Interaction between KDELR2 and HSP47 as a Key Determinant in Osteogenesis Imperfecta Caused by Bi-allelic Variants in KDELR2.

Osteogenesis imperfecta (OI) is characterized primarily by susceptibility to fractures with or without bone deformation. OI is genetically heterogeneous: over 20 genetic causes are recognized. We identified bi-allelic pathogenic KDELR2 variants as a cause of OI in four families. KDELR2 encodes KDEL endoplasmic reticulum protein retention receptor 2, which recycles ER-resident proteins with a KDEL-like peptide from the cis-Golgi to the ER through COPI retrograde transport. Analysis of patient primary fibroblasts showed intracellular decrease of HSP47 and FKBP65 along with reduced procollagen type I in culture media. Electron microscopy identified an abnormal quality of secreted collagen fibrils with increased amount of HSP47 bound to monomeric and multimeric collagen molecules. Mapping the identified KDELR2 variants onto the crystal structure of G. gallus KDELR2 indicated that these lead to an inactive receptor resulting in impaired KDELR2-mediated Golgi-ER transport. Therefore, in KDELR2-deficient individuals, OI most likely occurs because of the inability of HSP47 to bind KDELR2 and dissociate from collagen type I. Instead, HSP47 remains bound to collagen molecules extracellularly, disrupting fiber formation. This highlights the importance of intracellular recycling of ER-resident molecular chaperones for collagen type I and bone metabolism and a crucial role of HSP47 in the KDELR2-associated pathogenic mechanism leading to OI.

EspH is a hypervirulence factor for Mycobacterium marinum and essential for the secretion of the ESX-1 substrates EspE and EspF.

The pathogen Mycobacterium tuberculosis employs a range of ESX-1 substrates to manipulate the host and build a successful infection. Although the importance of ESX-1 secretion in virulence is well established, the characterization of its individual components and the role of individual substrates is far from complete. Here, we describe the functional characterization of the Mycobacterium marinum accessory ESX-1 proteins EccA1, EspG1 and EspH, i.e. proteins that are neither substrates nor structural components. Proteomic analysis revealed that EspG1 is crucial for ESX-1 secretion, since all detectable ESX-1 substrates were absent from the cell surface and culture supernatant in an espG1 mutant. Deletion of eccA1 resulted in minor secretion defects, but interestingly, the severity of these secretion defects was dependent on the culture conditions. Finally, espH deletion showed a partial secretion defect; whereas several ESX-1 substrates were secreted in normal amounts, secretion of EsxA and EsxB was diminished and secretion of EspE and EspF was fully blocked. Interaction studies showed that EspH binds EspE and therefore could function as a specific chaperone for this substrate. Despite the observed differences in secretion, hemolytic activity was lost in all M. marinum mutants, implying that hemolytic activity is not strictly correlated with EsxA secretion. Surprisingly, while EspH is essential for successful infection of phagocytic host cells, deletion of espH resulted in a significantly increased virulence phenotype in zebrafish larvae, linked to poor granuloma formation and extracellular outgrowth. Together, these data show that different sets of ESX-1 substrates play different roles at various steps of the infection cycle of M. marinum.

Mycobacteria employ two different mechanisms to cross the blood-brain barrier.

Central nervous system (CNS) infection by Mycobacterium tuberculosis is one of the most devastating complications of tuberculosis, in particular in early childhood. In order to induce CNS infection, M. tuberculosis needs to cross specialised barriers protecting the brain. How M. tuberculosis crosses the blood-brain barrier (BBB) and enters the CNS is not well understood. Here, we use transparent zebrafish larvae and the closely related pathogen Mycobacterium marinum to answer this question. We show that in the early stages of development, mycobacteria rapidly infect brain tissue, either as free mycobacteria or within circulating macrophages. After the formation of a functionally intact BBB, the infiltration of brain tissue by infected macrophages is delayed, but not blocked, suggesting that crossing the BBB via phagocytic cells is one of the mechanisms used by mycobacteria to invade the CNS. Interestingly, depletion of phagocytic cells did not prevent M. marinum from infecting the brain tissue, indicating that free mycobacteria can independently cause brain infection. Detailed analysis showed that mycobacteria are able to cause vasculitis by extracellular outgrowth in the smaller blood vessels and by infecting endothelial cells. Importantly, we could show that this second mechanism is an active process that depends on an intact ESX-1 secretion system, which extends the role of ESX-1 secretion beyond the macrophage infection cycle.

