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The non-canonical Wnt signalling receptor ROR1 is aberrantly expressed in numerous cancers, including ovarian and endometrial cancer. We previously reported that silencing ROR1 could inhibit the proliferation and metastatic potential of ovarian and endometrial cancer cells in vitro. Zilovertamab is an ROR1-targeting humanised monoclonal antibody, with demonstrated safety and efficacy in clinical trials of several ROR1-related malignancies. The aim of this study was to investigate the potential of zilovertamab alone, or in combination with commonly utilised gynaecological cancer therapies (cisplatin, paclitaxel and the PARP inhibitor—Olaparib) on high-grade serous ovarian cancer (HGSOC), including models of platinum resistance and homologous recombination deficiency (CaOV3, CaOV3CisR, PEO1 and PEO4) and endometrial cancer (EC) cell lines (Ishikawa and KLE). The effect of zilovertamab (at 25 µg/mL or 50 µg/mL) +/− agents was investigated using the IncuCyte S3 Live Cell imaging system. Zilovertamab alone inhibited the proliferation of HGSOC and EC cells in vitro, including in models of platinum resistance and homologous recombination deficiency. In general, the addition of commonly used chemotherapies to a fixed dose of zilovertamab did not enhance the observed anti-proliferative activity. This study supports the potential of zilovertamab, or other ROR1-targeting therapies, for treating women with HGSOC and EC. [ABSTRACT FROM AUTHOR]

Highlights: [•] Quorum quenching could be an alternative to or act synergistically with antibiotics. [•] Prophylactic quorum quenching approaches have demonstrated efficacy in vivo. [•] Quorum sensing antigens might warrant inclusion in microbial vaccination strategies. [•] Neutralization of quorum sensing molecules may restore proper host immune function. [•] Development of drug-resistance against quorum quenching strategies remains unclear. [Copyright &y& Elsevier]

Identification of cellular processes modulated by microbial organisms that undermine and disarm mammalian host defences against bacterial invaders has been the focus of significant biomedical research. In this microreview we will illustrate the role of bacterial N-acyl homoserine lactones ( AHL) as a strategy utilized by Gram-negative bacterial pathogens to enable colonization of the host through AHL-mediated inhibition of inflammation induced via innate immune receptor mechanisms. We will also highlight some of the signalling pathways in which the study of AHL-mediated effects on mammalian cells might lead to the discovery of global underlying principles linking inflammation and immunity to many chronic human diseases, including cancer and obesity. [ABSTRACT FROM AUTHOR]

Environmental and commensal microbes that live within, on and around us have an enormous impact on human health. Recent progress in studies of prokaryotic interplay as well as host–bacteria interactions suggests that secreted microbial products, including quorum sensing signals (QSS), are important mediators of these intrakingdom and interkingdom relations. Reports have assigned QSS diverse and sometimes seemingly contradictory effects on mammalian cell physiology ranging from either blunting of the immune response or exerting pro-inflammatory activities to inducing cellular stress pathways and ultimately apoptosis. Thus, it is still unclear whether microbes utilize QSS to establish and maintain infections via modulation of host signaling pathways or if the eukaryotic host uses the conserved microbial QSS structures as molecular danger beacons to detect and fight infections. Along the same lines exactly how and under what circumstances QSS are detected by host cells remains a mystery, especially considering the distinct chemical properties of the QSS classes with some being small enough to passively diffuse across membranes while others most likely require extracellular recognition mechanisms. [Copyright &y& Elsevier]

In Pseudomonas aeruginosa, quorum-sensing (QS) signaling regulates the expression of virulence factors and thus represents an attractive new target for anti-infective therapy. In the present study, we investigated whether solenopsin A, a venom alkaloid from the fire ant, possessed agonistic or antagonistic QS signaling activity in P. aeruginosa.We evaluated the modulation of virulence factor expression and transcriptional levels of QS-regulated genes in P. aeruginosa by solenopsinAand demonstrated that solenopsin A efficiently disrupted QS signaling. Interestingly, exogenously added C4-homoserine lactone (HSL), but not 3-oxo-C12-HSL, restored P. aeruginosa QS signaling, suggesting that solenopsin A targets the C4-HSL- dependent rhl QS system. [ABSTRACT FROM AUTHOR]

The control of innate immune responses through activation of the nuclear transcription factor NF-B is essential for the elimination of invading microbial pathogens. We showed that the bacterial N-(3-oxo-dodecanoyl) homoserine lactone (C12) selectively impairs the regulation of NF-κB functions in activated mammalian cells. The consequence is specific repression of stimulus-mediated induction of NF-κB-responsive genes encoding inflammatory cytokines and other immune regulators. These findings uncover a strategy by which C12-producing opportunistic pathogens, such as Pseudomonas aeruginosa, attenuate the innate immune system to establish and maintain local persistent infection in humans, for example, in cystic fibrosis patients. [ABSTRACT FROM AUTHOR]

Abstract: The Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, uses acyl-homoserine lactone-based quorum sensing systems to control its pathogenicity. One of its quorum sensing factors, N-3-oxo-dodecanoyl-homoserine lactone, has been shown not only to mediate bacterial quorum sensing but also to exert cytotoxic effects on mammalian cells. The monoclonal antibody RS2-1G9 generated against a 3-oxo-dodecanoyl-homoserine lactone analogue hapten was able to protect murine bone marrow-derived macrophages from the cytotoxic effects and also prevented the activation of the mitogen-activated protein kinase p38. These data demonstrate that an immunopharmacotherapeutic approach to combat P. aeruginosa infections might be a viable therapeutic option as the monoclonal antibody RS2-1G9 can readily sequester bacterial N-3-oxo-dodecanoyl-homoserine lactone molecules, thus interfering with their biological effects in prokaryotic and eukaryotic systems. [Copyright &y& Elsevier]

