Priming with DNA Expressing Trimeric HIV V1V2 Alters the Immune Hierarchy Favoring the Development of V2-Specific Antibodies in Rhesus Macaques.

The RV144 vaccine trial revealed a correlation between reduced risk of HIV infection and the level of nonneutralizing-antibody (Ab) responses targeting specific epitopes in the second variable domain (V2) of the HIV gp120 envelope (Env) protein, suggesting this region as a target for vaccine development. To favor induction of V2-specific Abs, we developed a vaccine regimen that included priming with DNA expressing an HIV V1V2 trimeric scaffold immunogen followed by booster immunizations with a combination of DNA and protein in rhesus macaques. Priming vaccination with DNA expressing the HIV recombinant subtype CRF01_AE V1V2 scaffold induced higher and broader V2-specific Ab responses than vaccination with DNA expressing CRF01_AE gp145 Env. Abs recognizing the V2 peptide that was reported as a critical target in RV144 developed only after the priming immunization with V1V2 DNA. The V2-specific Abs showed several nonneutralizing Fc-mediated functions, including ADCP and C1q binding. Importantly, robust V2-specific Abs were maintained upon boosting with gp145 DNA and gp120 protein coimmunization. In conclusion, priming with DNA expressing the trimeric V1V2 scaffold alters the hierarchy of humoral immune responses to V2 region epitopes, providing a method for more efficient induction and maintenance of V2-specific Env Abs associated with reduced risk of HIV infection.IMPORTANCE The aim of this work was to design and test a vaccine regimen focusing the immune response on targets associated with infection prevention. We demonstrated that priming with a DNA vaccine expressing only the HIV Env V1V2 region induces Ab responses targeting the critical region in V2 associated with protection. This work shows that V1V2 scaffold DNA priming immunization provides a method to focus immune responses to the desired target region, in the absence of immune interference by other epitopes. This induced immune responses with improved recognition of epitopes important for protective immunity, namely, V2-specific humoral immune responses inversely correlating with HIV risk of infection in the RV144 trial.

Control of Heterologous Simian Immunodeficiency Virus SIVsmE660 Infection by DNA and Protein Coimmunization Regimens Combined with Different Toll-Like-Receptor-4-Based Adjuvants in Macaques.

We developed a method of simultaneous vaccination with DNA and protein resulting in robust and durable cellular and humoral immune responses with efficient dissemination to mucosal sites and protection against simian immunodeficiency virus (SIV) infection. To further optimize the DNA-protein coimmunization regimen, we tested a SIVmac251-based vaccine formulated with either of two Toll-like receptor 4 (TLR4) ligand-based liposomal adjuvant formulations (TLR4 plus TLR7 [TLR4+7] or TLR4 plus QS21 [TLR4+QS21]) in macaques. Although both vaccines induced humoral responses of similar magnitudes, they differed in their functional quality, including broader neutralizing activity and effector functions in the TLR4+7 group. Upon repeated heterologous SIVsmE660 challenge, a trend of delayed viral acquisition was found in vaccinees compared to controls, which reached statistical significance in animals with the TRIM-5α-resistant (TRIM-5α R) allele. Vaccinees were preferentially infected by an SIVsmE660 transmitted/founder virus carrying neutralization-resistant A/K mutations at residues 45 and 47 in Env, demonstrating a strong vaccine-induced sieve effect. In addition, the delay in virus acquisition directly correlated with SIVsmE660-specific neutralizing antibodies. The presence of mucosal V1V2 IgG binding antibodies correlated with a significantly decreased risk of virus acquisition in both TRIM-5α R and TRIM-5α-moderate/sensitive (TRIM-5α M/S) animals, although this vaccine effect was more prominent in animals with the TRIM-5α R allele. These data support the combined contribution of immune responses and genetic background to vaccine efficacy. Humoral responses targeting V2 and SIV-specific T cell responses correlated with viremia control. In conclusion, the combination of DNA and gp120 Env protein vaccine regimens using two different adjuvants induced durable and potent cellular and humoral responses contributing to a lower risk of infection by heterologous SIV challenge.IMPORTANCE An effective AIDS vaccine continues to be of paramount importance for the control of the pandemic, and it has been proven to be an elusive target. Vaccine efficacy trials and macaque challenge studies indicate that protection may be the result of combinations of many parameters. We show that a combination of DNA and protein vaccinations applied at the same time provides rapid and robust cellular and humoral immune responses and evidence for a reduced risk of infection. Vaccine-induced neutralizing antibodies and Env V2-specific antibodies at mucosal sites contribute to the delay of SIVsmE660 acquisition, and genetic makeup (TRIM-5α) affects the effectiveness of the vaccine. These data are important for the design of better vaccines and may also affect other vaccine platforms.