Infection of zebrafish embryos with live fluorescent Streptococcus pneumoniae as a real-time pneumococcal meningitis model.

BACKGROUND: Streptococcus pneumoniae is one of the most important causes of bacterial meningitis, an infection where unfavourable outcome is driven by bacterial and host-derived toxins. In this study, we developed and characterized a pneumococcal meningitis model in zebrafish embryos that allows for real-time investigation of early host-microbe interaction. METHODS: Zebrafish embryos were infected in the caudal vein or hindbrain ventricle with green fluorescent wild-type S. pneumoniae D39 or a pneumolysin-deficient mutant. The kdrl:mCherry transgenic zebrafish line was used to visualize the blood vessels, whereas phagocytic cells were visualized by staining with far red anti-L-plastin or in mpx:GFP/mpeg1:mCherry zebrafish, that have green fluorescent neutrophils and red fluorescent macrophages. Imaging was performed by fluorescence confocal and time-lapse microscopy. RESULTS: After infection by caudal vein, we saw focal clogging of the pneumococci in the blood vessels and migration of bacteria through the blood-brain barrier into the subarachnoid space and brain tissue. Infection with pneumolysin-deficient S. pneumoniae in the hindbrain ventricle showed attenuated growth and migration through the brain as compared to the wild-type strain. Time-lapse and confocal imaging revealed that the initial innate immune response to S. pneumoniae in the subarachnoid space mainly consisted of neutrophils and that pneumolysin-mediated cytolytic activity caused a marked reduction of phagocytes. CONCLUSIONS: This new meningitis model permits detailed analysis and visualization of host-microbe interaction in pneumococcal meningitis in real time and is a very promising tool to further our insights in the pathogenesis of pneumococcal meningitis.

Genome-wide transposon mutagenesis indicates that Mycobacterium marinum customizes its virulence mechanisms for survival and replication in different hosts.

The interaction of environmental bacteria with unicellular eukaryotes is generally considered a major driving force for the evolution of intracellular pathogens, allowing them to survive and replicate in phagocytic cells of vertebrate hosts. To test this hypothesis on a genome-wide level, we determined for the intracellular pathogen Mycobacterium marinum whether it uses conserved strategies to exploit host cells from both protozoan and vertebrate origin. Using transposon-directed insertion site sequencing (TraDIS), we determined differences in genetic requirements for survival and replication in phagocytic cells of organisms from different kingdoms. In line with the general hypothesis, we identified a number of general virulence mechanisms, including the type VII protein secretion system ESX-1, biosynthesis of polyketide lipids, and utilization of sterols. However, we were also able to show that M. marinum contains an even larger set of host-specific virulence determinants, including proteins involved in the modification of surface glycolipids and, surprisingly, the auxiliary proteins of the ESX-1 system. Several of these factors were in fact counterproductive in other hosts. Therefore, M. marinum contains different sets of virulence factors that are tailored for specific hosts. Our data imply that although amoebae could function as a training ground for intracellular pathogens, they do not fully prepare pathogens for crossing species barriers.

Analysis of SecA2-dependent substrates in Mycobacterium marinum identifies protein kinase G (PknG) as a virulence effector.

The pathogenicity of mycobacteria is closely associated with their ability to export virulence factors. For this purpose, mycobacteria possess different protein secretion systems, including the accessory Sec translocation pathway, SecA2. Although this pathway is associated with intracellular survival and virulence, the SecA2-dependent effector proteins remain largely undefined. In this work, we studied a Mycobacterium marinum secA2 mutant with an impaired capacity to initiate granuloma formation in zebrafish embryos. By comparing the proteomic profile of cell envelope fractions from the secA2 mutant with wild type M. marinum, we identified putative SecA2-dependent substrates. Immunoblotting procedures confirmed SecA2-dependent membrane localization for several of these proteins, including the virulence factor protein kinase G (PknG). Interestingly, phenotypical defects of the secA2 mutant are similar to those described for ΔpknG, including phagosomal maturation. Overexpression of PknG in the secA2 mutant restored its localization to the cell envelope. Importantly, PknG-overexpression also partially restored the virulence of the secA2 mutant, as indicated by enhanced infectivity in zebrafish embryos and restored inhibition of phagosomal maturation. These results suggest that SecA2-dependent membrane localization of PknG is an important determinant for M. marinum virulence.

