The zebrafish mutants for the V-ATPase subunits d, ac45, E, H and c and their variable pigment dilution phenotype

Ramos-Balderas, Jose Luis; Carrillo-Rosas, Samantha; Guzmán, Adrián; Navarro, Rosa E.; Maldonado, Ernesto

doi:10.1186/1756-0500-6-39

Cited by 11 publications

(9 citation statements)

References 49 publications

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“…The efficacy of atp6v0ca knockdown was confirmed at the genetic level (Figure S5A). Furthermore, knockdown larvae displayed characteristic pigment dilution (Figure S5B-C) as has been observed in insertional mutants of v-ATPase subunits [37]. It has been previously demonstrated in zebrafish that morpholino-mediated atp6v0ca knockdown results in inhibition of lysosomal acidification [38].…”

Section: Phagosomal Acidification Within Macrophages Issupporting

confidence: 61%

Phagosomal Acidification Is Required to Kill Streptococcus pneumoniae in a Zebrafish Model

Prajsnar

Michno

Pooranachandran

et al. 2022

Cellular Microbiology

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Streptococcus pneumoniae (the pneumococcus) is a major human pathogen causing invasive disease, including community-acquired bacteraemia, and remains a leading cause of global mortality. Understanding the role of phagocytes in killing bacteria is still limited, especially in vivo. In this study, we established a zebrafish model to study the interaction between intravenously administered pneumococci and professional phagocytes such as macrophages and neutrophils, to unravel bacterial killing mechanisms employed by these immune cells. Our model confirmed the key role of polysaccharide capsule in promoting pneumococcal virulence through inhibition of phagocytosis. Conversely, we show pneumococci lacking a capsule are rapidly internalised by macrophages. Low doses of encapsulated S. pneumoniae cause near 100% mortality within 48 hours postinfection (hpi), while 50 times higher doses of unencapsulated pneumococci are easily cleared. Time course analysis of in vivo bacterial numbers reveals that while encapsulated pneumococcus proliferates to levels exceeding 105 CFU at the time of host death, unencapsulated bacteria are unable to grow and are cleared within 20 hpi. Using genetically induced macrophage depletion, we confirmed an essential role for macrophages in bacterial clearance. Additionally, we show that upon phagocytosis by macrophages, phagosomes undergo rapid acidification. Genetic and chemical inhibition of vacuolar ATPase (v-ATPase) prevents intracellular bacterial killing and induces host death indicating a key role of phagosomal acidification in immunity to invading pneumococci. We also show that our model can be used to study the efficacy of antimicrobials against pneumococci in vivo. Collectively, our data confirm that larval zebrafish can be used to dissect killing mechanisms during pneumococcal infection in vivo and highlight key roles for phagosomal acidification in macrophages for pathogen clearance.

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Section: Phagosomal Acidification Within Macrophages Issupporting

confidence: 61%

Phagosomal Acidification Is Required to Kill Streptococcus pneumoniae in a Zebrafish Model

Prajsnar

Michno

Pooranachandran

et al. 2022

Cellular Microbiology

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“…It has been shown that V-ATPase is mostly found in acidic premelanosomes, but not in mature and highly pigmented melanosomes [ 93 ]. Moreover, several studies using zebrafish mutants in V-ATPase subunits strongly suggested that V-ATPase expression is required for melanosome biogenesis and/or to sustain normal melanosome morphology and stability [ 94 , 95 ]. Conceivably, the overexpression of Atp6v0d2 and other V-ATPase subunits in MGRN1-depleted cells might, therefore, be related with the increased number of melanosomes in these cells rather with the high specific activity of TYR in these melanosomes.…”

Section: Discussionmentioning

confidence: 99%

Mahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells

Sirés-Campos

Lambertos

Delevoye

et al. 2021

Cell. Mol. Life Sci.

