Carbonic anhydrase related proteins (CARPs) X and XI are highly conserved across species and are predominantly expressed in neural tissues. The biological role of these proteins is still an enigma. Ray-finned fish have lost the CA11 gene, but instead possess two co-orthologs of CA10. We analyzed the expression pattern of zebrafish ca10a and ca10b genes during embryonic development and in different adult tissues, and studied 61 CARP X/XI-like sequences to evaluate their phylogenetic relationship. Sequence analysis of zebrafish ca10a and ca10b reveals strongly predicted signal peptides, N-glycosylation sites, and a potential disulfide, all of which are conserved, suggesting that all of CARP X and XI are secretory proteins and potentially dimeric. RT-qPCR showed that zebrafish ca10a and ca10b genes are expressed in the brain and several other tissues throughout the development of zebrafish. Antisense morpholino mediated knockdown of ca10a and ca10b showed developmental delay with a high rate of mortality in larvae. Zebrafish morphants showed curved body, pericardial edema, and abnormalities in the head and eye, and there was increased apoptotic cell death in the brain region. Swim pattern showed abnormal movement in morphant zebrafish larvae compared to the wild type larvae. The developmental phenotypes of the ca10a and ca10b morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system. In conclusion, we introduce a novel zebrafish model to investigate the mechanisms of CARP Xa and CARP Xb functions. Our data indicate that CARP Xa and CARP Xb have important roles in zebrafish development and suppression of ca10a and ca10b expression in zebrafish larvae leads to a movement disorder.

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.

Tuberculosis is a chronic bacterial disease with a complex pathogenesis. An effective immunity against Mycobacterium tuberculosis requires both the innate and adaptive immune responses, including proper T helper (Th) type 1 cell function. FURIN is a proprotein convertase subtilisin/kexin (PCSK) enzyme, which is highly expressed in Th1 type cells. FURIN expression in T cells is essential for maintaining peripheral immune tolerance, but its role in the innate immunity and infections has remained elusive. Here, we utilized Mycobacterium marinum infection models in zebrafish (Danio rerio) to investigate how furin regulates host responses against mycobacteria. In steady-state furinA td204e/؉ fish reduced furinA mRNA levels associated with low granulocyte counts and elevated Th cell transcription factor expressions. Silencing furin genes reduced the survival of M. marinuminfected zebrafish embryos. A mycobacterial infection upregulated furinA in adult zebrafish, and infected furinA td204e/؉ mutants exhibited a proinflammatory phenotype characterized by elevated tumor necrosis factor a (tnfa), lymphotoxin alpha (lta) and interleukin 17a/f3 (il17a/f3) expression levels. The enhanced innate immune response in the furinA td204e/؉ mutants correlated with a significantly decreased bacterial burden in a chronic M. marinum infection model. Our data show that upregulated furinA expression can serve as a marker for mycobacterial disease, since it inhibits early host responses and consequently promotes bacterial growth in a chronic infection.T uberculosis (TB) is an epidemic infectious disease caused by the mycobacterial species Mycobacterium tuberculosis (1, 2). Circa 13% of the individuals with active TB were simultaneous carriers of the human immunodeficiency virus (HIV), and almost one-third of TB-associated deaths occurred among HIV ϩ patients, demonstrating the critical role of cluster of differentiation 4 (CD4 ϩ ) T lymphocyte-mediated immunity in the control of M. tuberculosis infection (3, 4). More specifically, the adaptive immunity against TB is primarily mediated by T helper (Th) type 1 cells, as is suggested by the gene expression profile upon infection (5), as well as the infection-induced mortality of gamma interferon-deficient (6, 7) and interleukin-12 (IL-12)-deficient (8) mice.The proprotein convertase subtilisin/kexin (PCSK) enzymes are a family of serine endoproteases with nine members in humans: PCSK1 and -2, FURIN, PCSK4 to -7, membrane-bound transcription factor peptidase site 1 (MBTPS1), and PCSK9 (9). Typically, PCSKs convert precursor proteins (proproteins) into their biologically active forms by cleaving them at specific target motifs made up of the basic amino acids lysine and arginine (9, 10). FURIN was the first identified mammalian PCSK and is present in vertebrates and many invertebrates (11,12). A series of in vitro experiments have suggested a central role for FURIN in host defense because it proteolytically activates several immunoregulatory proproteins, such as membrane-inserted matrix metallo...

Bradavidin II is a biotin-binding protein from Bradyrhizobium japonicum that resembles chicken avidin and bacterial streptavidin. A biophysical characterization was carried out using dynamic light scattering, native mass spectrometry, differential scanning calorimetry, and isothermal titration calorimetry combined with structural characterization using X-ray crystallography. These observations revealed that bradavidin II differs from canonical homotetrameric avidin protein family members in its quaternary structure. In contrast with the other avidins, bradavidin II appears to have a dynamic (transient) oligomeric state in solution. It is monomeric at low protein concentrations but forms higher oligomeric assemblies at higher concentrations. The crystal structure of bradavidin II revealed an important role for Phe42 in shielding the bound ligand from surrounding water molecules, thus functionally replacing the L7,8 loop essential for tight ligand binding in avidin and streptavidin. This bradavidin II characterization opens new avenues for oligomerizationindependent biotin-binding protein development.