A tripartite cytolytic toxin formed by Vibrio cholerae proteins with flagellum-facilitated secretion.

The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD, and MakE. MakA, MakB, and MakE were readily detected in culture supernatants of wild-type V. cholerae, whereas secretion was very much reduced from a flagellum-deficient mutant. Crystal structures of MakA, MakB, and MakE revealed a structural relationship to a superfamily of bacterial pore-forming toxins. Expression of MakA/B/E in Escherichia coli resulted in toxicity toward Caenorhabditis elegans used as a predatory model organism. None of these Mak proteins alone or in pairwise combinations were cytolytic, but an equimolar mixture of MakA, MakB, and MakE acted as a tripartite cytolytic toxin in vitro, causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining the assembly of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity toward C. elegans Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen Vibrio anguillarum We suggest that the hitherto-unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms.

Vibrio cholerae cytotoxin MakA induces noncanonical autophagy resulting in the spatial inhibition of canonical autophagy.

Autophagy plays an essential role in the defense against many microbial pathogens as a regulator of both innate and adaptive immunity. Some pathogens have evolved sophisticated mechanisms that promote their ability to evade or subvert host autophagy. Here, we describe a novel mechanism of autophagy modulation mediated by the recently discovered Vibrio cholerae cytotoxin, motility-associated killing factor A (MakA). pH-dependent endocytosis of MakA by host cells resulted in the formation of a cholesterol-rich endolysosomal membrane aggregate in the perinuclear region. Aggregate formation induced the noncanonical autophagy pathway driving unconventional LC3 (herein referring to MAP1LC3B) lipidation on endolysosomal membranes. Subsequent sequestration of the ATG12-ATG5-ATG16L1 E3-like enzyme complex, required for LC3 lipidation at the membranous aggregate, resulted in an inhibition of both canonical autophagy and autophagy-related processes, including the unconventional secretion of interleukin-1ß (IL-1ß). These findings identify a novel mechanism of host autophagy modulation and immune modulation employed by V. cholerae during bacterial infection.

Phosphatidic acid-mediated binding and mammalian cell internalization of the Vibrio cholerae cytotoxin MakA.

Vibrio cholerae is a noninvasive intestinal pathogen extensively studied as the causative agent of the human disease cholera. Our recent work identified MakA as a potent virulence factor of V. cholerae in both Caenorhabditis elegans and zebrafish, prompting us to investigate the potential contribution of MakA to pathogenesis also in mammalian hosts. In this study, we demonstrate that the MakA protein could induce autophagy and cytotoxicity of target cells. In addition, we observed that phosphatidic acid (PA)-mediated MakA-binding to the host cell plasma membranes promoted macropinocytosis resulting in the formation of an endomembrane-rich aggregate and vacuolation in intoxicated cells that lead to induction of autophagy and dysfunction of intracellular organelles. Moreover, we functionally characterized the molecular basis of the MakA interaction with PA and identified that the N-terminal domain of MakA is required for its binding to PA and thereby for cell toxicity. Furthermore, we observed that the ΔmakA mutant outcompeted the wild-type V. cholerae strain A1552 in the adult mouse infection model. Based on the findings revealing mechanistic insights into the dynamic process of MakA-induced autophagy and cytotoxicity we discuss the potential role played by the MakA protein during late stages of cholera infection as an anti-colonization factor.

Suppression of ß-catenin signaling in colon carcinoma cells by a bacterial protein.

Colorectal cancer is one of the leading causes of cancer-related death worldwide. The adenomatous polyposis coli (APC) gene is mutated in hereditary colorectal tumors and in more than 80% of sporadic colorectal tumors. APC mutations impair ß-catenin degradation, leading to its permanent stabilization and increased transcription of cancer-driving target genes. In colon cancer, impairment of ß-catenin degradation leads to its cytoplasmic accumulation, nuclear translocation, and subsequent activation of tumor cell proliferation. Suppressing ß-catenin signaling in cancer cells therefore appears to be a promising strategy for new anticancer strategies. Recently, we discovered a novel Vibrio cholerae cytotoxin, motility-associated killing factor A (MakA), that affects both invertebrate and vertebrate hosts. It promotes bacterial survival and proliferation in invertebrate predators but has unknown biological role(s) in mammalian hosts. Here, we report that MakA can cause lethality of tumor cells via induction of apoptosis. Interestingly, MakA exhibited potent cytotoxic activity, in particular against several tested cancer cell lines, while appearing less toxic toward nontransformed cells. MakA bound to the tumor cell surface became internalized into the endolysosomal compartment and induced leakage of endolysosomal membranes, causing cytosolic release of cathepsins and activation of proapoptotic proteins. In addition, MakA altered ß-catenin integrity in colon cancer cells, partly through a caspase- and proteasome-dependent mechanism. Importantly, MakA inhibited ß-catenin-mediated tumor cell proliferation. Remarkably, intratumor injection of MakA significantly reduced tumor development in a colon cancer murine solid tumor model. These data identify MakA as a novel candidate to be considered in new strategies for development of therapeutic agents against colon cancer.