The blue-emissive antibody EP2-19G2 that has been elicited against trans-stilbene has unprecedented ability to produce bright luminescence and has been used as a biosensor in various applications. We show that the prolonged luminescence is not stilbene fluorescence. Instead, the emissive species is a charge-transfer excited complex of an anionic stilbene and a cationic, parallel π-stacked tryptophan. Upon charge recombination, this complex generates exceptionally bright blue light. Complex formation is enabled by a deeply penetrating ligand-binding pocket, which in turn results from a noncanonical interface between the two variable domains of the antibody. [ABSTRACT FROM AUTHOR]

Abstract: A large number of Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signaling molecules in quorum sensing, which is a population density-dependent mechanism to coordinate gene expression. Antibody RS2-1G9 was elicited against a lactam mimetic of the N-acyl homoserine lactone and represents the only reported monoclonal antibody that recognizes the naturally-occuring N-acyl homoserine lactone with high affinity. Due to its high cross-reactivity, RS2-1G9 showed remarkable inhibition of quorum sensing signaling in Pseudomonas aeruginosa, a common opportunistic pathogen in humans. The crystal structure of Fab RS2-1G9 in complex with a lactam analog revealed complete encapsulation of the polar lactam moiety in the antibody-combining site. This mode of recognition provides an elegant immunological solution for tight binding to an aliphatic, lipid-like ligand with a small head group lacking typical haptenic features, such as aromaticity or charge, which are often incorporated into hapten design to generate high-affinity antibodies. The ability of RS2-1G9 to discriminate between closely related AHLs is conferred by six hydrogen bonds to the ligand. Conversely, cross-reactivity of RS2-1G9 towards the lactone is likely to originate from conservation of these hydrogen bonds as well as an additional hydrogen bond to the oxygen of the lactone ring. A short, narrow tunnel exiting at the protein surface harbors a portion of the acyl chain and would not allow entry of the head group. The crystal structure of the antibody without its cognate lactam or lactone ligands revealed a considerably altered antibody-combining site with a closed binding pocket. Curiously, a completely buried ethylene glycol molecule mimics the lactam ring and, thus, serves as a surrogate ligand. The detailed structural delineation of this quorum-quenching antibody will aid further development of an antibody-based therapy against bacterial pathogens by interference with quorum sensing. [Copyright &y& Elsevier]

Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas double-stranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2α (eIF2α). Here we have shown that p38 and eIF2α phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl)homoserine lactones, the secreted products of a number of Gram-negative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl)homoserine lactone induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl)homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors, or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl)homoserine lactone on host cells and its role in persistent airway infections caused by P. aeruginosa. [ABSTRACT FROM AUTHOR]

Cocaine is a highly addictive drug, and despite intensive efforts, effective therapies for cocaine craving and addiction remain elusive. In recent years, we and others have reported advances in anti-cocaine immunopharmacotherapy based on specific antibodies capable of sequestering the drug before it reaches the brain. In an effort to obtain high affinity therapeutic anti-cocaine antibodies, either whole IgGs or other antibody constructs, fluorescence spectroscopic techniques could provide a means of assisting selection and engineering strategies. We report the synthesis of a series of cocaine-fluorophore conjugates (GNC-F1, GNC-F2, GNC-1) and the functional evaluation of these compounds against single-chain Fv antibodies obtained via crystallographic analysis/engineering and against commercially available anti-cocaine monoclonal antibodies with a wide range of cocaine-binding affinities. From these studies, we determined that the GNC-F2 fluorophore reproduced affinity constants obtained using [³H]-labeled cocaine. We anticipate that the readily synthesized and nonradioactive GNC-F2 will find use both as a tool for bioimaging and in the high-throughput selection and engineering of potential therapeutic antibodies against cocaine. [ABSTRACT FROM AUTHOR]

Bacteria use small diffusible molecules to exchange information in a process called quorum sensing An important class of autoinducers used by Gram-negative bacteria is the family of N-acylhomoserine lactones. Here, we report the discovery of a previously undescribed nonenzymatically formed product from N-(3-oxodo- decanoyl)-L-homoserine lactone; both the N-acylhomoserine and its novel tetramic acid degradation product, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione, are potent antibacterial agents. Bactericidal activity was observed against all tested Gram-positive bacterial strains, whereas no toxicity was seen against Gram-negative bacteria. We propose that Pseudomonas aeruginosa utilizes this tetramic acid as an interference strategy to preclude encroachment by competing bacteria. Additionally, we have discovered that this tetramic acid binds iron with comparable affinity to known bacterial siderophores, possibly providing an unrecognized mechanism for iron solubilization. These findings merit new attention such that other previously identified autoinducers be reevaluated for additional biological functions. [ABSTRACT FROM AUTHOR]

Cocaine addiction continues to be a major health and social problem in the United States and other countries. Currently used pharmacological agents for treating cocaine abuse have proved inadequate, leaving few treatment options. An alternative is to use protein-based therapeutics that can eliminate the load of cocaine, thereby attenuating its effects. This approach is especially attractive because the therapeutic agents exert no pharmacodynamic action of their own and therefore have little potential for side effects. The effectiveness of these agents, however, is limited by their inability to act directly within the CNS. Bacteriophage have the capacity to penetrate the CNS when administered intranasally. Here, a method is presented for engineering filamentous bacteriophage to display cocaine-binding proteins on its surface that sequester cocaine in the brain. These antibody-displaying constructs were examined by using a locomotor activity rodent model to assess the ability of the phage-displayed proteins to block the psychoactive effects of cocaine. Results presented demonstrate a strategy in the continuing efforts to find effective treatments for cocaine addiction and suggest the application of this protein-based treatment for other drug abuse syndromes. [ABSTRACT FROM AUTHOR]

The article discusses the interference of N-Acyl L-homoserine lactone-based (AHL) quorum sensing signaling as a new approach for anti-infective therapy in the U.S. The interference focused on synthesizing a set of molecules highly congruent in structure to AHL. This paper investigates the stability of several AHL under physiological conditions.

