Bacterial pseudaminic acid binding to Siglec-10 induces a macrophage interleukin-10 response and suppresses phagocytosis.

Pseudaminic acid (Pse) on pathogenic bacteria exopolysaccharide engages with the sialic acid-binding immunoglobulin-type lectin (Siglec)-10 receptor on macrophages via the critical 7-N-acetyl group. This binding stimulates macrophages to secrete interleukin 10 that suppresses phagocytosis against bacteria, but can be reverted by blocking Pse-Siglec-10 interaction with Pse-binding protein as a promising therapy.

Klebsiella pneumoniae K2 capsular polysaccharide degradation by a bacteriophage depolymerase does not require trimer formation.

K2-capsular Klebsiella pneumoniae is a hypervirulent pathogen that causes fatal infections. Here, we describe a phage tailspike protein, named K2-2, that specifically depolymerizes the K2 capsular polysaccharide (CPS) of K. pneumoniae into tetrasaccharide repeating units. Nearly half of the products contained O-acetylation, which was thought crucial to the immunogenicity of CPS. The product-bound structures of this trimeric enzyme revealed intersubunit carbohydrate-binding grooves, each accommodating three tetrasaccharide units of K2 CPS. The catalytic residues and the key interactions responsible for K2 CPS recognition were identified and verified by site-directed mutagenesis. Further biophysical and functional characterization, along with the structure of a tetrameric form of K2-2, demonstrated that the formation of intersubunit catalytic center does not require trimerization, which could be nearly completely disrupted by a single-residue mutation in the C-terminal domain. Our findings regarding the assembly and catalysis of K2-2 provide cues for the development of glycoconjugate vaccines against K. pneumoniae infection. IMPORTANCE: Generating fragments of capsular polysaccharides from pathogenic bacteria with crucial antigenic determinants for vaccine development continues to pose challenges. The significance of the C-terminal region of phage tailspike protein (TSP) in relation to its folding and trimer formation remains largely unexplored. The polysaccharide depolymerase described here demonstrates the ability to depolymerize the K2 CPS of K. pneumoniae into tetrasaccharide fragments while retaining the vital O-acetylation modification crucial for immunogenicity. By carefully characterizing the enzyme, elucidating its three-dimensional structures, conducting site-directed mutagenesis, and assessing the antimicrobial efficacy of the mutant enzymes against K2 K. pneumoniae, we offer valuable insights into the mechanism by which this enzyme recognizes and depolymerizes the K2 CPS. Our findings, particularly the discovery that trimer formation is not required for depolymerizing activity, challenge the current understanding of trimer-dependent TSP activity and highlight the catalytic mechanism of the TSP with an intersubunit catalytic center.

The deficiency of poly-ß-1,6-N-acetyl-glucosamine deacetylase trigger A. baumannii to convert to biofilm-independent colistin-tolerant cells.

Acinetobacter baumannii is a nosocomial pathogen that can be resistant to antibiotics by rapidly modulating its anti-drug mechanisms. The multidrug-resistant A. baumannii has been considered one of the most threatening pathogens to our society. Biofilm formation and persistent cells within the biofilm matrix are recognized as intractable problems, especially in hospital-acquired infections. Poly-ß-1,6-N-acetyl-glucosamine (PNAG) is one of the important building blocks in A. baumannii's biofilm. Here, we discover a protein phosphoryl-regulation on PNAG deacetylase, AbPgaB1, in which residue Ser411 was phosphorylated. The phosphoryl-regulation on AbPgaB1 modulates the product turnover rate in which deacetylated PNAG is produced and reflected in biofilm production. We further uncovered the PgaB deficient A. baumannii strain shows the lowest level of biofilm production but has a high minimal inhibition concentration to antibiotic colistin and tetracycline. Based on bactericidal post-antibiotic effects and time-dependent killing assays with antibacterial drugs, we claim that the PgaB-deficient A. baumannii converts to colistin-tolerant cells. This study utilizes a biofilm-independent colistin-tolerant model of A. baumannii to further investigate its characteristics and mechanisms to better understand clinical outcomes.

The Biosynthetic Gene Cluster of Mushroom-Derived Antrocin Encodes Two Dual-Functional Haloacid Dehalogenase-like Terpene Cyclases.