Mannan core branching of lipo(arabino)mannan is required for mycobacterial virulence in the context of innate immunity.

The causative agent of tuberculosis (TB), Mycobacterium tuberculosis, remains an important worldwide health threat. Although TB is one of the oldest infectious diseases of man, a detailed understanding of the mycobacterial mechanisms underlying pathogenesis remains elusive. Here, we studied the role of the α(1→2) mannosyltransferase MptC in mycobacterial virulence, using the Mycobacterium marinum zebrafish infection model. Like its M. tuberculosis orthologue, disruption of M. marinum mptC (mmar_3225) results in defective elongation of mannose caps of lipoarabinomannan (LAM) and absence of α(1→2)mannose branches on the lipomannan (LM) and LAM mannan core, as determined by biochemical analysis (NMR and GC-MS) and immunoblotting. We found that the M. marinum mptC mutant is strongly attenuated in embryonic zebrafish, which rely solely on innate immunity, whereas minor virulence defects were observed in adult zebrafish. Strikingly, complementation with the Mycobacterium smegmatis mptC orthologue, which restored mannan core branching but not cap elongation, was sufficient to fully complement the virulence defect of the mptC mutant in embryos. Altogether our data demonstrate that not LAM capping, but mannan core branching of LM/LAM plays an important role in mycobacterial pathogenesis in the context of innate immunity.

Cyanovirin-N inhibits mannose-dependent Mycobacterium-C-type lectin interactions but does not protect against murine tuberculosis.

Cyanovirin-N (CV-N) is a mannose-binding lectin that inhibits HIV-1 infection by blocking mannose-dependent target cell entry via C-type lectins. Like HIV-1, Mycobacterium tuberculosis expresses mannosylated surface structures and exploits C-type lectins to gain cell access. In this study, we investigated whether CV-N, like HIV-1, can inhibit M. tuberculosis infection. We found that CV-N specifically interacted with mycobacteria by binding to the mannose-capped lipoglycan lipoarabinomannan. Furthermore, CV-N competed with the C-type lectins DC-SIGN and mannose receptor for ligand binding and inhibited the binding of M. tuberculosis to dendritic cells but, unexpectedly, not to macrophages. Subsequent in vivo infection experiments in a mouse model demonstrated that, despite its activity, CV-N did not inhibit or delay M. tuberculosis infection. This outcome argues against a critical role for mannose-dependent C-type lectin interactions during the initial stages of murine M. tuberculosis infection and suggests that, depending on the circumstances, M. tuberculosis can productively infect cells using different modes of entry.

Unexpected link between lipooligosaccharide biosynthesis and surface protein release in Mycobacterium marinum.

The mycobacterial cell envelope is characterized by the presence of a highly impermeable second membrane, which is composed of mycolic acids intercalated with different unusual free lipids, such as lipooligosaccharides (LOS). Transport across this cell envelope requires a dedicated secretion system for extracellular proteins, such as PE_PGRS proteins, which are specific mycobacterial proteins with polymorphic GC-rich sequence (PGRS). In this study, we set out to identify novel components involved in the secretion of PE_PGRS proteins by screening Mycobacterium marinum transposon mutants for secretion defects. Interestingly, most mutants were not affected in secretion but in the release of PE_PGRS proteins from the cell surface. These mutants had insertions in a gene cluster associated with LOS biosynthesis. Lipid analysis of these mutants revealed a role at different stages of LOS biosynthesis for 10 novel genes. Furthermore, we show that regulatory protein WhiB4 is involved in LOS biosynthesis. The absence of the most extended LOS molecule, i.e. LOS-IV, and a concomitant accumulation of LOS-III was already sufficient to reduce the release of PE_PGRS proteins from the mycobacterial cell surface. A similar effect was observed for major surface protein EspE. These results show that the attachment of surface proteins is strongly influenced by the glycolipid composition of the mycobacterial cell envelope. Finally, we tested the virulence of a LOS-IV-deficient mutant in our zebrafish embryo infection model. This mutant showed a marked increase in virulence as compared with the wild-type strain, suggesting that LOS-IV plays a role in the modulation of mycobacterial virulence.

