Cloning, Characterization, and Sulfonamide and Thiol Inhibition Studies of an α‑Carbonic Anhydrase from <i>Trypanosoma cruzi</i>, the Causative Agent of Chagas Disease

An α-carbonic anhydrase (CA, EC 4.2.1.1) has been identified, cloned, and characterized from the unicellular protozoan <i>Trypanosoma cruzi</i>, the causative agent of Chagas disease. The enzyme (TcCA) has a very high catalytic activity for the CO₂ hydration reaction, being similar kinetically to the human (h) isoform hCA II, although it is devoid of the His64 proton shuttle. A large number of aromatic/heterocyclic sulfonamides and some 5-mercapto-1,3,4-thiadiazoles were investigated as TcCA inhibitors. The aromatic sulfonamides were weak inhibitors (<i>K</i>_I values of 192 nM to 84 μM), whereas some heterocyclic compounds inhibited the enzyme with <i>K</i>_I values in the range 61.6–93.6 nM. The thiols were the most potent in vitro inhibitors (<i>K</i>_I values of 21.1–79.0 nM), and some of them also inhibited the epimastigotes growth of two <i>T. cruzi</i> strains in vivo

Phenotypes of zebrafish larvae knocked down with different concentrations of <i>ca10a</i> and <i>ca10b</i> antisense morpholinos.

<p>Phenotypes of zebrafish larvae knocked down with different concentrations of <i>ca10a</i> and <i>ca10b</i> antisense morpholinos.</p

Inactivation of <i>ca10a</i> and <i>ca10b</i> Genes Leads to Abnormal Embryonic Development and Alters Movement Pattern in Zebrafish

<div><p>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 <i>CA11</i> gene, but instead possess two co-orthologs of <i>CA10</i>. We analyzed the expression pattern of zebrafish <i>ca10a</i> and <i>ca10b</i> 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 <i>ca10a</i> and <i>ca10b</i> 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 <i>ca10a</i> and <i>ca10b </i>genes are expressed in the brain and several other tissues throughout the development of zebrafish. Antisense morpholino mediated knockdown of <i>ca10a</i> and <i>ca10b</i> 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 <i>ca10a</i> and <i>ca10b</i> 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 <i>ca10a</i> and <i>ca10b</i> expression in zebrafish larvae leads to a movement disorder.</p></div

Silencing of <i>ca10a</i> and <i>ca10b</i> in zebrafish larvae.

<p><b>A)</b> Schematic presentation of matured <i>ca10a</i> mRNA showing the site of translational blocking with MO1 at the translation start site (arrow). <b>B)</b> Schematic structure of unprocessed mRNA for <i>ca10a</i> with a target region (horizontal bar) for a splice site blocking morpholino (MO2) which knocks down the exon eight. <b>C)</b> Schematic depiction of unprocessed mRNA for <i>ca10b</i> and target sites for splice site interfering morpholinos, MO1 and MO2 (black horizontal bars) and gRNA target regions (red horizontal bars). <b>D)</b> Gel electrophoresis showing RT-PCR analysis of <i>ca10a</i> morphant mRNA injected with MO2. <b>E)</b> RT-PCR gel image of <i>ca10b</i> morphant zebrafish mRNA injected with MO1 and MO2 targeting different exons. The images show the reduction in the length of the mRNAs (Lane 2 in <b>D</b> and Lane 3 and 4 in <b>E)</b> compared with wild type mRNAs of <i>ca10a</i> and <i>ca10b</i> in wild type fish (Lane 3 in <b>D</b> and lane 2 <b>E</b>). <b>F)</b> The efficiency of the CRISPR/Cas9 mediated mutagenesis in zebrafish embryos was evaluated with a T7 endonuclease assay. For both <i>ca10a</i> and <i>ca10b</i>, uninjected and gRNA control fish are shown and as well as two individual embryos with a mutated target site and a pool of 5–10 mutated embryos. Representative cleaved PCR products of the expected sizes are shown as arrow heads. Cleavage percentage was calculated from the band intensities of each lane.</p

Expression levels of <i>ca10a</i> and <i>ca10b</i> genes in adult zebrafish tissues.

<p>A quantitative analysis of <i>ca10a</i> and <i>ca10b</i> genes was made for 14 adult zebrafish tissues using RT-qPCR. <b>A</b>, expression of <i>ca10a</i> in zebrafish tissues; <b>B</b>, expression of <i>ca10b</i> in zebrafish tissues. The values were normalized to the beta actin control according to the Pfaffl´s equation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134263#pone.0134263.ref034" target="_blank">34</a>]. The expression of the <i>ca10a</i> gene in brain and <i>ca10b</i> in the ovary assigned a relative value of 1.</p

Displacement patterns of the morphant and control fish.

<p>Representative displacement trajectories of the movement pattern are shown for <b>A)</b> larvae injected with <i>ca10a</i>-MO2, <b>B)</b> larvae injected with <i>ca10b-</i>MO2 <b>C)</b> larvae injected with RC-MOs and, <b>D)</b> wildtype <b>(</b>uninjected) larvae. Groups of 13 to 22 fish were video recorded in 90mm petri dishes over a 2 minute time period. ImageJ and MtrackJ plugin were used to track the paths of all fish [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134263#pone.0134263.ref039" target="_blank">39</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0134263#pone.0134263.ref040" target="_blank">40</a>].</p

Morpholino sequences used for knockdown of <i>ca10a</i> and <i>ca10b</i> genes.

<p>Morpholino sequences used for knockdown of <i>ca10a</i> and <i>ca10b</i> genes.</p

Partial rescue of <i>ca10a</i> and <i>ca10b</i> zebrafish morphants.

<p><b>A)</b> The <i>ca10a</i> morphant (5dpf) embryos; <b>B)</b> The 5 dpf zebrafish <i>ca10a</i> morphant embryos rescued with injection of <i>CA10</i> mRNA; <b>C)</b> The <i>ca10b</i> morphant (5dpf) embryos; <b>D)</b> Partially rescued 5dpf embryos with <i>CA11</i> mRNA.</p

Sequences of CARP X/XI family.

<p>Sequences of CARP X/XI family.</p

Comparisons of CARP X-like proteins as identity percentages between aligned protein sequences.

<p>Comparisons of CARP X-like proteins as identity percentages between aligned protein sequences.</p