(-)-Antrocin (1), produced by the medicinal mushroom Antrodia cinnamomea, is a potent antiproliferative compound. The biosynthetic gene cluster of 1 was identified, and the pathway was characterized by heterologous expression. We characterized a haloacid dehalogenase-like terpene cyclase AncC that biosynthesizes the drimane-type sesquiterpene (+)-albicanol (2) from farnesyl pyrophosphate (FPP). Biochemical characterization of AncC, including kinetic studies and mutagenesis, demonstrated the functions of two domains: a terpene cyclase (TC) and a pyrophosphatase (PPase). The TC domain first cyclizes FPP to albicanyl pyrophosphate, and the PPase domain then removes the pyrophosphate to form 2. Intriguingly, AncA (94 % sequence identity to AncC), in the same gene cluster, converts FPP into (R)-trans-γ-monocyclofarnesol instead of 2. Notably, Y283/F375 in the TC domain of AncA serve as a gatekeeper in controlling the formation of a cyclofarnesoid rather than a drimane-type scaffold.

An Immunological Polysaccharide from Tremella fuciformis: Essential Role of Acetylation in Immunomodulation.

The edible fungus Tremella fuciformis was shown to have a high molecular weight (1.87 × 103 kDa) bioactive polysaccharide, denoted as TFP-F1. Monosaccharide composition and NMR analysis of the polysaccharide and its derivatives indicated it contained fucose (Fucp), xylose (Xylp), mannose (Manp), and glucuronic acid (GlcAp) in a ratio of 0.9:1.0:3.2:1.2. Using IR, NMR, and GC-MS spectroscopic data, the structure of TFP-F1 was elucidated as {→3)-[ß-D-GlcAp-(1→2)]-α-D-Manp-(1→3)-α-D-Manp-(1→3)-[α-L-Fucp-(1→2)-ß-D-Xylp-(1→2)]-α-D-Manp-(1→}n, with partial acetylation of C6-OH in mannoses. Furthermore, at a concentration of 1 µg/mL, TFP-F1 was found to stimulate the secretion of TNF-α and IL-6 in J774A.1 macrophage cells in vitro via interaction with toll-like receptor 4 (TLR4). The removal of O-acetyl groups led to the loss of immunomodulatory activities, demonstrating that O-acetyl groups play an essential role in enhancing the production of pro-inflammatory cytokines.

High-efficiency decomposition of eggshell membrane by a keratinase from Meiothermus taiwanensis.

Eggshell membrane (ESM), a plentiful biological waste, consists of collagen-like proteins and glycosaminoglycans (GAGs) such as hyaluronic acid (HA). Here we used a keratinase (oeMtaker)-mediated system to decompose ESM. The best reaction condition was established by incubating the solution containing oeMtaker, sodium sulfite, and ESM with a weight ratio of 1:120:600. ESM enzymatic hydrolysate (ESM-EH) showed a high proportion of essential amino acids and type X collagen peptides with 963-2259 Da molecular weights. The amounts of GAGs and sulfated GAGs in ESM-EH were quantified as 6.4% and 0.7%, respectively. The precipitated polysaccharides with an average molecular weight of 1300-1700 kDa showed an immunomodulatory activity by stimulating pro-inflammatory cytokines (IL-6 and TNF-α) production. In addition, a microorganism-based system was established to hydrolyze ESM by Meiothermus taiwanensis WR-220. The amounts of GAGs and sulfated GAGs in the system were quantified as 0.9% and 0.1%, respectively. Based on our pre-pilot tests, the system shows great promise in developing into a low-cost and high-performance process. These results indicate that the keratinase-mediated system could hydrolyze ESM more efficiently and produce more bioactive substances than ever for therapeutical applications and dietary supplements.

Synergic action of an inserted carbohydrate-binding module in a glycoside hydrolase family 5 endoglucanase.