The article discusses a review on quenching of bacterial quorum sensing (QS) signaling by macromolecules and summarizes research on QS attenuation by three types of proteins namely enzymes, antibodies that scavenge quorum sensing molecules (QSMs) and decoy receptors. It defines quorum sensing as the ability of unicellular bacteria to act as a single multicellular organism in a cell-density-dependent manner. It suggests that quorum quenching (QQ) macromolecules can function as molecular tools to study intercellular signaling in bacterial populations.

Summary: Quorum sensing (QS) is the process through which bacteria communicate utilizing small diffusible molecules termed autoinducers. It has been demonstrated that QS controls a plethora of microbial processes including the expression of virulence factors. Here we report an immunopharmacotherapeutic approach for the attenuation of QS in the Gram-positive human pathogen Staphylococcus aureus. An anti-autoinducer monoclonal antibody, AP4-24H11, was elicited against a rationally designed hapten, and efficiently inhibited QS in vitro through the sequestration of the autoinducing peptide (AIP)-4 produced by S. aureus RN4850. Importantly, AP4-24H11 suppressed S. aureus pathogenicity in an abscess formation mouse model in vivo and provided complete protection against a lethal S. aureus challenge. These findings provide a strong foundation for further investigations of immunopharmacotherapy for the treatment of bacterial infections in which QS controls the expression of virulence factors. [Copyright &y& Elsevier]

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa. Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL). [ABSTRACT FROM AUTHOR]

Bioconjugate formats provide alternative strategies for antigen targeting with bispecific antibodies. Here, PSMA-targeted Fab conjugates were generated using different bispecific formats. Interchain disulfide bridging of an αCD3 Fab enabled installation of either the PSMA-targeting small molecule DUPA (SynFab) or the attachment of an αPSMA Fab (BisFab) by covalent linkage. Optimization of the reducing conditions was critical for selective interchain disulfide reduction and good bioconjugate yield. Activity of αPSMA/CD3 Fab conjugates was tested by in vitro cytotoxicity assays using prostate cancer cell lines. Both bispecific formats demonstrated excellent potency and antigen selectivity. [ABSTRACT FROM AUTHOR]

Bispecific antibodies (BsAbs) are designed to engage two antigens simultaneously, thus, effectively expanding the ability of antibody-based therapeutics to target multiple pathways within the same cell, engage two separate soluble antigens, bind the same antigen with distinct paratopes, or crosslink two different cell types. Many recombinant BsAb formats have emerged, however, expression and purification of such constructs can often be challenging. To this end, we have developed a chemical strategy for generating BsAbs using native IgG2 architecture. Full-length antibodies can be conjugated via disulfide bridging with linkers bearing orthogonal groups to produce BsAbs. We report that an αHER2/EGFR BsAb was successfully generated by this approach and retained the ability to bind both antigens with no significant loss of potency. [ABSTRACT FROM AUTHOR]

The article describes the discovery of synthetic 4,5-dihydroxy-2,3-pentanedione (DPD) analogues due to an unexpected switch in the modulation of a class of synergistic compounds toward the AI-2-based quorum sensing (QS) of Vibrio harveyi. According to the authors, DPD analogues can elicit different biological effects in two different species with known AI-3 QS systems. They add that the difference in activity was revealed through chemical synthesis and the analysis of bacterial phenotypes.

This article presents information about a study which describes the preparation and kinetics of the first catalytic phage-displayed therapeutic with suitable rates to treat cocaine addiction. Cocaine esterase (cocE) is a globular enzyme with a molecular weight of 65 kDa and is the most efficient protein catalyst for the hydrolysis of cocaine characterized to date. The size and catalytic efficiency of cocE make it an ideal candidate for an improved cocaine therapy. Displaying cocE on the phage surface may overcome the inherent disadvantages of natural enzyme and endow it with more favorable immuno/proteodynamics.

Reported herein is that (4 S)-4,5-dihydroxy-2,3-pentanedione (DPD) can undergo a previously undocumented non-enzymatic glycation reaction. Incubation of DPD with viral DNA or the antibiotic gramicidin S resulted in significant biochemical alterations. A protein-labeling method was consequently developed that facilitated the identification of unrecognized glycation targets of DPD in a prokaryotic system. These results open new avenues toward tracking and understanding the fate and function of the elusive quorum-sensing signaling molecule. [ABSTRACT FROM AUTHOR]

Reported herein is that (4 S)-4,5-dihydroxy-2,3-pentanedione (DPD) can undergo a previously undocumented non-enzymatic glycation reaction. Incubation of DPD with viral DNA or the antibiotic gramicidin S resulted in significant biochemical alterations. A protein-labeling method was consequently developed that facilitated the identification of unrecognized glycation targets of DPD in a prokaryotic system. These results open new avenues toward tracking and understanding the fate and function of the elusive quorum-sensing signaling molecule. [ABSTRACT FROM AUTHOR]

Summary: Small molecule probes have been used extensively to explore biologic systems and elucidate cellular signaling pathways. In this study, we use an inhibitor of bacterial communication to monitor changes in the proteome of Salmonella enterica serovar Typhimurium with the aim of discovering unrecognized processes regulated by AI-2-based quorum-sensing (QS), a mechanism of bacterial intercellular communication that allows for the coordination of gene expression in a cell density-dependent manner. In S. typhimurium, this system regulates the uptake and catabolism of intercellular signals and has been implicated in pathogenesis, including the invasion of host epithelial cells. We demonstrate that our QS antagonist is capable of selectively inhibiting the expression of known QS-regulated proteins in S. typhimurium, thus attesting that QS inhibitors may be used to confirm proposed and elucidate previously unidentified QS pathways without relying on genetic manipulation. [Copyright &y& Elsevier]

