marinum is a close relative of M. tuberculosis and a natural zebrafish pathogen. It causes a systemic infection in zebrafish. The M. marinum infection in zebrafish larvae is an especially well-established tuberculosis model. Thus our group considered the M. marinum infection in adult zebrafish as an advantageous tuberculosis model. The first aim of this thesis was to study the progression of M. marinum infection in adult zebrafish with histological methods and transcriptional analysis. The second aim was to study the role of interleukin 10 (il10) during this infection. The final aim was to perform a forward genetic screen to identify host genes having a role in the defense against mycobacterial infection in zebrafish. To reach the first aim, we used Ziehl-Neelsen staining to identify M. marinum in longitudinal tissue sections taken from adult zebrafish at different time-points of the infection. Importantly, analysis showed that in low-dose M. marinum infection, the number of granulomas and affected organs remained stable from four weeks post infection onwards, whereas in a high-dose infection the number of affected organs was higher and granuloma numbers kept rising along the course of infection. To further characterize the immune response to M. marinum, we did a whole genome- level transcriptome analysis from the kidney (the main hematopoietic organ of fish) samples. Analysis revealed that at 14 days post infection (dpi), innate immune response related processes were enriched among the upregulated genes and many lipid metabolism-related processes were enriched among the downregulated genes. To study the role of il10 during M. marinum infection in zebrafish, we used a mutant zebrafish line, il10e46, having a non-sense mutation resulting in a truncated protein product. We showed that these homozygous mutated fish had improved survival during M. marinum infection compared to wild type (WT) control fish and decreased bacterial burden at 8/9 weeks post low-dose infection, indicating improved resistance against M. marinum. In addition, il10e46/e46 mutants had decreased expression of the pro-inflammatory cytokines tnfb and il1b possibly reflecting a slower progression of infection. Most importantly however, il10e46/e46 mutants showed improved interferon gamma 1 (ifng1) expression and a shift towards a T helper 1 (Th1) cell response, which provides a potential explanation for the improved resistance. In order to identify genes participating in the immune defense against mycobacterial infection in adult zebrafish, we performed a forward genetic screen. We identified ten mutant zebrafish lines with impaired survival and one line with improved survival following low-dose infection with M. marinum. The most susceptible line, mutant463, showed a significantly higher bacterial burden compared to WT control fish at 14 dpi, indicating impaired resistance rather than tolerance against M. marinum. We also included mutant463 in the transcriptome analysis we performed. The results showed impaired expression of 27 genes in zebrafish kidney cells at 14 dpi compared to WT. These included seven genes with known or predicted immunological roles. In the first original publication, we showed that the histological features of M. marinum infection in adult zebrafish are similar to tuberculosis. Results from the experiments with il10 mutants in the second original publication are in line with earlier literature highlighting the role of the Th1 response in resistance against mycobacterial infection. This further validates the feasibility of our model in studying tuberculosis infection. Moreover, in the third original communication we found genes potentially required for defense against mycobacterial infection. Further studies are warranted to confirm and characterize the roles of these genes in more detail

Intelectin 3 is dispensable for resistance against a mycobacterial infection in zebrafish (Danio rerio)

Abstract Tuberculosis is a multifactorial bacterial disease, which can be modeled in the zebrafish (Danio rerio). Abdominal cavity infection with Mycobacterium marinum, a close relative of Mycobacterium tuberculosis, leads to a granulomatous disease in adult zebrafish, which replicates the different phases of human tuberculosis, including primary infection, latency and spontaneous reactivation. Here, we have carried out a transcriptional analysis of zebrafish challenged with low-dose of M. marinum, and identified intelectin 3 (itln3) among the highly up-regulated genes. In order to clarify the in vivo significance of Itln3 in immunity, we created nonsense itln3 mutant zebrafish by CRISPR/Cas9 mutagenesis and analyzed the outcome of M. marinum infection in both zebrafish embryos and adult fish. The lack of functional itln3 did not affect survival or the mycobacterial burden in the zebrafish. Furthermore, embryonic survival was not affected when another mycobacterial challenge responsive intelectin, itln1, was silenced using morpholinos either in the WT or itln3 mutant fish. In addition, M. marinum infection in dexamethasone-treated adult zebrafish, which have lowered lymphocyte counts, resulted in similar bacterial burden in both WT fish and homozygous itln3 mutants. Collectively, although itln3 expression is induced upon M. marinum infection in zebrafish, it is dispensable for protective mycobacterial immune response