Most known cellulase-associated carbohydrate-binding modules (CBMs) are attached to the N- or C-terminus of the enzyme or are expressed separately and assembled into multi-enzyme complexes (for example to form cellulosomes), rather than being an insertion into the catalytic domain. Here, by solving the crystal structure, it is shown that MtGlu5 from Meiothermus taiwanensis WR-220, a GH5-family endo-ß-1,4-glucanase (EC 3.2.1.4), has a bipartite architecture consisting of a Cel5A-like catalytic domain with a (ß/α)8 TIM-barrel fold and an inserted CBM29-like noncatalytic domain with a ß-jelly-roll fold. Deletion of the CBM significantly reduced the catalytic efficiency of MtGlu5, as determined by isothermal titration calorimetry using inactive mutants of full-length and CBM-deleted MtGlu5 proteins. Conversely, insertion of the CBM from MtGlu5 into TmCel5A from Thermotoga maritima greatly enhanced the substrate affinity of TmCel5A. Bound sugars observed between two tryptophan side chains in the catalytic domains of active full-length and CBM-deleted MtGlu5 suggest an important stacking force. The synergistic action of the catalytic domain and CBM of MtGlu5 in binding to single-chain polysaccharides was visualized by substrate modeling, in which additional surface tryptophan residues were identified in a cross-domain groove. Subsequent site-specific mutagenesis results confirmed the pivotal role of several other tryptophan residues from both domains of MtGlu5 in substrate binding. These findings reveal a way to incorporate a CBM into the catalytic domain of an existing enzyme to make a robust cellulase.

Development of Klebsiella pneumoniae Capsule Polysaccharide-Conjugated Vaccine Candidates Using Phage Depolymerases.

Klebsiella pneumoniae is an important pathogen associated with nosocomial infection and has developed increasing resistance to antibiotics such as extended-spectrum ß-lactams and carbapenem. In recent years, K. pneumoniae isolates have emerged as a major cause of global community-acquired infections such as pneumonia and pyogenic liver abscess. Although serotypes K1 and K2 have been identified as the predominant capsular types associated with invasive infections, no K. pneumoniae vaccine is commercially available, probably due to immunogenicity loss in the traditional depolymerization method to obtain capsule polysaccharide (CPS) for the preparation of conjugated vaccine. In this study, we successfully retained immunogenicity by using K1 (K1-ORF34) and K2 (K2-ORF16) CPS depolymerases that were identified from phages to cleave K1 and K2 CPSs into intact structural units of oligosaccharides with intact modifications. The obtained K1 and K2 oligosaccharides were separately conjugated with CRM197 carrier protein to generate CPS-conjugated vaccines. Immunization experiments of mice showed both K1 and K2 CPS-conjugated vaccines induced anti-CPS antibodies with 128-fold and 64-fold increases of bactericidal activities, respectively, compare to mice without vaccinations. Challenge tests indicated that K1 or K2 CPS-conjugated vaccine and divalent vaccine (a mixture of K1 and K2 CPS-conjugated vaccines) protected mice from subsequent infection of K. pneumoniae by the respective capsular type. Thus, we demonstrated K1 and K2 CPS-conjugated vaccines prepared by CPS depolymerases is a promising candidate for developing vaccines against human K. pneumoniae infections.

Structural and biological insights into Klebsiella pneumoniae surface polysaccharide degradation by a bacteriophage K1 lyase: implications for clinical use.

BACKGROUND: K1 capsular polysaccharide (CPS)-associated Klebsiella pneumoniae is the primary cause of pyogenic liver abscesses (PLA) in Asia. Patients with PLA often have serious complications, ultimately leading to a mortality of ~ 5%. This K1 CPS has been reported as a promising target for development of glycoconjugate vaccines against K. pneumoniae infection. The pyruvylation and O-acetylation modifications on the K1 CPS are essential to the immune response induced by the CPS. To date, however, obtaining the fragments of K1 CPS that contain the pyruvylation and O-acetylation for generating glycoconjugate vaccines still remains a challenge. METHODS: We analyzed the digested CPS products with NMR spectroscopy and mass spectrometry to reveal a bacteriophage-derived polysaccharide depolymerase specific to K1 CPS. The biochemical and biophysical properties of the enzyme were characterized and its crystal structures containing bound CPS products were determined. We also performed site-directed mutagenesis, enzyme kinetic analysis, phage absorption and infectivity studies, and treatment of the K. pneumoniae-infected mice with the wild-type and mutant enzymes. RESULTS: We found a bacteriophage-derived polysaccharide lyase that depolymerizes the K1 CPS into fragments of 1-3 repeating trisaccharide units with the retention of the pyruvylation and O-acetylation, and thus the important antigenic determinants of intact K1 CPS. We also determined the 1.46-Å-resolution, product-bound crystal structure of the enzyme, revealing two distinct carbohydrate-binding sites in a trimeric ß-helix architecture, which provide the first direct evidence for a second, non-catalytic, carbohydrate-binding site in bacteriophage-derived polysaccharide depolymerases. We demonstrate the tight interaction between the pyruvate moiety of K1 CPS and the enzyme in this second carbohydrate-binding site to be crucial to CPS depolymerization of the enzyme as well as phage absorption and infectivity. We also demonstrate that the enzyme is capable of protecting mice from K1 K. pneumoniae infection, even against a high challenge dose. CONCLUSIONS: Our results provide insights into how the enzyme recognizes and depolymerizes the K1 CPS, and demonstrate the potential use of the protein not only as a therapeutic agent against K. pneumoniae, but also as a tool to prepare structurally-defined oligosaccharides for the generation of glycoconjugate vaccines against infections caused by this organism.