ESX-5-deficient Mycobacterium marinum is hypervirulent in adult zebrafish.

ESX-5 is a mycobacterial type VII protein secretion system responsible for transport of numerous PE and PPE proteins. It is involved in the induction of host cell death and modulation of the cytokine response in vitro. In this work, we studied the effects of ESX-5 in embryonic and adult zebrafish using Mycobacterium marinum. We found that ESX-5-deficient M. marinum was slightly attenuated in zebrafish embryos. Surprisingly, the same mutant showed highly increased virulence in adult zebrafish, characterized by increased bacterial loads and early onset of granuloma formation with rapid development of necrotic centres. This early onset of granuloma formation was accompanied by an increased expression of pro-inflammatory cytokines and tissue remodelling genes in zebrafish infected with the ESX-5 mutant. Experiments using RAG-1-deficient zebrafish showed that the increased virulence of the ESX-5 mutant was not dependent on the adaptive immune system. Mixed infection experiments with wild-type and ESX-5 mutant bacteria showed that the latter had a specific advantage in adult zebrafish and outcompeted wild-type bacteria. Together our experiments indicate that ESX-5-mediated protein secretion is used by M. marinum to establish a moderate and persistent infection.

Mycobacterial secretion systems ESX-1 and ESX-5 play distinct roles in host cell death and inflammasome activation.

During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5--two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators--during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1ß activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread.

Identification of a glycosyltransferase from Mycobacterium marinum involved in addition of a caryophyllose moiety in lipooligosaccharides.

Deletion of Mycobacterium marinum MMAR2333 resulted in the loss of three of four subclasses of lipooligosaccharides (LOSs). The mutant was unable to extend an intermediate (LOS-II*) by addition of caryophyllose. These data and the predicted domain structure suggest that MMAR2333 is a glycosyltransferase involved in the generation of a lipid-linked caryophyllose donor.

IL-1R1-Dependent Signals Improve Control of Cytosolic Virulent Mycobacteria In Vivo.

Mycobacterium tuberculosis infections claim more than a million lives each year, and better treatments or vaccines are required. A crucial pathogenicity factor is translocation from phagolysosomes to the cytosol upon phagocytosis by macrophages. Translocation from the phagolysosome to the cytosol is an ESX-1-dependent process, as previously shown in vitro Here, we show that in vivo, mycobacteria also translocate to the cytosol but mainly when host immunity is compromised. We observed only low numbers of cytosolic bacilli in mice, armadillos, zebrafish, and patient material infected with M. tuberculosis, M. marinum, or M. leprae In contrast, when innate or adaptive immunity was compromised, as in severe combined immunodeficiency (SCID) or interleukin-1 receptor 1 (IL-1R1)-deficient mice, significant numbers of cytosolic M. tuberculosis bacilli were detected in the lungs of infected mice. Taken together, in vivo, translocation to the cytosol of M. tuberculosis is controlled by adaptive immune responses as well as IL-1R1-mediated signals.IMPORTANCE For decades, Mycobacterium tuberculosis has been one of the deadliest pathogens known. Despite infecting approximately one-third of the human population, no effective treatment or vaccine is available. A crucial pathogenicity factor is subcellular localization, as M. tuberculosis can translocate from phagolysosome to the cytosol in macrophages. The situation in vivo is more complicated. In this study, we establish that high-level cytosolic escape of mycobacteria can indeed occur in vivo but mainly when host resistance is compromised. The IL-1 pathway is crucial for the control of the number of cytosolic mycobacteria. The establishment that immune signals result in the clearance of cells containing cytosolic mycobacteria connects two important fields, cell biology and immunology, which is vital for the understanding of the pathology of M. tuberculosis.

Quantification of Natural Growth of Two Strains of Mycobacterium Marinum for Translational Antituberculosis Drug Development.