Scalable, cGMP-compatible purification of extracellular vesicles carrying bioactive human heterodimeric IL-15/lactadherin complexes.

The development of extracellular vesicles (EV) for therapeutic applications is contingent upon the establishment of reproducible, scalable, and high-throughput methods for the production and purification of clinical grade EV. Methods including ultracentrifugation (U/C), ultrafiltration, immunoprecipitation, and size-exclusion chromatography (SEC) have been employed to isolate EV, each facing limitations such as efficiency, particle purity, lengthy processing time, and/or sample volume. We developed a cGMP-compatible method for the scalable production, concentration, and isolation of EV through a strategy involving bioreactor culture, tangential flow filtration (TFF), and preparative SEC. We applied this purification method for the isolation of engineered EV carrying multiple complexes of a novel human immunostimulatory cytokine-fusion protein, heterodimeric IL-15 (hetIL-15)/lactadherin. HEK293 cells stably expressing the fusion cytokine were cultured in a hollow-fibre bioreactor. Conditioned medium was collected and EV were isolated comparing three procedures: U/C, SEC, or TFF + SEC. SEC demonstrated comparable particle recovery, size distribution, and hetIL-15 density as U/C purification. Relative to U/C, SEC preparations achieved a 100-fold reduction in ferritin concentration, a major protein-complex contaminant. Comparative proteomics suggested that SEC additionally decreased the abundance of cytoplasmic proteins not associated with EV. Combination of TFF and SEC allowed for bulk processing of large starting volumes, and resulted in bioactive EV, without significant loss in particle yield or changes in size, morphology, and hetIL-15/lactadherin density. Taken together, the combination of bioreactor culture with TFF + SEC comprises a scalable, efficient method for the production of highly purified, bioactive EV carrying hetIL-15/lactadherin, which may be useful in targeted cancer immunotherapy approaches.

HIV Env conserved element DNA vaccine alters immunodominance in macaques.

Sequence diversity and immunodominance are major obstacles in the design of an effective vaccine against HIV. HIV Env is a highly-glycosylated protein composed of 'conserved' and 'variable' regions. The latter contains immunodominant epitopes that are frequently targeted by the immune system resulting in the generation of immune escape variants. This work describes 12 regions in HIV Env that are highly conserved throughout the known HIV M Group sequences (Env CE), and are poorly immunogenic in macaques vaccinated with full-length Env expressing DNA vaccines. Two versions of plasmids encoding the 12 Env CE were generated, differing by 0-5 AA per CE to maximize the inclusion of commonly detected variants. In contrast to the full-length env DNA vaccine, vaccination of macaques with a combination of these 2 Env CE DNA induced robust, durable cellular immune responses with a significant fraction of CD8+ T cells with cytotoxic phenotype (Granzyme B+ and CD107a+). Although inefficient in generating primary responses to the CE, boosting of the Env CE DNA primed macaques with the intact env DNA vaccine potently augmented pre-existing immunity, increasing magnitude, breadth and cytotoxicity of the cellular responses. Fine mapping showed that 7 of the 12 CE elicited T cell responses. Env CE DNA also induced humoral responses able to recognize the full-length Env. Env CE plasmids are therefore capable of inducing durable responses to highly conserved regions of Env that are frequently absent after Env vaccination or immunologically subdominant. These modified antigens are candidates for use as prophylactic and therapeutic HIV vaccines.

DNA Prime-Boost Vaccine Regimen To Increase Breadth, Magnitude, and Cytotoxicity of the Cellular Immune Responses to Subdominant Gag Epitopes of Simian Immunodeficiency Virus and HIV.