In enteric bacteria, the kinase LsrK catalyzes the phosphorylation of the C5-hydroxyl group in the linear form of 4,5-dihydroxy-2,3-pentanedione (DPD), the precursor of the type II bacterial quorum sensing molecule (AI-2). This phosphorylation is required for AI-2 sequestration in the cytoplasm and subsequent derepression of AI-2-related genes necessary for quorum development. While LsrK is a critical enzyme within the DPD quorum sensing relay system, kinetic details of this kinase have yet to be reported. A continuous UV–vis spectrophotometric assay was developed that allowed steady-state kinetic analysis of LsrK to be undertaken with the substrates ATP and DPD. The data was most consistent with a rapid equilibrium ordered mechanism with ATP binding first: kcat (7.4 ± 0.6 s-1), Km,ATP (150 ± 30 µM) and Km(app),DPD (1.0 ± 0.2 mM). The assay also allowed a DPD substrate profile to be conducted, which provided an unexpected biochemical disconnect between the previous agonist/antagonist cell-based reporter assay and the LsrK assay presented herein. Together these findings raise the importance of LsrK and lay the foundation not only for further understanding of this enzyme and its critical biological role but also for the rational design of regulatory molecules targeting AI-2 quorum sensing in pathogenic bacteria. [ABSTRACT FROM AUTHOR]

The opportunistic bacterial pathogen Pseudomonas aeruginosa causes chronic lung infections in cystic fibrosis (CF) patients. Importantly, virulence factor expression and biofilm formation in P. aeruginosa is coordinated by quorum sensing (QS) and one of the key QS signaling molecules is 3-oxo-C12-HSL. Remarkably, a tetramic acid, (C12-TA), with antibacterial properties is formed spontaneously from 3-oxo-C12-HSL under physiological conditions. Seeking to better understand this relationship, we sought to investigate whether 3-oxo-C12-HSL and C12-TA may be contributing factors to the overall pathogenicity of P. aeruginosa in CF individuals and if their detection and quantitation in sputum samples might be used as an indicator to assess disease states and monitor therapy success in CF patients. To this end, 3-oxo-C12-HSL and C12-TA concentrations were initially analyzed in P. aeruginosa flow cell biofilms using liquid chromatography coupled with mass spectrometry (LC-MS). A liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method was then developed and validated for their detection and quantification in the sputa of CF patients. To the best of our knowledge, this is the first report to show the presence of both the quorum sensing molecule (3-oxo-C12-HSL) and its rearranged product (C12-TA) in human clinical samples such as sputum. A total of 47 sputum samples from 20 CF and 2 non-CF individuals were analyzed. 3-Oxo-C12-HSL was detected and quantified in 45 samples with concentrations ranging from 20 to >1000 nM; C12-TA was found in 14 samples (13-900 nM). On the basis of our findings, quorum sensing autoinducers merit further investigation as biomarkers for infectious disease states. [ABSTRACT FROM AUTHOR]

Flagella are cell surface appendages involved in a number of bacterial behaviors, such as motility, biofilm formation, and chemotaxis. Despite these important functions, flagella can pose a liability to a bacterium when serving as potent immunogens resulting in the stimulation of the innate and adaptive immune systems. Previous work showing appendage overexpression, referred to as attenuating gene expression (AGE), was found to enfeeble wild-type Salmonella. Thus, this approach was adapted to discern whether flagella overexpression could induce similar attenuation. To test its feasibility, flagellar filament subunit FliC and flagellar regulon master regulator FlhDC were overexpressed in Salmonella enterica serovar Typhimurium wild-type strain H71. The results show that the expression of either FliC or FlhDC alone, and co- expression of the two, significantly attenuates Salmonella. The flagellated bacilli were unable to replicate within macrophages and thus were not lethal to mice. In-depth investigation suggests that flagellum-mediated AGE was due to the disruptive effects of flagella on the bacterial membrane, resulting in heightened susceptibilities to hydrogen peroxide and bile. Furthermore, flagellum-attenuated Salmonella elicited elevated immune responses to Salmonella presumably via FliC's adjuvant effect and conferred robust protection against wild-type Salmonella challenge. [ABSTRACT FROM AUTHOR]

Chronic respiratory infections are a major cause of morbidity and mortality, most particularly in Cystic Fibrosis (CF) patients. The recent finding that gastro-esophageal reflux (GER) frequently occurs in CF patients led us to investigate the impact of bile on the behaviour of Pseudomonas aeruginosa and other CF-associated respiratory pathogens. Bile increased biofilm formation, Type Six Secretion, and quorum sensing in P. aeruginosa, all of which are associated with the switch from acute to persistent infection. Furthermore, bile negatively influenced Type Three Secretion and swarming motility in P. aeruginosa, phenotypes associated with acute infection. Bile also modulated biofilm formation in a range of other CF-associated respiratory pathogens, including Burkholderia cepacia and Staphylococcus aureus. Therefore, our results suggest that GER-derived bile may be a host determinant contributing to chronic respiratory infection. [ABSTRACT FROM AUTHOR]

Bacterial infections are a primary cause of morbidity and mortality worldwide. Bacteremia is a particular concern owing to the possibility of septic shock and the development of metastatic infections. Treatment of bacteremia is increasingly compromised by the emergence of antibiotic resistant strains, creating an urgent need for alternative therapy. Here, we introduce a method for in vivo photoacoustic (PA) detection and photothermal (PT) eradication of Staphylococcus aureus in tissue and blood. We show that this method could be applicable for label-free diagnosis and treatment of in the bloodstream using intrinsic near-infrared absorption of endogenous carotenoids with nonlinear PA and PT contrast enhancement. To improve sensitivity and specificity for detection of circulating bacteria cells (CBCs), two-color gold and multilayer magnetic nanoparticles with giant amplifications of PA and PT contrasts were functionalized with an antibody cocktail for molecular targeting of S. aureus surface-associated markers such as protein A and lipoprotein. With a murine model, the utility of this approach was demonstrated for ultrasensitive detection of CBCs with threshold sensitivity as low as 0.5 CBCs/mL, in vivo magnetic enrichment of CBCs, PT eradication of CBCs, and real-time monitoring of therapeutic efficacy by CBC counting. Our PA-PT nano-theranostic platform, which integrates in vivo multiplex targeting, magnetic enrichment, signal amplification, multicolor recognition, and feedback control, could be used as a biological tool to gain insights on dissemination pathways of CBCs, infection progression by bacteria re-seeding, and sepsis development and treatment, and could potentially be feasible in humans, especially using bypass schematic. [ABSTRACT FROM AUTHOR]