The zebrafish infected with Mycobacterium marinum (M. marinum) is an attractive tuberculosis disease model, showing similar pathogenesis to Mycobacterium tuberculosis (M. tuberculosis) infections in humans. To translate pharmacological findings from this disease model to higher vertebrates, a quantitative understanding of the natural growth of M. marinum in comparison to the natural growth of M. tuberculosis is essential. Here, the natural growth of two strains of M. marinum, E11 and MUSA , is studied over an extended period using an established model-based approach, the multistate tuberculosis pharmacometric (MTP) model, for comparison to that of M. tuberculosis. Poikilotherm-derived strain E11 and human-derived strain MUSA were grown undisturbed up to 221 days and viability of cultures (colony forming unit (CFU)/mL) was determined by plating at different time points. Nonlinear mixed effects modeling using the MTP model quantified the bacterial growth, the transfer among fast, slow, and non-multiplying states, and the inoculi. Both strains showed initial logistic growth, reaching a maximum after 20-25 days for E11 and MUSA , respectively, followed by a decrease to a new plateau. Natural growth of both E11 and MUSA was best described with Gompertz growth functions. For E11, the inoculum was best described in the slow-multiplying state, for MUSA in the fast-multiplying state. Natural growth of E11 was most similar to that of M. tuberculosis, whereas MUSA showed more aggressive growth behavior. Characterization of natural growth of M. marinum and quantitative comparison with M. tuberculosis brings the zebrafish tuberculosis disease model closer to the quantitative translational pipeline of antituberculosis drug development.

CSN5 inhibition triggers inflammatory signaling and Rho/ROCK-dependent loss of endothelial integrity.

RhoGTPases regulate cytoskeletal dynamics, migration and cell-cell adhesion in endothelial cells. Besides regulation at the level of guanine nucleotide binding, they also undergo post-translational modifications, for example ubiquitination. RhoGTPases are ubiquitinated by Cullin RING ligases which are in turn regulated by neddylation. Previously we showed that inhibition of Cullin RING ligase activity by the neddylation inhibitor MLN4924 is detrimental for endothelial barrier function, due to accumulation of RhoB and the consequent induction of contractility. Here we analyzed the effect of pharmacological activation of Cullin RING ligases on endothelial barrier integrity in vitro and in vivo. CSN5i-3 induced endothelial barrier disruption and increased macromolecule leakage in vitro and in vivo. Mechanistically, CSN5i-3 strongly induced the expression and activation of RhoB and to lesser extent of RhoA in endothelial cells, which enhanced cell contraction. Elevated expression of RhoGTPases was a consequence of activation of the NF-κB pathway. In line with this notion, CSN5i-3 treatment decreased IκBα expression and increased NF-κB-mediated ICAM-1 expression and consequent adhesion of neutrophils to endothelial cells. This study shows that sustained neddylation of Cullin RING-ligases leads to activation the NF-κB pathway in endothelial cells, elevated expression of RhoGTPases, Rho/ROCK-dependent activation of MLC and disruption of the endothelial barrier.

Type VII Secretion Substrates of Pathogenic Mycobacteria Are Processed by a Surface Protease.

Tuberculosis, one of the world's most severe infectious diseases, is caused by Mycobacterium tuberculosis A major weapon of this pathogen is a unique cell wall that protects the pathogen from eradication by the immune system. Mycobacteria have specialized secretion systems, e.g., type VII secretion or ESX systems, to transport substrates across this cell wall. The largest group of proteins that are secreted by these ESX systems are the PE proteins. Previously, it was shown that the N-terminal PE domain of about 100 amino acids is required for secretion. Here, we describe the identification of an aspartic protease, designated PecA, that removes (part of) this PE domain at the cell surface. Nearly all of the observed PE_PGRS proteins are processed by PecA. Interestingly, the protease itself is also a secreted PE protein and subject to self-cleavage. Furthermore, a defect in surface processing has no effect on the activity of the PE lipase protein LipY but does seem to affect the functioning of other virulence factors, as a pecA mutant strain of Mycobacterium marinum shows moderate attenuation in zebrafish larvae. In conclusion, our results reveal the presence of a functional aspartic acid protease in M. marinum that cleaves LipY, itself as well as other members of the PE_PGRS family. Finally, mutants lacking PecA show growth attenuation in vivo, suggesting that PecA plays a role during infection.IMPORTANCE Aspartic proteases are common in eukaryotes and retroviruses but are relatively rare among bacteria (N. D. Rawlings and A. Bateman, BMC Genomics 10:437, 2009, https://doi.org/10.1186/1471-2164-10-437). In contrast to eukaryotic aspartic proteases, bacterial aspartic proteases are generally located in the cytoplasm. We have identified a surface-associated mycobacterial aspartic protease, PecA, which cleaves itself and many other type VII secretion substrates of the PE_PGRS family. PecA is present in most pathogenic mycobacterial species, including M. tuberculosis In addition, pathogenicity of M. marinum is reduced in the ΔpecA mutant, indicating that PecA contributes to virulence.