HIV sequence diversity and the propensity of eliciting immunodominant responses targeting variable regions of the HIV proteome are hurdles in the development of an effective AIDS vaccine. An HIV-derived conserved element (CE) p24gag plasmid DNA (pDNA) vaccine is able to redirect immunodominant responses to otherwise subdominant and often more vulnerable viral targets. By homology to the HIV immunogen, seven CE were identified in SIV p27Gag Analysis of 31 rhesus macaques vaccinated with full-length SIV gag pDNA showed inefficient induction (58% response rate) of cellular responses targeting these CE. In contrast, all 14 macaques immunized with SIV p27CE pDNA developed robust T cell responses recognizing CE. Vaccination with p27CE pDNA was also critical for the efficient induction and increased the frequency of Ag-specific T cells with cytotoxic potential (granzyme B+ CD107a+) targeting subdominant CE epitopes, compared with the responses elicited by the p57gag pDNA vaccine. Following p27CE pDNA priming, two booster regimens, gag pDNA or codelivery of p27CE+gag pDNA, significantly increased the levels of CE-specific T cells. However, the CE+gag pDNA booster vaccination elicited significantly broader CE epitope recognition, and thus, a more profound alteration of the immunodominance hierarchy. Vaccination with HIV molecules showed that CE+gag pDNA booster regimen further expanded the breadth of HIV CE responses. Hence, SIV/HIV vaccine regimens comprising CE pDNA prime and CE+gag pDNA booster vaccination significantly increased cytotoxic T cell responses to subdominant highly conserved Gag epitopes and maximized response breadth.

Comparison of intradermal and intramuscular delivery followed by in vivo electroporation of SIV Env DNA in macaques.

A panel of SIVmac251 transmitted Env sequences were tested for expression, function and immunogenicity in mice and macaques. The immunogenicity of a DNA vaccine cocktail expressing SIVmac239 and three transmitted SIVmac251 Env sequences was evaluated upon intradermal or intramuscular injection followed by in vivo electroporation in macaques using sequential vaccination of gp160, gp120 and gp140 expressing DNAs. Both intradermal and intramuscular vaccination regimens using the gp160 expression plasmids induced robust humoral immune responses, which further improved using the gp120 expressing DNAs. The responses showed durability of binding and neutralizing antibody titers and high avidity for>1 y. The intradermal DNA delivery regimen induced higher cross-reactive responses able to neutralize the heterologous tier 1B-like SIVsmE660_CG7V. Analysis of cellular immune responses showed induction of Env-specific memory responses and cytotoxic granzyme B(+) T cells in both vaccine groups, although the magnitude of the responses were ~10x higher in the intramuscular/electroporation group. The cellular responses induced by both regimens were long lasting and could be detected ~1 y after the last vaccination. These data show that both DNA delivery methods are able to induce robust and durable immune responses in macaques.

The p40 subunit of interleukin (IL)-12 promotes stabilization and export of the p35 subunit: implications for improved IL-12 cytokine production.

IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

Comparison of immune responses generated by optimized DNA vaccination against SIV antigens in mice and macaques.

Optimized DNA vectors were constructed comprising the proteome of SIV including the structural, enzymatic, regulatory, and accessory proteins. In addition to native antigens as produced by the virus, fusion proteins and modified antigens with altered secretion, cellular localization and stability characteristics were generated. The DNA vectors were tested for expression upon transfection in human cells. In addition, the vectors were tested either alone or in combinations in mice and macaques, which provided an opportunity to compare immune responses in two animal models. DNA only immunization using intramuscular injection in the absence or presence of in vivo electroporation did not alter the phenotype of the induced T cell responses in mice. Although several fusion proteins induced immune responses to all the components of a polyprotein, we noted fusion proteins that abrogated immune response to some of the components. Since the expression levels of such fusion proteins were not affected, these data suggest that the immune recognition of certain components was altered by the fusion. Testing different DNA vectors in mice and macaques revealed that a combination of DNAs producing different forms of the same antigen generated more balanced immune responses, a desirable feature for an optimal AIDS vaccine.

Differential expression and molecular associations of Syk in systemic lupus erythematosus T cells.

