Intracellular ion channels are essential regulators of organellar and cellular function, yet the molecular identity and physiological role of many of these channels remains elusive. In particular, no ion channel has been characterized in melanosomes, organelles that produce and store the major mammalian pigment melanin. Defects in melanosome function cause albinism, characterized by vision and pigmentation deficits, impaired retinal development, and increased susceptibility to skin and eye cancers. The most common form of albinism is caused by mutations in oculocutaneous albinism II (OCA2), a melanosome-specific transmembrane protein with unknown function. Here we used direct patch-clamp of skin and eye melanosomes to identify a novel chloride-selective anion conductance mediated by OCA2 and required for melanin production. Expression of OCA2 increases organelle pH, suggesting that the chloride channel might regulate melanin synthesis by modulating melanosome pH. Thus, a melanosomal anion channel that requires OCA2 is essential for skin and eye pigmentation.DOI: http://dx.doi.org/10.7554/eLife.04543.001

Intracellular organelles mediate complex cellular functions that often require ion transport across their membranes. Melanosomes are organelles responsible for the synthesis of the major mammalian pigment melanin. Defects in melanin synthesis result in pigmentation defects, visual deficits, and increased susceptibility to skin and eye cancers. Although genes encoding putative melanosomal ion transporters have been identified as key regulators of melanin synthesis, melanosome ion transport and its contribution to pigmentation remain poorly understood. Here we identify two-pore channel 2 (TPC2) as the first reported melanosomal cation conductance by directly patch-clamping skin and eye melanosomes. TPC2 has been implicated in human pigmentation and melanoma, but the molecular mechanism mediating this function was entirely unknown. We demonstrate that the vesicular signaling lipid phosphatidylinositol bisphosphate PI(3,5)P2 modulates TPC2 activity to control melanosomal membrane potential, pH, and regulate pigmentation.

SLC45A2 encodes a putative transporter expressed primarily in pigment cells. SLC45A2 mutations cause oculocutaneous albinism type IV (OCA4) and polymorphisms are associated with pigmentation variation, but the localization, function, and regulation of SLC45A2 and its variants remain unknown. We show that SLC45A2 localizes to a cohort of mature melanosomes that only partially overlaps with those expressing the chloride channel OCA2. SLC45A2 expressed ectopically in HeLa cells localizes to lysosomes and raises lysosomal pH, suggesting that in melanocytes, SLC45A2 expression, like OCA2 expression, results in the deacidification of maturing melanosomes to support melanin synthesis. Interestingly, OCA2 overexpression compensates for loss of SLC45A2 expression in pigmentation. Analyses of SLC45A2- and OCA2-deficient mouse melanocytes show that SLC45A2 likely functions later during melanosome maturation than OCA2. Moreover, the light skin-associated SLC45A2 allelic F374 variant restores only moderate pigmentation to SLC45A2-deficient melanocytes due to rapid proteasome-dependent degradation resulting in lower protein expression levels in melanosomes than the dark skin-associated allelic variant. Our data suggest that SLC45A2 maintains melanosome neutralization initially orchestrated by transient OCA2 activity to support melanization at late stages of melanosome maturation, and that a common variant imparts reduced activity due to protein instability.

Multidrug-resistant pathogens pose a serious threat to human health. For decades, the antibiotic vancomycin has been a potent option when treating Gram-positive multidrug-resistant infections. Nonetheless, in recent decades, we have begun to see an increase in vancomycin-resistant bacteria. Here, we show that the nuclear factor-kappa B (NF-κB) inhibitor N-[3,5-Bis(trifluoromethyl)phenyl]-5-chloro-2-hydroxybenzamide (IMD0354) was identified as a positive hit through a Caenorhabditis elegans–methicillin-resistant Staphylococcus aureus (MRSA) infection screen. IMD0354 was a potent bacteriostatic drug capable of working at a minimal inhibitory concentration (MIC) as low as 0.06 µg/mL against various vancomycin-resistant strains. Interestingly, IMD0354 showed no hemolytic activity at concentrations as high as 16 µg/mL and is minimally toxic to C. elegans in vivo with 90% survival up to 64 µg/mL. In addition, we demonstrated that IMD0354′s mechanism of action at high concentrations is membrane permeabilization. Lastly, we found that IMD0354 is able to inhibit vancomycin-resistant Staphylococcus aureus (VRSA) initial cell attachment and biofilm formation at sub-MIC levels and above. Our work highlights that the NF-κB inhibitor IMD0354 has promising potential as a lead compound and an antimicrobial therapeutic candidate capable of combating multidrug-resistant bacteria.