Burkholderia pseudomallei, a Gram-negative saprophytic bacterium, is the causative agent of the potentially fatal melioidosis disease in humans. In this study, environmental parameters including temperature, nutrient content, pH and the presence of glucose were shown to play a role in in vitro biofilm formation by 28 B. pseudomallei clinical isolates, including four isolates with large colony variants (LCVs) and small colony variants (SCVs) morphotypes. Enhanced biofilm formation was observed when the isolates were tested in LB medium, at 307deg;C, at pH 7.2, and in the presence of as little as 2 mM glucose respectively. It was also shown that all SVCs displayed significantly greater capacity to form biofilms than the corresponding LCVs when cultured in LB at 37°C. In addition, octanoyl-homoserine lactone (C8-HSL), a quorum sensing molecule, was identified by mass spectrometry analysis in bacterial isolates referred to as LCV CTH, LCV VIT, SCV TOM, SCV CTH, 1 and 3, and the presence of other AHL's with higher masses; decanoyl-homoserine lactone (C10-HSL) and dodecanoyl-homoserine lactone (C12-HSL) were also found in all tested strain in this study. Last but not least, we had successfully acquired two Bacillus sp. soil isolates, termed KW and SA respectively, which possessed strong AHLs degradation activity. Biofilm formation of B. pseudomallei isolates was significantly decreased after treated with culture supernatants of KW and SA strains, demonstrating that AHLs may play a role in B. pseudomallei biofilm formation. [ABSTRACT FROM AUTHOR]

The most feared complication following intestinal resection is anastomotic leakage. In high risk areas (esophagus/rectum) where neoadjuvant chemoradiation is used, the incidence of anastomotic leaks remains unacceptably high (∼10%) even when performed by specialist surgeons in high volume centers. The aims of this study were to test the hypothesis that anastomotic leakage develops when pathogens colonizing anastomotic sites become in vivo transformed to express a tissue destroying phenotype. We developed a novel model of anastomotic leak in which rats were exposed to pre-operative radiation as in cancer surgery, underwent distal colon resection and then were intestinally inoculated with Pseudomonas aeruginosa, a common colonizer of the radiated intestine. Results demonstrated that intestinal tissues exposed to preoperative radiation developed a significant incidence of anastomotic leak (>60%; p<0.01) when colonized by P. aeruginosa compared to radiated tissues alone (0%). Phenotype analysis comparing the original inoculating strain (MPAO1-termed P1) and the strain retrieved from leaking anastomotic tissues (termed P2) demonstrated that P2 was altered in pyocyanin production and displayed enhanced collagenase activity, high swarming motility, and a destructive phenotype against cultured intestinal epithelial cells (i.e. apoptosis, barrier function, cytolysis). Comparative genotype analysis between P1 and P2 revealed a single nucleotide polymorphism (SNP) mutation in the mexT gene that led to a stop codon resulting in a non-functional truncated protein. Replacement of the mutated mexT gene in P2 with mexT from the original parental strain P1 led to reversion of P2 to the P1 phenotype. No spontaneous transformation was detected during 20 passages in TSB media. Use of a novel virulence suppressing compound PEG/Pi prevented P. aeruginosa transformation to the tissue destructive phenotype and prevented anastomotic leak in rats. This work demonstrates that in vivo transformation of microbial pathogens to a tissue destroying phenotype may have important implications in the pathogenesis of anastomotic leak. [ABSTRACT FROM AUTHOR]

Growth-dependent cell-cell communication termed quorum sensing is a key regulatory system in bacteria for controlling gene expression including virulence factors. In this study five potential bacterial pathogens including Bacillus sp. W2.2, Klebsiella sp. W4.2, Pseudomonas sp. W3 and W3.1 and Serratia sp. W2.3 were isolated from diseased Tilapia fish in Malaysia, supplied by the leading global fish supplier. Proteolytic activity assays confirmed that with the exception of Klebsiella sp. W4.2, all isolates showed distinct proteolytic activity. Furthermore Bacillus sp. W2.2 and Pseudomonas sp. strains W3 and W3.1 also displayed haemolytic activity. By using high resolution liquid chromatography mass spectrometry, we revealed the presence of unusually long- chain N-(3-oxohexadecanoyl)-homoserine lactone (3-oxo-C16-HSL) from Pseudomonas sp. W3.1 and N-dodecanoyl-homoserine lactone (C12-HSL) from Serratia sp. W2.3, respectively. Interestingly, Pseudomonas sp. W3.1 also produced a wide range of Pseudomonas quinolone signalling (PQS) molecules. Pseudomonas sp. W3 did not show any quorum sensing properties but possessed quorum quenching activity that inactivated AHLs. This study is the first documentation that shows unusual long- chain AHLs production in Serratia sp. and Pseudomonas sp. isolated from diseased fish and the latter also produce a wide range of PQS molecules. [ABSTRACT FROM AUTHOR]