Diminished expression of TCR zeta and reciprocal up-regulation and association of FcRgamma with the TCR/CD3 complex is a hallmark of systemic lupus erythematosus (SLE) T cells. In this study we explored whether differential molecular associations of the spleen tyrosine kinase Syk that preferentially binds to FcRgamma contribute to pathological amplification of signals downstream of this "rewired TCR" in SLE. We detected higher amounts of Syk expression and activity in SLE compared with normal T cells. Selective inhibition of the activity of Syk reduced the strength of TCR-induced calcium responses and slowed the rapid kinetics of actin polymerization exclusively in SLE T cells. Syk and ZAP-70 also associated differently with key molecules involved in cytoskeletal and calcium signaling in SLE T cells. Thus, while Vav-1 and LAT preferentially bound to Syk, phospholipase C-gamma1 bound to both Syk and ZAP-70. Our results show that differential associations of Syk family kinases contribute to the enhanced TCR-induced signaling responses in SLE T cells. Thus, we propose molecular targeting of Syk as a measure to control abnormal T cell responses in SLE.

The RNA-stabilizing protein HuR regulates the expression of zeta chain of the human T cell receptor-associated CD3 complex.

T cell dysfunction is crucial to the pathogenesis of systemic lupus erythematosus (SLE); however, the molecular mechanisms involved in the deficient expression of the T cell receptor-associated CD3zeta chain in SLE are not clear. SLE T cells express abnormally increased levels of an alternatively spliced isoform of CD3zeta that lacks a 562-bp region in its 3'-untranslated region (UTR). We showed previously that two adenosine/uridine-rich elements (ARE) in this splice-deleted region of CD3zeta transcript are critical for the mRNA stability and protein expression of CD3zeta. In this study we show for the first time that the mRNA-stabilizing protein HuR binds to these two ARE bearing regions of CD3zeta 3'-UTR. Knockdown of HuR resulted in decreased expression of the CD3zeta chain, whereas overexpression led to the increase of CD3zeta chain levels. Additionally, overexpression of HuR in human T cells resulted in increased mRNA stability of CD3zeta. Our results identify the 3'-UTR of CD3zeta as a novel target for the mRNA-stabilizing protein HuR. Thus, the absence of two critical AREs in the alternatively spliced CD3zeta 3'-UTR found in SLE T cells may result in decreased HuR binding, representing a possible molecular mechanism contributing to the reduced stability and expression of CD3zeta in SLE.

Stability and translation of TCR zeta mRNA are regulated by the adenosine-uridine-rich elements in splice-deleted 3' untranslated region of zeta-chain.

Systemic lupus erythematosus (SLE) T cells display reduced expression of TCR zeta protein. Recently, we reported that in SLE T cells, the residual TCR zeta protein is predominantly derived from an alternatively spliced form that undergoes splice deletion of 562 nt (from 672 to 1233 bases) within the 3' untranslated region (UTR) of TCR zeta mRNA. The stability and translation of the alternatively spliced form of TCR zeta mRNA are low compared with that of the wild-type TCR zeta mRNA. We report that two adenosine-uridine-rich sequence elements (AREs), defined by the splice-deleted 3' UTR region, but not an ARE located upstream are responsible for securing TCR zeta mRNA stability and translation. The stabilizing effect of the splice-deleted region-defined AREs extended to the luciferase mRNA and was not cell type-specific. The findings demonstrate distinct sequences within the splice-deleted region 672 to 1233 of the 3' UTR, which regulate the transcription, mRNA stability, and translation of TCR zeta mRNA. The absence of these sequences represents a molecular mechanism that contributes to altered TCR zeta-chain expression in lupus.

Interaction of uteroglobin with lipocalin-1 receptor suppresses cancer cell motility and invasion.

