CARD19, the protein formerly known as BinCARD, is a mitochondrial protein that does not regulate Bcl10-dependent NF-κB activation after TCR engagement.

After T cell receptor (TCR) engagement, the CARD11-Bcl10-Malt1 (CBM) complex oligomerizes to transduce NF-κB activating signals. Bcl10 is then degraded to limit NF-κB activation. The cDNA AK057716 (BinCARD-1) was reported to encode a novel CARD protein that interacts with Bcl10 and modestly inhibits NF-κB activation. In a later study, a second isoform, BinCARD-2, was identified. Here, we report that the cDNA AK057716 (BinCARD-1) is an incompletely spliced derivative of the gene product of C9orf89, whereas CARD19 (BinCARD-2) represents the properly spliced isoform, with conservation across diverse species. Immunoblotting revealed expression of CARD19 in T cells, but no evidence of BinCARD-1 expression, and microscopy demonstrated that endogenous CARD19 localizes to mitochondria. Although we confirmed that both BinCARD-1 and CARD19 can inhibit NF-κB activation and promote Bcl10 degradation when transiently overexpressed in HEK293T cells, loss of endogenous CARD19 expression had little effect on Bcl10-dependent NF-κB activation, activation of Malt1 protease function, or Bcl10 degradation after TCR engagement in primary murine CD8 T cells. Together, these data indicate that the only detectable translated product of C9orf89 is the mitochondrial protein CARD19, which does not play a discernible role in TCR-dependent, Bcl10-mediated signal transduction to Malt1 or NF-κB.

Hyphae of Rhizopus arrhizus and Lichtheimia corymbifera Are More Virulent and Resistant to Antifungal Agents Than Sporangiospores In Vitro and in Galleria mellonella.

Mucorales species cause debilitating, life-threatening sinopulmonary diseases in immunocompromised patients and penetrating wounds in trauma victims. Common antifungal agents against mucormycosis have significant toxicity and are often ineffective. To evaluate treatments against mucormycosis, sporangiospores are typically used for in vitro assays and in pre-clinical animal models of pulmonary infections. However, in clinical cases of wound mucormycosis caused by traumatic inoculation, hyphal elements found in soil are likely the form of the inoculated organism. In this study, Galleria mellonella larvae were infected with either sporangiospores or hyphae of Rhizopus arrhizus and Lichtheimia corymbifera. Hyphal infections resulted in greater and more rapid larval lethality than sporangiospores, with an approximate 10-16-fold decrease in LD50 of hyphae for R. arrhizus (p = 0.03) and L. corymbifera (p = 0.001). Liposomal amphotericin B, 10 mg/kg, was ineffective against hyphal infection, while the same dosage was effective against infections produced by sporangiospores. Furthermore, in vitro, antifungal susceptibility studies show that minimum inhibitory concentrations of several antifungal agents against hyphae were higher when compared to those of sporangiospores. These findings support using hyphal elements of Mucorales species for virulence testing and antifungal drug screening in vitro and in G. mellonella for studies of wound mucormycosis.

Impact of Blast Overpressure on the Pharmacokinetics of Various Antibiotics in Sprague Dawley Rats.

INTRODUCTION: Combat injuries are complex and multimodal. Most injuries to the extremities occur because of explosive devices such as improvised explosive devices. Blast exposure dramatically increases the risk of infection in combat wounds, and there is limited available information on the best antibiotic treatments for these injuries. We previously demonstrated that mice exposed to blast displayed a delayed clearance of cefazolin from the plasma and liver; further semi-mechanistic modeling determined that cefazolin concentrations in the skin of these mice were reduced. Our objective was to investigate the effects of blast on the pharmacokinetics of antibiotics of different types used for the treatment of combat wounds in the rat model. MATERIALS AND METHODS: Male Sprague Dawley rats were exposed to blast overpressure followed by injection of a bolus of animal equivalent doses of an antibiotic (cefazolin, cefepime, ertapenem, or clindamycin) into the tail vein at 1-hour post-blast exposure. Blood was collected at predetermined time points via repeated sampling from the tail vein. Animals were also euthanized at predetermined time points, at which time liver, kidney, skin, and blood via cardiac puncture were collected. Antibiotic concentrations were determined by ultra-performance liquid chromatography-tandem mass spectrometry. RESULTS: Blast-exposed rats exhibited a similar rate of clearance compared to non-blasted rats in the blood, liver, kidney, and skin, which is inconsistent with the data regarding cefazolin in blast-exposed mice. CONCLUSIONS: Our results in rats do not recapitulate our previous observation of delayed cefazolin clearance in mice following the blast overpressure exposure. Although using rats permitted us to collect multiple blood samples from the same animals, rats may not be a suitable model for measuring the pharmacokinetics of antibiotics following blast. The interpretation of the results may be challenging because of variation in data among rat subjects in the same sample groups.

CARD19 Interacts with Mitochondrial Contact Site and Cristae Organizing System Constituent Proteins and Regulates Cristae Morphology.

CARD19 is a mitochondrial protein of unknown function. While CARD19 was originally reported to regulate TCR-dependent NF-κB activation via interaction with BCL10, this function is not recapitulated ex vivo in primary murine CD8+ T cells. Here, we employ a combination of SIM, TEM, and confocal microscopy, along with proteinase K protection assays and proteomics approaches, to identify interacting partners of CARD19 in macrophages. Our data show that CARD19 is specifically localized to the outer mitochondrial membrane. Through deletion of functional domains, we demonstrate that both the distal C-terminus and transmembrane domain are required for mitochondrial targeting, whereas the CARD is not. Importantly, mass spectrometry analysis of 3×Myc-CARD19 immunoprecipitates reveals that CARD19 interacts with the components of the mitochondrial intermembrane bridge (MIB), consisting of mitochondrial contact site and cristae organizing system (MICOS) components MIC19, MIC25, and MIC60, and MICOS-interacting proteins SAMM50 and MTX2. These CARD19 interactions are in part dependent on a properly folded CARD. Consistent with previously reported phenotypes upon siRNA silencing of MICOS subunits, absence of CARD19 correlates with irregular cristae morphology. Based on these data, we propose that CARD19 is a previously unknown interacting partner of the MIB and the MIC19-MIC25-MIC60 MICOS subcomplex that regulates cristae morphology.