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Mahogunin Ring Finger 1 (MGRN1) is an E3-ubiquitin ligase absent in dark-furred mahoganoid mice. We investigated the mechanisms of hyperpigmentation in Mgrn1-null melan-md1 melanocytes, Mgrn1-KO cells obtained by CRISPR-Cas9-mediated knockdown of Mgrn1 in melan-a6 melanocytes, and melan-a6 cells depleted of MGRN1 by siRNA treatment. Mgrn1-deficient melanocytes showed higher melanin content associated with increased melanosome abundance and higher fraction of melanosomes in highly melanized maturation stages III–IV. Expression, post-translational processing and enzymatic activity of the rate-limiting melanogenic enzyme tyrosinase measured in cell-free extracts were comparable in control and MGRN1-depleted cells. However, tyrosinase activity measured in situ in live cells and expression of genes associated with regulation of pH increased upon MGRN1 repression. Using pH-sensitive fluorescent probes, we found that downregulation of MGRN1 expression in melanocytes and melanoma cells increased the pH of acidic organelles, including melanosomes, strongly suggesting a previously unknown role of MGRN1 in the regulation of melanosomal pH. Among the pH regulatory genes upregulated by Mgrn1 knockdown, we identified those encoding several subunits of the vacuolar adenosine triphosphatase V-ATPase (mostly Atp6v0d2) and a calcium channel of the transient receptor potential channel family, Mucolipin 3 (Mcoln3). Manipulation of expression of the Mcoln3 gene showed that overexpression of Mcoln3 played a significant role in neutralization of the pH of acidic organelles and activation of tyrosinase in MGRN1-depleted cells. Therefore, lack of MGRN1 led to cell-autonomous stimulation of pigment production in melanocytes mostly by increasing tyrosinase specific activity through neutralization of the melanosomal pH in a MCOLN3-dependent manner.

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“…In mammals, mature melanosomes are transported from melanocytes to keratinocytes [43] . Furthermore, mutations in V-ATPase subunits produce pigment dilution phenotypes in Drosophila , zebrafish, mice and humans [44] , [45] . Since V-ATPase function has been shown to be essential for melanosome biogenesis, we hypothesize that the pigmented phenotype of CALM may be a consequence of increased expression of ATP6V0B and an increase in the number of mature melanosomes produced in melanocytes (or heightened pigmentation) and/or transported to surrounding keratinocytes.…”

Section: Discussionmentioning

confidence: 99%

Genetic Modifiers of Neurofibromatosis Type 1-Associated Café-au-Lait Macule Count Identified Using Multi-platform Analysis

et al. 2014

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Neurofibromatosis type 1 (NF1) is an autosomal dominant, monogenic disorder of dysregulated neurocutaneous tissue growth. Pleiotropy, variable expressivity and few NF1 genotype-phenotype correlates limit clinical prognostication in NF1. Phenotype complexity in NF1 is hypothesized to derive in part from genetic modifiers unlinked to the NF1 locus. In this study, we hypothesized that normal variation in germline gene expression confers risk for certain phenotypes in NF1. In a set of 79 individuals with NF1, we examined the association between gene expression in lymphoblastoid cell lines with NF1-associated phenotypes and sequenced select genes with significant phenotype/expression correlations. In a discovery cohort of 89 self-reported European-Americans with NF1 we examined the association between germline sequence variants of these genes with café-au-lait macule (CALM) count, a tractable, tumor-like phenotype in NF1. Two correlated, common SNPs (rs4660761 and rs7161) between DPH2 and ATP6V0B were significantly associated with the CALM count. Analysis with tiled regression also identified SNP rs4660761 as significantly associated with CALM count. SNP rs1800934 and 12 rare variants in the mismatch repair gene MSH6 were also associated with CALM count. Both SNPs rs7161 and rs4660761 (DPH2 and ATP6V0B) were highly significant in a mega-analysis in a combined cohort of 180 self-reported European-Americans; SNP rs1800934 (MSH6) was near-significant in a meta-analysis assuming dominant effect of the minor allele. SNP rs4660761 is predicted to regulate ATP6V0B, a gene associated with melanosome biology. Individuals with homozygous mutations in MSH6 can develop an NF1-like phenotype, including multiple CALMs. Through a multi-platform approach, we identified variants that influence NF1 CALM count.

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The zebrafish mutants for the V-ATPase subunits d, ac45, E, H and c and their variable pigment dilution phenotype

Cited by 11 publications

References 49 publications

Phagosomal Acidification Is Required to Kill Streptococcus pneumoniae in a Zebrafish Model

Phagosomal Acidification Is Required to Kill Streptococcus pneumoniae in a Zebrafish Model

Mahogunin Ring Finger 1 regulates pigmentation by controlling the pH of melanosomes in melanocytes and melanoma cells

Genetic Modifiers of Neurofibromatosis Type 1-Associated Café-au-Lait Macule Count Identified Using Multi-platform Analysis

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