A hexasaccharide from capsular polysaccharide of carbapenem-resistant Klebsiella pneumoniae KN2 is a ligand of Toll-like receptor 4.

Klebsiella pneumoniae serotype KN2 is a carbapenem-resistant strain and leads to the health care-associated infections, such as bloodstream infections. Its capsular polysaccharide (CPS) was isolated and cleaved by a specific enzyme from a bacteriophage into a hexasaccharide-repeating unit. With GC-MS, NMR, and Mass analyses, the structure of KN2 CPS was determined to be {→3)-ß-D-Glcp-(1→3)-[α-D-GlcpA-(1→4)-ß-D-Glcp-(1→6)]-α-D-Galp-(1→6)-ß-D-Galp-(1→3)-ß-D-Galp-(1→}n. We demonstrated that 1 µg/mL CPS could stimulate J774A.1 murine macrophages to release tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in vitro. Also, we proved that KN2 CPS induced the immune response through Toll-like receptor 4 (TLR4) in the human embryonic kidney (HEK)-293 cells. Strikingly, the hexasaccharide alone shows the same immune response as the CPS, suggesting that the hexasaccharide can shape the adaptive immunity to be a potential vaccine adjuvant. The glucuronic acid (GlcA) on other polysaccharides can affect the immune response, but the GlcA-reduced KN2 CPS and hexasaccharide still maintain their immunomodulatory activities.

A GalNAc/Gal-specific lectin modulates immune responses via toll-like receptor 4 independently of carbohydrate-binding ability.

Toll-like receptor 4 (TLR4) recognizes various protein ligands; however, the protein-TLR4 binding model is unclear. Here we demonstrate a Crenomytilus grayanus lectin (CGL)-TLR4/MD2 model to show that CGL interacts with a TLR4/myeloid differentiation factor 2 (MD2) complex independently of sugar-binding properties. CGL could suppress lipopolysaccharide-induced immune responses significantly, suggesting that TLR4 itself has potential as a therapeutic target.

An Enzyme-Mediated Aza-Michael Addition Is Involved in the Biosynthesis of an Imidazoyl Hybrid Product of Conidiogenone B.

Meleagrin B is a terpene-alkaloid hybrid natural product that contains both the conidiogenone and meleagrin scaffold. Their derivatives show diverse biological activities. We characterized the biosynthesis of (-)-conidiogenone B (1), which involves a diterpene synthase and a P450 monooxygenase. In addition, an α,ß-hydrolase (Con-ABH) was shown to catalyze an aza-Michael addition between 1 and imidazole to give 3S-imidazolyl conidiogenone B (6). Compound 6 was more potent than 1 against Staphylococcus aureus strains.

New Hydroquinone Monoterpenoid and Cembranoid-Related Metabolites from the Soft Coral Sarcophyton tenuispiculatum.

Chemical investigation of the marine soft coral Sarcophyton tenuispiculatum resulted in the isolation of a 1,4-dihydrobenzoquinone, sarcotenuhydroquinone (1), three new cembranoids, sarcotenusenes A‒C (2‒4), and ten previously reported metabolites 5-14. The chemical structures of all isolated metabolites were determined by detailed spectroscopic analyses. In biological assays, anti-inflammatory, cytotoxic, and peroxisome proliferator-activated receptor γ (PPAR-γ) transcription factor assays of all compounds were performed. None of the isolated compounds were found to exhibit activity in the PPAR-γ transcription factor assay. The anti-inflammatory assays showed that (+)-7α,8ß-dihydroxydeepoxysarcophine (13) inhibited the production of IL-1ß to 56 ± 1% at a concentration of 30 µM in lipopolysaccharide (LPS)-stimulated J774A.1 macrophage cells. In addition, 1 and 2 were found to exhibit cytotoxicity towards a panel of cancer cell lines.