Background: Quorum sensing (QS) in Sinorhizobium meliloti involves at least half a dozen different N-acyl homoserine lactone (AHL) signals. These signals are produced by SinI, the sole AHL synthase in S. meliloti Rm8530. The sinI gene is regulated by two LuxR-type transcriptional regulators, SinR and ExpR. Mutations in sinI, sinR and expR abolish the production of exopolysaccharide II (EPS II). Methodology/Principal Findings: This study investigated a new type of coordinated surface spreading of Rm8530 that can be categorized as swarming. Motility assays on semi-solid surfaces revealed that both flagella and EPS II are required for this type of motility. The production of EPS II depends on AHLs produced by SinI. Of these AHLs, only C16:1- and 3-oxo-C16:1-homoserine lactones (HSLs) stimulated swarming in an ExpR-dependent manner. These two AHLs induced the strongest response in the wggR reporter fusions. WggR is a positive regulator of the EPS II biosynthesis gene expression. The levels of the wggR activation correlated with the extent of swarming. Furthermore, swarming of S. meliloti required the presence of the high molecular weight (HMW) fraction of EPS II. Within swarming colonies, a recombinase-based RIVET reporter in the wggR gene was resolved in 30% of the cells, indicating an enhanced regulation of EPS II production in the subpopulation of cells, which was sufficient to support swarming of the entire colony. Conclusions/Significance: Swarming behavior of S. meliloti Rm8530 on semi-solid surfaces is found to be dependent on the functional QS regulatory cascades. Even though multiple AHL signals are produced by the bacterium, only two AHLs species, C16:1- and 3-oxo- C16:1-HSLs, affected swarming by up-regulating the expression of wggR. While EPS II is produced by Rm8530 as high and low molecular weight fractions, only the HMW EPS II facilitated initial stages of swarming, thus, suggesting a function for this polymer. [ABSTRACT FROM AUTHOR]

The synthesis of virulence factors by pathogenic bacteria is highly regulated and occurs in response to diverse environmental cues. An array of two component systems (TCSs) serves to link perception of different cues to specific changes in gene expression and/or bacterial behaviour. Those TCSs that regulate functions associated with virulence represent attractive targets for interference in anti-infective strategies for disease control. We have previously identified PA2572 as a putative response regulator required for full virulence of Pseudomonas aeruginosa, the opportunistic human pathogen, to Galleria mellonella (Wax moth) larvae. Here we have investigated the involvement of candidate sensors for signal transduction involving PA2572. Mutation of PA2573, encoding a probable methyl-accepting chemotaxis protein, gave rise to alterations in motility, virulence, and antibiotic resistance, functions which are also controlled by PA2572. Comparative transcriptome profiling of mutants revealed that PA2572 and PA2573 regulate expression of a common set of 49 genes that are involved in a range of biological functions including virulence and antibiotic resistance. Bacterial two-hybrid analysis indicated a REC-dependent interaction between PA2572 and PA2573 proteins. Finally expression of PA2572 in the PA2573 mutant background restored virulence to G. mellonella towards wild- type levels. The findings indicate a role for the orphan chemotaxis sensor PA2573 in the regulation of virulence and antibiotic tolerance in P. aeruginosa and indicate that these effects are exerted in part through signal transduction involving PA2572. [ABSTRACT FROM AUTHOR]

The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate- contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroAR.aquatilis, was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroAE.coli), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroAR.aquatilis were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroAE.coli. To probe the sites contributing to increased tolerance to glyphosate, mutant R.aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R.aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious. [ABSTRACT FROM AUTHOR]

Stereochemistry is a key aspect of molecular recognition for biological systems. As such, receptors and enzymes are often highly stereospecific, only recognizing one stereoisomer of a ligand. Recently, the quorum sensing signaling molecules used by the nosocomial opportunistic pathogen, Acinetobacter baumannii, were identified, and the primary signaling molecule isolated from this species was N-(3-hydroxydodecanoyl)-L-homoserine lactone. A plethora of bacterial species have been demonstrated to utilize 3-hydroxy-acylhomoserine lactone autoinducers, and in virtually all cases, the (R)-stereoisomer was identified as the natural ligand and exhibited greater autoinducer activity than the corresponding (S)-stereoisomer. Using chemical synthesis and biochemical assays, we have uncovered a case of stereochemical insignificance in A. baumannii and provide a unique example where stereochemistry appears nonessential for acylhomoserine lactone-mediated quorum sensing signaling. Based on previously reported phylogenetic studies, we suggest that A. baumannii has evolutionarily adopted this unique, yet promiscuous quorum sensing system to ensure its survival, particularly in the presence of other proteobacteria. [ABSTRACT FROM AUTHOR]

Multivalency is a common principle in the recognition of cellular receptors, and multivalent agonists and antagonists have played a major role in understanding mammalian cell receptor biology. The study of bacterial cell receptors using similar approaches, however, has lagged behind. Herein we describe our efforts toward the development of a dendrimer-based multivalent probe for studying AI-2 quorum-sensing receptors. From these studies, we have discovered a chemical probe specific for Lsr-type AI-2 quorum-sensing receptors with the potential for enabling the identification of new bacterial species that utilize AI-2 as a quorum-sensing signaling molecule. [ABSTRACT FROM AUTHOR]

In the postantibiotic era, available treatment options for severe bacterial infections caused by methicillin-resistant Staphylococcus aureus have become limited. Therefore, new and innovative approaches are needed to combat such life-threatening infections. Virulence factor expression in S. aureus is regulated in a cell density-dependent manner using "quorum sensing," which involves generation and secretion of autoinducing peptides (AIPs) into the surrounding environment to activate a bacterial sensor kinase at a particular threshold concentration. Mouse monoclonal antibody AP4-24H11 was shown previously to blunt quorum sensing-mediated changes in gene expression in vitro and protect mice from a lethal dose of S: aureus by sequestering the AIP signal. We have elucidated the crystal structure of the AP4-24H11 Fab in complex with AIP-4 at 2.5 Å resolution to determine its mechanism of ligand recognition. A key GluH95 provides much of the binding specificity through formation of hydrogen bonds with each of the four amide nitrogens in the AIP-4 macrocyclic ring. Importantly, these structural data give clues as to the interactions between the cognate staphylococcal AIP receptors AgrC and the AIPs, as AP4-24H11.AIP-4 binding recapitulates features that have been proposed for AgrC-AIP recognition. Additionally, these structural insights may enable the engineering of AlP cross-reactive antibodies or quorum quenching vaccines for use in active or passive immunotherapy for prevention or treatment of S. aureus infections. [ABSTRACT FROM AUTHOR]