Cellular migration and invasion are critical for important biological processes including cancer metastasis. We previously reported that uteroglobin (UG), a multifunctional secreted protein, binds to several cell types inhibiting migration and invasion [G.C. Kundu, A.K. Z. Zhang Mandal, G. Mantile-Selvaggi, A.B. Mukherjee (1998) Uteroglobin (UG) suppresses extracellular matrix invasion by normal and cancer cells that express the high affinity UG-binding proteins. J Biol Chem. 273: 22819-22824]. More recently, we reported that HTB-81 adenocarcinoma cells, which do not bind UG, are refractory to UG-mediated inhibition of migration and invasion [Z. Zhang, G.C. Kundu, D. Panda, A.K. Mandal et al. (1999) Loss of transformed phenotype in cancer cells by overexpression of the uteroglobin gene. Proc Natl Acad Sci U S A. 96, 3963-3968]. Since UG shares several biological properties with lipocalin-1 that mediates some of its biological effects via its receptor (Lip-1R), we sought to determine whether UG might interact with Lip-1R and inhibit migration and invasion of HTB-81 cells. To address this question, we first transfected COS-1 cells, which do not bind UG, with a Lip-1R-cDNA construct and performed binding assays using 125I-human UG (hUG). The results show that hUG binds Lip-1R on these cells with high specificity. Further, transfection of HTB-81 cells with the same construct yielded 125I-hUG binding with high affinity (Kd=18 nM) and specificity. The hUG-Lip-1R interaction was further confirmed by transfecting HTB-81, HTB-30 and HTB-174 cells, which are refractory to UG-binding, with a green fluorescent protein (GFP)-Lip-1R-cDNA construct and testing for Lip-1R-hUG colocalization by fluorescence microscopy. Finally, we demonstrate that Lip-1R-hUG interaction on Lip-1R transfected HTB-81 cells renders them fully responsive to hUG-mediated inhibition of migration and invasion. Taken together, these results suggest that Lip-1R is at least one of the UG-binding proteins through which UG exerts anti-motility and anti-invasive effects.

Uteroglobin inhibits prostaglandin F2alpha receptor-mediated expression of genes critical for the production of pro-inflammatory lipid mediators.

Prematurity is one of the leading causes of infant mortality. It may result from intrauterine infection, which mediates premature labor by stimulating the production of inflammatory lipid mediators such as prostaglandin F2alpha (PGF2alpha). The biological effects of PGF2alpha are mediated via the G protein-coupled receptor FP; however, the molecular mechanism(s) of FP signaling that mediates inflammatory lipid mediator production remains unclear. We reported previously that in the human uterus, a composite organ in which fibroblast, epithelial, and smooth muscle cells are the major constituents, an inverse relationship exists between the levels of PGF2alpha and a steroid-inducible anti-inflammatory protein, uteroglobin. Here we report that, in NIH 3T3 fibroblasts and human uterine smooth muscle cells, FP signaling is mediated via multi-kinase pathways in a cell type-specific manner to activate NF-kappaB, thus stimulating the expression of cyclooxygenase-2. Cyclooxygenase-2 is a critical enzyme for the production of prostaglandins from arachidonic acid, which is released from membrane phospholipids by phospholipase A2, the expression of which is also stimulated by PGF2alpha. Most importantly, uteroglobin inhibits FP-mediated NF-kappaB activation and cyclooxygenase-2 gene expression by binding and most likely by sequestering PGF2alpha into its central hydrophobic cavity, thereby preventing FP-PGF2alpha interaction and suppressing the production of inflammatory lipid mediators. We propose that uteroglobin plays important roles in maintaining homeostasis in organs that are vulnerable to inadvertent stimulation of FP-mediated inflammatory response.

Increased caspase-3 expression and activity contribute to reduced CD3zeta expression in systemic lupus erythematosus T cells.

T cells isolated from patients with systemic lupus erythematosus (SLE) express low levels of CD3zeta-chain, a critical molecule involved in TCR-mediated signaling, but the involved mechanisms are not fully understood. In this study we examined caspase-3 as a candidate for cleaving CD3zeta in SLE T cells. We demonstrate that SLE T cells display increased expression and activity of caspase-3. Treatment of SLE T cells with the caspase-3 inhibitor Z-Asp-Glu-Val-Asp-FMK reduced proteolysis of CD3zeta and enhanced its expression. In addition, Z-Asp-Glu-Val-Asp-FMK treatment increased the association of CD3zeta with lipid rafts and simultaneously reversed the abnormal lipid raft preclustering, heightened TCR-induced calcium responses, and reduced the expression of FcRgamma-chain exclusively in SLE T cells. We conclude that caspase-3 inhibitors can normalize SLE T cell function by limiting the excessive digestion of CD3zeta-chain and suggest that such molecules can be considered in the treatment of this disease.