Interleukin 10 mutant zebrafish have an enhanced interferon gamma response and improved survival against a Mycobacterium marinum infection

Abstract Tuberculosis ranks as one of the world’s deadliest infectious diseases causing more than a million casualties annually. IL10 inhibits the function of Th1 type cells, and IL10 deficiency has been associated with an improved resistance against Mycobacterium tuberculosis infection in a mouse model. Here, we utilized M. marinum infection in the zebrafish (Danio rerio) as a model for studying Il10 in the host response against mycobacteria. Unchallenged, nonsense il10e46/e46 mutant zebrafish were fertile and phenotypically normal. Following a chronic mycobacterial infection, il10e46/e46 mutants showed enhanced survival compared to the controls. This was associated with an increased expression of the Th cell marker cd4-1 and a shift towards a Th1 type immune response, which was demonstrated by the upregulated expression of tbx21 and ifng1, as well as the down-regulation of gata3. In addition, at 8 weeks post infection il10e46/e46 mutant zebrafish had reduced expression levels of proinflammatory cytokines tnfb and il1b, presumably indicating slower progress of the infection. Altogether, our data show that Il10 can weaken the immune defense against M. marinum infection in zebrafish by restricting ifng1 response. Importantly, our findings support the relevance of M. marinum infection in zebrafish as a model for tuberculosis

Mycobacterium marinum Causes a Latent Infection that Can Be Reactivated by Gamma Irradiation in Adult Zebrafish

The mechanisms leading to latency and reactivation of human tuberculosis are still unclear, mainly due to the lack of standardized animal models for latent mycobacterial infection. In this longitudinal study of the progression of a mycobacterial disease in adult zebrafish, we show that an experimental intraperitoneal infection with a low dose (~35 bacteria) of Mycobacterium marinum, results in the development of a latent disease in most individuals. The infection is characterized by limited mortality (25%), stable bacterial loads 4 weeks following infection and constant numbers of highly organized granulomas in few target organs. The majority of bacteria are dormant during a latent mycobacterial infection in zebrafish, and can be activated by resuscitation promoting factor ex vivo. In 5–10% of tuberculosis cases in humans, the disease is reactivated usually as a consequence of immune suppression. In our model, we are able to show that reactivation can be efficiently induced in infected zebrafish by γ-irradiation that transiently depletes granulo/monocyte and lymphocyte pools, as determined by flow cytometry. This immunosuppression causes reactivation of the dormant mycobacterial population and a rapid outgrowth of bacteria, leading to 88% mortality in four weeks. In this study, the adult zebrafish presents itself as a unique non-mammalian vertebrate model for studying the development of latency, regulation of mycobacterial dormancy, as well as reactivation of latent or subclinical tuberculosis. The possibilities for screening for host and pathogen factors affecting the disease progression, and identifying novel therapeutic agents and vaccine targets make this established model especially attractive.Public Library of Science open acces