Abstract: Alkynyl- and azido-tagged 3-oxo-C12-acylhomoserine lactone probes have been synthesized to examine their potential utility as probes for discovering the mammalian protein target of the Pseudomonas aeruginosa autoinducer, 3-oxo-C12-acylhomoserine lactone. Although such substitutions are commonly believed to be quite conservative, from these studies, we have uncovered a drastic difference in activity between the alkynyl- and azido-modified compounds, and provide an example where such structural modification has proved to be much less than conservative. [Copyright &y& Elsevier]

Onchocerciasis, or river blindness, is a neglected tropical disease caused by the filarial nematode Onchocerca volvulus that affects more than 37 million people, mainly in third world countries. Currently, the only approved drug available for mass treatment is ivermectin, however, drug resistance is beginning to emerge, thus, new therapeutic targets and agents are desperately needed to treat and cure this devastating disease. Chitin metabolism plays a central role in invertebrate biology due to the critical structural function of chitin for the organism. Taken together with its absence in mammals, targeting chitin is an appealing therapeutic avenue. Importantly, the chitinase OvCHT1 from O. volvulus was recently discovered, however, its exact role in the worm's metabolism remains unknown. A screening effort against OvCHT1 was conducted using the Johns Hopkins Clinical Compound Library that contains over 1,500 existing drugs. Closantel, a veterinary anthelmintic with known proton ionophore activities, was identified as a potent and specific inhibitor of filarial chitinases, an activity not previously reported for this compound. Notably, closantel was found also to completely inhibit molting of O. volvulus infective L3 stage larvae. Closantel appears to target two important biochemical processes essential to filarial parasites. To begin to unravel closantel's effects, a retro-fragment-based study was used to define structural elements critical for closantel's chitinase inhibitor function. As resources towards the development of new agents that target neglected tropical diseases are scant, the finding of an existing drug with impact against O. volvulus provides promise in the hunt for new therapies against river blindness. [ABSTRACT FROM AUTHOR]

In nature, bacteria rarely exist as single, isolated entities, but rather as communities comprised of many other species including higher host organisms. To survive in these competitive environments, microorganisms have developed elaborate tactics such as the formation of biofilms and the production of antimicrobial toxins. Recently, it was discovered that the Gram-negative bacterium Pseudomonas aeruginosa, an opportunistic human pathogen, produces an antibiotic, 3-(1-hydroxydecylidene)-5-(2-hydroxyethyl)pyrrolidine-2,4-dione (C12-TA), derived from one of its quorum sensing molecules. Here, we present a comprehensive study of the expanded spectrum of C12-TA antibacterial activity against microbial competitors encountered by P. aeruginosa in nature as well as significant human pathogens. The mechanism of action of C12-TA was also elucidated, and C12-TA was found to dissipate both the membrane potential and the pH gradient of Gram-positive bacteria, correlating well with cell death. Notably, in stark contrast to its parent molecule 3-oxo-dodecanoyl homoserine lactone (3-oxo-C12-HSL), neither activation of cellular stress pathways nor cytotoxicity was observed in human cells treated with C12-TA. Our results suggest that the OS machinery of P. aeruginosa has evolved for a dual-function, both to signal others of the same species and also to defend against host immunity and competing bacteria. Because of the broad-spectrum antibacterial activity, established mode of action, lack of rapid resistance development, and tolerance by human cells, the C12-TA scaffold may also serve as a new lead compound for the development of antimicrobial therapeutics. [ABSTRACT FROM AUTHOR]

Abstract: Tumor targeting peptides are promising vehicles for site-directed cancer therapy. Pep42, a cyclic 13-mer oligopeptide that specifically binds to glucose-regulated protein 78 (GRP78) and internalized into cancer cells, represents an excellent vehicle for tumor cell-specific chemotherapy. Here, we report the synthesis and evaluation of Pep42-prodrug conjugates that contain a cathepsin B-cleavable linker, resulting in the traceless release of drug inside the cancer cells. [Copyright &y& Elsevier]

Abstract: Cocaine is a powerful and addictive stimulant whose abuse remains a prevalent health and societal crisis. Unfortunately, no pharmacological therapies exist and therefore alternative protein-based therapies have been examined. One such approach is immunopharmacotherapy, wherein antibodies are utilized to either bind or hydrolyze cocaine thereby blocking it from exerting its euphoric effect. Towards this end, antibodies capable of binding and hydrolyzing cocaine were identified by phage display from a biased single chain antibody library generated from the spleens of mice previously immunized with a cocaine phosphonate transition state analog hapten. Two classes of antibodies emerged based on sequence homology and mode of action. Alanine scanning mutagenesis and kinetic analysis revealed that residues H97, H99, and L96 are crucial for antibodies 3F5 and 3H9 to accelerate the hydrolysis of cocaine. Antibodies 3F1 through 3F4, which are similar to our previously identified 3A6 class of antibodies, catalyze hydrolysis through transition state stabilization by tyrosine or histidine residues H50 and L94. Mutation of either one or both tyrosine residues to histidine conferred hydrolytic activity on previously inactive antibody 3F4. Mutational analysis of residue H50 of antibody 3F3 resulted in a glutamine mutant with a rate enhancement three times greater than wild-type. A double mutant, containing glutamineH50 and lysineH52, showed a tenfold rate enhancement over wild-type. These results indicate the power of initial selection of catalytic antibodies from a biased antibody library in both rapid generation and screening of mutants for improved catalysis. [Copyright &y& Elsevier]