Gastrointestinally distributed UDP-glucuronosyltransferase 1A10, which metabolizes estrogens and nonsteroidal anti-inflammatory drugs, depends upon phosphorylation.

Among gastrointestinal distributed isozymes encoded at the UGT1 locus, UDP-glucuronosyltransferase 1A10 (UGT1A10) metabolizes a number of important chemicals. Similar to broad conversion of phytoestrogens (Basu, N. K., Ciotti, M., Hwang, M. S., Kole, L., Mitra, P. S., Cho, J. W., and Owens, I. S. (2004) J. Biol. Chem. 279, 1429-1441), UGT1A10 metabolized estrogens and their derivatives, whereas UGT1A1, -1A3, -1A7, and -1A8 differentially exhibited reduced activity toward the same. UGT1A10 compared with UGT1A7, -1A8, and -1A3 generally exhibited high activity toward acidic nonsteroidal anti-inflammatory drugs and natural benzaldehyde derivatives, while UGT1A3 metabolized most efficiently aromatic transcinnamic acids known to be generated from flavonoid glycosides by microflora in the lower gastrointestinal tract. Finally UGT1A10, -1A7, -1A8, and -1A3 converted plant-based salicylic acids; methylsalicylic acid was transformed at high levels, and acetylsalicylic (aspirin) and salicylic acid were transformed at moderate to low levels. Atypically UGT1A10 transformed estrogens between pH 6 and 8 but acidic structures preferentially at pH 6.4. Furthermore evidence indicates UGT1A10 expressed in COS-1 cells depends upon phosphorylation; UGT1A10 versus its single, double, and triple mutants at three predicted protein kinase C phosphorylation sites incorporated [(33)P]-orthophosphate and showed a progressive decrease with no detectable label or activity for the triple T73A/T202A/S432G-1A10 mutant. Single and double mutants revealed either null/full activity or null/additive activity, respectively. Additionally UGT1A10-expressing cultures glucuronidated 17beta-[(14)C]estradiol, whereas cultures containing null mutants at protein kinase C sites showed no estrogen conversion. Importantly UGT1A10 in cells supported 10-fold higher glucuronidation of 17beta-estradiol than UGT1A1. In summary, our results suggest gastrointestinally distributed UGT1A10 is important for detoxifying estrogens/phytoestrogens and aromatic acids with complementary activity by UGT1A7, -1A8, -1A3, and/or -1A1 evidently dependent upon phosphorylation.

Use of resistant mutants to characterize the target of mycobacillin in Aspergillus niger membranes.

The mycobacillin-sensitive Aspergillus niger strain G3Br and resistant mutants of it did not show any differences in their total lipid content, although the amounts of phospholipids and sterols, particularly phosphatidylcholine and cholesterol, were lower in resistant cells. Mycobacillin resistance was accompanied by an increase in the phase-transition temperature of plasma membrane preparations. When exposed to mycobacillin, resistant and sensitive cells did not differ qualitatively with respect to most released materials (lysine, proline, Pi, Na+, K+, Ca2+); however, the release of ATP was completely inhibited in resistant cells unless they were exposed to concentrations of mycobacillin exceeding their respective MIC value. Resistant cells, under steady-state conditions, displayed greater uptake and release of the same specific materials--except ATP--as sensitive cells did under similar conditions. Thus release and uptake of those materials except ATP are not implicated in the mode of action of mycobacillin. The inhibiting action of mycobacillin (at concentrations higher than the MIC) on sensitive or resistant cells was completely antagonized by ATP (which did not form any complex with mycobacillin) but not by any of the releasable components, either alone or in combination. This observation, coupled with the authors' recent findings on ATP release, indicates that the fungistatic action of mycobacillin is due to excessive ATP release, leading to energy starvation. Interestingly, ATP release during the first 2 h of incubation with mycobacillin was minimal, but increased to over 96% during the next 48 h. Release and uptake of ATP via liposomes, prepared with lipid and protein isolated from membranes of the mycobacillin-sensitive parent and resistant mutants, showed that mycobacillin action could be inhibited either by resistant protein or by resistant lipid. The mycobacillin target appears to be a lipid-protein site on the membrane of sensitive A. niger G3Br.