Identification and real-time imaging of a myc-expressing neutrophil population involved in inflammation and mycobacterial granuloma formation in zebrafish.

By enhancer trap screening we identified a transgenic zebrafish line showing leukocyte-specific YFP expression during late embryo and early larval development. Its enhancer detection insertion was mapped near a novel member of the myc proto-oncogene family, encoding transcription factors known to be important for regulating human myelopoiesis. Characterization of the zebrafish myc family showed that only this particular myc gene is strongly expressed in leukocytes. To identify the myc/YFP-expressing cell type, we re-examined specificity of described myeloid markers by multiplex fluorescent in situ hybridization, showing that lcp1 can be considered as a general leukocyte marker, csf1r as a macrophage-specific marker, and mpx and lyz as neutrophil-specific markers. Subsequent colocalization analysis defined the YFP-positive cells as a subset of the neutrophil population. Using real-time confocal imaging we demonstrate that these cells migrate to sites of inflammation and are involved in innate immune responses towards infections, including Mycobacterium marinum-induced granuloma formation.

Transmission of Mycobacterium marinum from fish to a very young child.

Mycobacterium marinum causes tuberculosis in fish and can cause skin infections in humans who swim in contaminated water or who have direct contact with infected fish. We report the case study of an 18-month-old girl with M. marinum abscesses, who acquired the infection through indirect contact with a contaminated bucket. Appropriate cleaning of aquarium equipment is very important, especially with young children in the household.

Identification and High-Resolution Imaging of α-Tocopherol from Human Cells to Whole Animals by TOF-SIMS Tandem Mass Spectrometry.

A unique method for identification of biomolecular components in different biological specimens, while preserving the capability for high speed 2D and 3D molecular imaging, is employed to investigate cellular response to oxidative stress. The employed method enables observing the distribution of the antioxidant α-tocopherol and other molecules in cellular structures via time-of-flight secondary ion mass spectrometry (TOF-SIMS (MS1)) imaging in parallel with tandem mass spectrometry (MS2) imaging, collected simultaneously. The described method is employed to examine a network formed by neuronal cells differentiated from human induced pluripotent stem cells (iPSCs), a model for investigating human neurons in vitro. The antioxidant α-tocopherol is identified in situ within different cellular layers utilizing a 3D TOF-SIMS tandem MS imaging analysis. As oxidative stress also plays an important role in mediating inflammation, the study was expanded to whole body tissue sections of M. marinum-infected zebrafish, a model organism for tuberculosis. The TOF-SIMS tandem MS imaging results reveal an increased presence of α-tocopherol in response to the pathogen. Graphical Abstract ᅟ.

Transcriptome profiling of adult zebrafish at the late stage of chronic tuberculosis due to Mycobacterium marinum infection.

The Mycobacterium marinum-zebrafish infection model was used in this study for analysis of a host transcriptome response to mycobacterium infection at the organismal level. RNA isolated from adult zebrafish that showed typical signs of fish tuberculosis due to a chronic progressive infection with M. marinum was compared with RNA from healthy fish in microarray analyses. Spotted oligonucleotide sets (designed by Sigma-Compugen and MWG) and Affymetrix GeneChips were used, in total comprising 45,465 zebrafish transcript annotations. Based on a detailed comparative analysis and quantitative reverse transcriptase-PCR analysis, we present a validated reference set of 159 genes whose regulation is strongly affected by mycobacterial infection in the three types of microarrays analyzed. Furthermore, we analyzed the separate datasets of the microarrays with special emphasis on the expression profiles of immune-related genes. Upregulated genes include many known components of the inflammatory response and several genes that have previously been implicated in the response to mycobacterial infections in cell cultures of other organisms. Different marker genes of the myeloid lineage that have been characterized in zebrafish also showed increased expression. Furthermore, the zebrafish homologs of many signal transduction genes with relationship to the immune response were induced by M. marinum infection. Future functional analysis of these genes may contribute to understanding the mechanisms of mycobacterial pathogenesis. Since a large group of genes linked to immune responses did not show altered expression in the infected animals, these results suggest specific responses in mycobacterium-induced disease.