Peptidic ligands can be used for specific cell targeting and the delivery of payloads into the target cell. Here we describe the screening of a pool of cyclic peptide phage display libraries using whole-cell panning against human melanoma cell line Me6652/4. This strategy resulted in the selection of the cyclic 13-mer Pep42, CTVALPGGYVRVC, which showed preferential internalization into melanoma cell line Me6652/4 versus the reference cell line Me6652/56. This translocation is a receptor-mediated process that does not require electrostatic interactions nor does it involve transfer to the lysosomal compartment. The cellular receptor for Pep42 was identified as the surface membrane form of glucose-regulated protein 78 (GRP78), a member of the heat shock protein family and a marker on malignant cancer cells. The cellular uptake and intracellular trafficking of Pep42–Quantum Dot conjugates was monitored by confocal laser microscopy, and colocalization within the endoplasmic reticulum was observed. The uptake of Pep42 could be blocked by a monoclonal antibody against the identified receptor. Furthermore, Pep42 was shown to target specifically GRP78-expressing cancer cells. The in vitro cytotoxicity of a Pep42–Taxol conjugate was evaluated by flow cytometry wherein the conjugate was shown to induce apoptosis and was more effective in promoting programmed cell death in Me6652/4 cells. In summary, the data presented suggest that cyclic peptide Pep42 might be a powerful tool in the construction of drug conjugates designed to selectively kill malignant cancer cells. [ABSTRACT FROM AUTHOR]

Summary: Antibody 7A1 hydrolyzes cocaine to produce nonpsychoactive metabolites ecgonine methyl ester and benzoic acid. Crystal structures of 7A1 Fab′ and six complexes with substrate cocaine, the transition state analog, products ecgonine methyl ester and benzoic acid together and individually, as well as heptaethylene glycol have been analyzed at 1.5–2.3 Å resolution. Here, we present snapshots of the complete cycle of the cocaine hydrolytic reaction at atomic resolution. Significant structural rearrangements occur along the reaction pathway, but they are generally limited to the binding site, including the ligands themselves. Several interacting side chains either change their rotamers or alter their mobility to accommodate the different reaction steps. CDR loop movements (up to 2.3 Å) and substantial side chain rearrangements (up to 9 Å) alter the shape and size (∼320–500 Å3) of the antibody active site from “open” to “closed” to “open” for the substrate, transition state, and product states, respectively. [Copyright &y& Elsevier]

Overexpression of the cell-surface glycosphingolipid G[SUBM3] is associated with a number of different cancers, including those of the skin, colon, breast, and lung. Antibodies against the G[SUBM3] epitope have potential application as therapeutic agents in the treatment of these cancers. We describe the chemoenzymatic synthesis of two G[SUBM3]-derived reagents and their use in the panning of a phage-displayed human single-chain Fv (scFv) antibody library derived from the blood of cancer patients. Three scFv-phage clones, GM3A6, GM3AS, and GM3A15, were selected for recombinant expression and were characterized using BIAcore and flow cytometry. BIAcore measurements using the purified, soluble scFvs yielded dissociation constants (K[SUBd]) ranging from 4.2 × 10[SUP-7] to 2.1× 1[SUP-5] M. Flow cytometry was used to evaluate the ability of each scFv to discriminate between normal human cells (human dermal fibroblast, HDFa), melanoma cells (HMV-1, M21, and C-8161), and breast cancer cells (BCM-1, BCM-2, and BMS). GM3A6 displayed cross-reactivity with normal cells, as well as tumor cells, and GM3A15 possessed little or no binding activity toward any of the cell lines tested. However, GM3A8 bound to five of the six tumor cell lines and showed no measurable reactivity against the HDFa cells. Hence, we have demonstrated that a synthetic G[SUBM3] panning reagent can be used to isolate a fully human scFv that is highly specific for native G[SUBM3] on the surface of tumor cells. The result is a significant step toward effective immunotherapies for the treatment of cancer. [ABSTRACT FROM AUTHOR]

As a guide for chemical probe design, focused analogue synthetic studies were undertaken upon the lactone ring of 3-oxo-C12-homoserine lactone. We have concluded that hydrolytic instability of the heterocyclic ring is pivotal for its ability to modulate immune signaling and probe preparation was aligned with these findings. [ABSTRACT FROM AUTHOR]

Bacteria have developed cell-to-cell communication mechanisms, termed quorum sensing (QS), that regulate bacterial gene expression in a cell population-dependent manner. Autoinducer-2 (AI-2), a class of QS signaling molecules derived from (4S)-4,5-dihydroxy-2,3-pentanedione (DPD), has been identified in both Gram-negative and Gram-positive bacteria. Despite considerable interest in the AI-2 QS system, the biomolecular communication used by distinct bacterial species still remains shrouded. Herein, we report the synthesis and evaluation of a new class of DPD analogues, C4-alkoxy-5-hydroxy-2,3-pentanediones, termed C4-alkoxy-HPDs. Remarkably, two of the analogues were more potent QS agonists than the natural ligand, DPD, in Vibrio harveyi. The findings presented extend insights into ligand-receptor recognition/signaling in the AI-2 mediated QS system. [ABSTRACT FROM AUTHOR]

Abstract: Simultaneous activation of signaling pathways requires dynamic assembly of higher-order protein complexes at the cytoplasmic domains of membrane-associated receptors in a stimulus-specific manner. Here, using the paradigm of cellular activation through cytokine and innate immune receptors, we demonstrate the proof-of-principle application of small molecule probes for the dissection of receptor-proximal signaling processes, such as activation of the transcription factor NF-κB and the protein kinase p38. [Copyright &y& Elsevier]