Bacteriophage Tail-Spike Proteins Enable Detection of Pseudaminic-Acid-Coated Pathogenic Bacteria and Guide the Development of Antiglycan Antibodies with Cross-Species Antibacterial Activity.

Pseudaminic acid (Pse), a unique carbohydrate in surface-associated glycans of pathogenic bacteria, has pivotal roles in virulence. Owing to its significant antigenicity and absence in mammals, Pse is considered an attractive target for vaccination or antibody-based therapies against bacterial infections. However, a specific and universal probe for Pse, which could also be used in immunotherapy, has not been reported. In a prior study, we used a tail spike protein from a bacteriophage (ΦAB6TSP) that digests Pse-containing exopolysaccharide (EPS) from Acinetobacter baumannii strain 54149 (Ab-54149) to form a glycoconjugate for preparing anti-Ab-54149 EPS serum. We report here that a catalytically inactive ΦAB6TSP (I-ΦAB6TSP) retains binding ability toward Pse. In addition, an I-ΦAB6TSP-DyLight-650 conjugate (Dy-I-ΦAB6TSP) was more sensitive in detecting Ab-54149 than an antibody purified from anti- Ab-54149 EPS serum. Dy-I-ΦAB6TSP also cross-reacted with other pathogenic bacteria containing Pse on their surface polysaccharides (e.g., Helicobacter pylori and Enterobacter cloacae), revealing it to be a promising probe for detecting Pse across bacterial species. We also developed a detection method that employs I-ΦAB6TSP immobilized on microtiter plate. These results suggested that the anti-Ab-54149 EPS serum would exhibit cross-reactivity to Pse on other organisms. When this was tested, this serum facilitated complement-mediated killing of H. pylori and E. cloacae, indicating its potential as a cross-species antibacterial agent. This work opens new avenues for diagnosis and treatment of multidrug resistant (MDR) bacterial infections.

Effect of membrane fusion protein AdeT1 on the antimicrobial resistance of Escherichia coli.

Acinetobacter baumannii is a prevalent pathogen that can rapidly acquire resistance to antibiotics. Indeed, multidrug-resistant A. baumannii is a major cause of hospital-acquired infections and has been recognised by the World Health Organization as one of the most threatening bacteria to our society. Resistance-nodulation-division (RND) type multidrug efflux pumps have been demonstrated to convey antibiotic resistance to a wide range of pathogens and are the primary resistance mechanism employed by A. baumannii. A component of an RND pump in A. baumannii, AdeT1, was previously demonstrated to enhance the antimicrobial resistance of Escherichia coli. Here, we report the results of experiments which demonstrate that wild-type AdeT1 does not confer antimicrobial resistance in E. coli, highlighting the importance of verifying protein production when determining minimum inhibitory concentrations (MICs) especially by broth dilution. Nevertheless, using an agar-based MIC assay, we found that propionylation of Lys280 on AdeT1 renders E. coli cells more resistant to erythromycin.

Phosphoproteomics and Bioinformatics Analyses Reveal Key Roles of GSK-3 and AKAP4 in Mouse Sperm Capacitation.

Protein phosphorylation can induce signal transduction to change sperm motility patterns during sperm capacitation. However, changes in the phosphorylation of sperm proteins in mice are still incompletely understood. Here, capacitation-related phosphorylation in mouse sperms were firstly investigated by label-free quantitative (LFQ) phosphoproteomics coupled with bioinformatics analysis using ingenuity pathway analysis (IPA) methods such as canonical pathway, upstream regulator, and network analysis. Among 1632 phosphopeptides identified at serine, threonine, and tyrosine residues, 1050 novel phosphosites, corresponding to 402 proteins, were reported. Gene heatmaps for IPA canonical pathways showed a novel role for GSK-3 in GP6 signaling pathways associated with capacitation for 60 min. At the same time, the reduction of the abundant isoform-specific GSK-3α expression was shown by western blot (WB) while the LFQ pY of this isoform slightly decreased and then increased. The combined results from WB and LFQ methods explain the less inhibitory phosphorylation of GSK-3α during capacitation and also support the predicted increases in its activity. In addition, pAKAP4 increased at the Y156 site but decreased at the Y811 site in a capacitated state, even though IPA network analysis and WB analysis for overall pAKAP revealed upregulated trends. The potential roles of GSK-3 and AKAP4 in fertility are discussed.