Abnormal cerebellar development and ataxia in CARP VIII morphant zebrafish

Congenital ataxia and mental retardation are mainly caused by variations in the genes that affect brain development. Recent reports have shown that mutations in the CA8 gene are associated with mental retardation and ataxia in humans and ataxia in mice. The gene product, carbonic anhydrase-related protein VIII (CARP VIII), is predominantly present in cerebellar Purkinje cells, where it interacts with the inositol 1,4,5-trisphosphate receptor type 1, a calcium channel. In this study, we investigated the effects of the loss of function of CARP VIII during embryonic development in zebrafish using antisense morpholino oligonucleotides against the CA8 gene. Knockdown of CA8 in zebrafish larvae resulted in a curved body axis, pericardial edema and abnormal movement patterns. Histologic examination revealed gross morphologic defects in the cerebellar region and in the muscle. Electron microscopy studies showed increased neuronal cell death in developing larvae injected with CA8 antisense morpholinos. These data suggest a pivotal role for CARP VIII during embryonic development. Furthermore, suppression of CA8 expression leads to defects in motor and coordination functions, mimicking the ataxic human phenotype. This work reveals an evolutionarily conserved function of CARP VIII in brain development and introduces a novel zebrafish model in which to investigate the mechanisms of CARP VIII-related ataxia and mental retardation in humans

Characterization of the innate immune response to Streptococcus pneumoniae infection in zebrafish.

Streptococcus pneumoniae (pneumococcus) is one of the most frequent causes of pneumonia, sepsis and meningitis in humans, and an important cause of mortality among children and the elderly. We have previously reported the suitability of the zebrafish (Danio rerio) larval model for the study of the host-pathogen interactions in pneumococcal infection. In the present study, we characterized the zebrafish innate immune response to pneumococcus in detail through a whole-genome level transcriptome analysis and revealed a well-conserved response to this human pathogen in challenged larvae. In addition, to gain understanding of the genetic factors associated with the increased risk for severe pneumococcal infection in humans, we carried out a medium-scale forward genetic screen in zebrafish. In the screen, we identified a mutant fish line which showed compromised resistance to pneumococcus in the septic larval infection model. The transcriptome analysis of the mutant zebrafish larvae revealed deficient expression of a gene homologous for human C-reactive protein (CRP). Furthermore, knockout of one of the six zebrafish crp genes by CRISPR-Cas9 mutagenesis predisposed zebrafish larvae to a more severe pneumococcal infection, and the phenotype was further augmented by concomitant knockdown of a gene for another Crp isoform. This suggests a conserved function of C-reactive protein in anti-pneumococcal immunity in zebrafish. Altogether, this study highlights the similarity of the host response to pneumococcus in zebrafish and humans, gives evidence of the conserved role of C-reactive protein in the defense against pneumococcus, and suggests novel host genes associated with pneumococcal infection

Zebrafish mortality, the development of bacterial load and the number of lesions have dose-dependent patterns.

<p>Adult zebrafish were i.p. infected with either a low (34±15 cfu) (n = 180) or a high dose (2029±709 cfu) (n = 104) of <i>M. marinum</i>. (A) Survival was followed for 32 weeks. * P<0.05 (B) The figure shows the average loads for 5 fish (except 32 wk high dose, n = 2). Low-dose statistics: * sig. diff. from 1 wk, ** sig. diff. from 1 and 2 wk. High-dose statistics: *** sig. diff. from 1, 2, 8, 11 and 20 wk. Low-dose vs. high-dose statistics: loads at time-points marked with † are sig. diff. (C) By default, 4 individuals per dose were analyzed by Ziehl-Neelsen staining (except 20 wk high dose, n = 3) per time-point The gonads, pancreas, liver, muscle, mesentery, spleen, gut and kidney were assessed and the number of organs with visible bacteria was determined. *P<0.05. (D) The total number of granulomas in a sample set for each individual was counted. * P<0.05.</p

<i>M. marinum</i> induces the formation of granulomas that mature into well-defined structures during an infection.

<p>In fish infected with a low dose (34±15 cfu) of <i>M. marinum</i>, Ziehl-Neelsen staining at 2 wpi commonly reveals areas with free bacteria (C). Some slightly better formed and restricted areas containing bacteria, here referred to as early granulomas, are also seen (A), but as shown in (B) trichrome staining of the adjacent slide, encapsulation around the mycobacterial lesions is absent at the early stage of infection. At 20 weeks, fish that have survived have mature granulomas (D–F) many of which are multicentric surrounded by a fibrous capsule (D&E). (E) Trichrome staining shows the fibrous capsule in blue (F). The amount of bacteria inside granulomas has increased from the earliest time-points.</p