Staphylococcus aureus and Candida spp. are commonly linked with topical biofilm-associated infections such as those found on chronic wounds. These biofilms are notoriously difficult to treat, highlighting the grave need to discover and study new broad-spectrum agents to combat associated infections. Here we report that the kinase inhibitor Bay 11-7085 exhibited bactericidal activity against multidrug-resistant S. aureus with a minimum inhibitory concentration (MIC) of 4 μg/ml. In addition, S. aureus strain MW2 did not acquire resistance to antibiotic pressure. Furthermore, Bay 11-7085 exhibited potency against Candida albicans and the emerging pathogen Candida auris with a MIC of 0.5–1 μg/ml. Bay 11-7085 partially inhibited and eradicated biofilm formation of various pathogens, such as VRSA (vancomycin-resistant S. aureus), as well as antifungal-resistant Candida spp. isolates. Notably, Bay 11-7085 partially inhibited initial cell attachment and formation of a VRSA-C. albicans polymicrobial biofilm in vitro. In contrast to C. albicans, inhibition of VRSA biofilm was linked to initial cell attachment independent of its bactericidal activity. Finally, Bay 11-7085 was effective in vivo at increasing the lifespan of C. elegans during an S. aureus and a C. albicans infection. Our work proposes kinase inhibitor Bay 11-7085 as a potential compound capable of combating biofilms associated with primary multidrug-resistant bacteria and yeast pathogens associated with wound infections.

We previously reported the antibacterial activity of CD437, a known antitumor compound. It proved to be a potent antimicrobial agent effective against both growing and persister cells of methicillin-resistant Staphylococcus aureus (MRSA). Herein, we report the synthesis of a panel of analogs and their effect on both MRSA and cancer cells. The hydrophobic group of the parent compound was varied in steric bulk, and lipid-mimicking analogs were tested. Biological assessment confirmed that the adamantane moiety is the most effective substitution for antibacterial activity, and some preferential action in cancer over MRSA was achieved.

Sphingosine-1-phophate (S1P) is a sphingolipid-derived signaling molecule that controls diverse cellular functions including cell growth, homeostasis, and stress responses. In a variety of metazoans, cytosolic S1P is transported into the extracellular space where it activates S1P receptors in a concentration-dependent manner. In the free-living nematode Caenorhabditis elegans, the spin-2 gene, which encodes a S1P transporter, is activated during Gram-positive or Gram-negative bacterial infection of the intestine. However, the role during infection of spin-2 and three additional genes in the C. elegans genome encoding other putative S1P transporters has not been elucidated. Here, we report an evolutionally conserved function for S1P and a non-canonical role for S1P transporters in the C. elegans immune response to bacterial pathogens. We found that mutations in the sphingosine kinase gene (sphk-1) or in the S1P transporter genes spin-2 or spin-3 decreased nematode survival after infection with Pseudomonas aeruginosa or Enterococcus faecalis. In contrast to spin-2 and spin-3, mutating spin-1 leads to an increase in resistance to P. aeruginosa. Consistent with these results, when wild-type C. elegans were supplemented with extracellular S1P, we found an increase in their lifespan when challenged with P. aeruginosa and E. faecalis. In comparison, spin-2 and spin-3 mutations suppressed the ability of S1P to rescue the worms from pathogen-mediated killing, whereas the spin-1 mutation had no effect on the immune-enhancing activity of S1P. S1P demonstrated no antimicrobial activity toward P. aeruginosa and Escherichia coli and only minimal activity against E. faecalis MMH594 (40 µM). These data suggest that spin-2 and spin-3, on the one hand, and spin-1, on the other hand, transport S1P across cellular membranes in opposite directions. Finally, the immune modulatory effect of S1P was diminished in C. elegans sek-1 and pmk-1 mutants, suggesting that the immunomodulatory effects of S1P are mediated by the p38 MAPK signaling pathway.

Candida auris is an emerging healthcare-associated fungal pathogen that has become a serious global health threat. Current treatment options are limited due to drug resistance. New therapeutic strategies are required to target this organism and its pathogenicity. Plant polyphenols are structurally diverse compounds that present a vast range of biological properties. In the present study, plant-derived molecules ellagic acid (EA) and caffeic acid phenethyl ester (CAPE) were investigated for their antifungal and antivirulence activities against Candida auris. We also tested against C. albicans. The minimum inhibitory concentration (MIC) for EA ranged from 0.125 to 0.25 µg/mL and for CAPE ranged from 1 to 64 µg/mL against drug-resistant C. auris strains. Killing kinetics determined that after 4 h treatment with CAPE, there was a complete reduction of viable C. auris cells compared to fluconazole. Both compounds might act by modifying the fungal cell wall. CAPE significantly reduced the biomass and the metabolic activity of C. auris biofilm and impaired C. auris adhesion to cultured human epithelial cells. Furthermore, both compounds prolonged the survival rate of Galleria mellonella infected by C. auris (p = 0.0088 for EA at 32 mg/kg and p = 0.0028 for CAPE at 4 mg/kg). In addition, EA at 4 μg/mL prolonged the survival of C. albicans-infected Caenorhabditis elegans (p < 0.0001). CAPE was not able to prolong the survival of C. albicans-infected C. elegans. These findings highlight the antifungal and antivirulence effects of EA and CAPE against C. auris, and warrant further investigation as novel antifungal agents against drug-resistant infections.