Phagocytosis enhancement, endotoxin tolerance, and signal mechanisms of immunologically active glucuronoxylomannan from Auricularia auricula-judae.

Glucuronoxylomannan (AAPS) from the edible wood ear mushroom Auricularia auricula-judae has been demonstrated to exhibit immunostimulatory properties through its binding to TLR4. However, the mechanisms of immune modulation by AAPS in mammalian cells remains unclear. In the present study, we demonstrated that AAPS induced immunostimulatory effects were regulated by reactive oxygen species, mitogen-activated protein kinases, protein kinase C-α and NF-κB. AAPS remarkably increased the phagocytosis and bactericidal activity of macrophages. In lipopolysaccharide-activated macrophages, AAPS induced endotoxin tolerance like effect characterized by the downregulation of nitric oxide, interleukin-6 and TNF-α via the downregulation of NF-κB activation. Our findings provide firm scientific evidences for the immunoenhancing properties of wood ear mushroom, and the potential of AAPS to be strong candidates for the development of new carbohydrate-based nutraceutical supplements in the management of immunity related disorders in the future.

New Biscembranoids Sardigitolides A-D and Known Cembranoid-Related Compounds from Sarcophyton digitatum: Isolation, Structure Elucidation, and Bioactivities.

Chemical examination from the cultured soft coral Sarcophyton digitatum resulted in the isolation and structural identification of four new biscembranoidal metabolites, sardigitolides A-D (1-4), along with three previously isolated biscembranoids, sarcophytolide L (5), glaucumolide A (6), glaucumolide B (7), and two known cembranoids (8 and 9). The chemical structures of all isolates were elucidated on the basis of 1D and 2D NMR spectroscopic analyses. Additionally, in order to discover bioactivity of marine natural products, 1-8 were examined in terms of their inhibitory potential against the upregulation of inflammatory factor production in lipopolysaccharide (LPS)-stimulated murine macrophage J774A.1 cells and their cytotoxicities against a limited panel of cancer cells. The anti-inflammatory results showed that at a concentration of 10 µg/mL, 6 and 8 inhibited the production of IL-1ß to 68 ± 1 and 56 ± 1%, respectively, in LPS-stimulated murine macrophages J774A.1. Furthermore, sardigitolide B (2) displayed cytotoxicities toward MCF-7 and MDA-MB-231 cancer cell lines with the IC50 values of 9.6 ± 3.0 and 14.8 ± 4.0 µg/mL, respectively.

Multimeric TAT peptides are effective in vitro inhibitors of Staphylococcus saprophyticus.

TAT (48-60) is a tridecapeptide from the envelope protein of HIV that was previously shown to possess cell-penetrating properties and antibacterial activity, making it a potential drug delivery agent for anticancer drugs and as antibacterial compound. Previous reports indicated that dimerization enhances the desired bioactivity of TAT; hence, we sought to synthesize multimeric TAT peptides. Herein, we describe the effects of multimerization on the antibacterial activity and secondary structure of the peptide. Terminal modifications such as N-acetylation and C-amidation were employed in the design. TATp monomer, dimer, and tetramer were synthesized using solid-phase peptide synthesis, purified by reversed-phase HPLC, and then characterized by mass spectrometry. Multimerization of the peptide did not change the secondary structure conformation. The CD analysis revealed a polyproline-II conformation for all peptide designs. Thus, this study provides a method of increasing the biological activity of the peptide by multimerization while retaining the secondary conformation of its monomeric unit. Furthermore, the bacteria Staphylococcus saprophyticus was found to be susceptible to the dimer and tetramer, with MIC50 of 12.50 µm and <1.56 µm, respectively. This suggests a structure-activity relationship whereby the antibacterial activity increases with increase in valency.

Cyanine dye mediated mitochondrial targeting enhances the anti-cancer activity of small-molecule cargoes.

Organelle-specific delivery systems are of significant clinical interest. We demonstrate the use of common cyanine dyes Cy3 and Cy5 as vectors for targeting and delivering cargoes to mitochondria in cancer cells. Specifically, conjugation to the dyes can increase cytotoxicity by up to 1000-fold.