Molecular Basis of Acute Cystitis Reveals Susceptibility Genes and Immunotherapeutic Targets.

Tissue damage is usually regarded as a necessary price to pay for successful elimination of pathogens by the innate immune defense. Yet, it is possible to distinguish protective from destructive effects of innate immune activation and selectively attenuate molecular nodes that create pathology. Here, we identify acute cystitis as an Interleukin-1 beta (IL-1ß)-driven, hyper-inflammatory condition of the infected urinary bladder and IL-1 receptor blockade as a novel therapeutic strategy. Disease severity was controlled by the mechanism of IL-1ß processing and mice with intact inflammasome function developed a moderate, self-limiting form of cystitis. The most severe form of acute cystitis was detected in mice lacking the inflammasome constituents ASC or NLRP-3. IL-1ß processing was hyperactive in these mice, due to a new, non-canonical mechanism involving the matrix metalloproteinase 7- (MMP-7). ASC and NLRP-3 served as transcriptional repressors of MMP7 and as a result, Mmp7 was markedly overexpressed in the bladder epithelium of Asc-/- and Nlrp3-/- mice. The resulting IL-1ß hyper-activation loop included a large number of IL-1ß-dependent pro-inflammatory genes and the IL-1 receptor antagonist Anakinra inhibited their expression and rescued susceptible Asc-/- mice from bladder pathology. An MMP inhibitor had a similar therapeutic effect. Finally, elevated levels of IL-1ß and MMP-7 were detected in patients with acute cystitis, suggesting a potential role as biomarkers and immunotherapeutic targets. The results reproduce important aspects of human acute cystitis in the murine model and provide a comprehensive molecular framework for the pathogenesis and immunotherapy of acute cystitis, one of the most common infections in man. TRIAL REGISTRATION: The clinical studies were approved by the Human Ethics Committee at Lund University (approval numbers LU106-02, LU236-99 and Clinical Trial Registration RTP-A2003, International Committee of Medical Journal Editors, www.clinicaltrials.gov).

HAMLET - A protein-lipid complex with broad tumoricidal activity.

HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is a tumoricidal protein-lipid complex with broad effects against cancer cells of different origin. The therapeutic potential is emphasized by a high degree of specificity for tumor tissue. Here we review early studies of HAMLET, in collaboration with the Orrenius laboratory, and some key features of the subsequent development of the HAMLET project. The early studies focused on the apoptotic response that accompanies death in HAMLET treated tumor cells and the role of mitochondria in this process. In subsequent studies, we have identified a sequence of interactions that starts with the membrane integration of HAMLET and the activation of ion fluxes followed by HAMLET internalization, progressive inhibition of MAPK kinases and GTPases and sorting of HAMLET to different cellular compartments, including the nuclei. Therapeutic efficacy of HAMLET has been demonstrated in animal models of glioblastoma, bladder cancer and intestinal cancer. In clinical studies, HAMLET has been shown to target skin papillomas and bladder cancers. The findings identify HAMLET as a new drug candidate with promising selectivity for cancer cells and a strong therapeutic potential.

Prevention and treatment of colon cancer by peroral administration of HAMLET (human α-lactalbumin made lethal to tumour cells).

BACKGROUND: Most colon cancers start with dysregulated Wnt/ß-catenin signalling and remain a major therapeutic challenge. Examining whether HAMLET (human α-lactalbumin made lethal to tumour cells) may be used for colon cancer treatment is logical, based on the properties of the complex and its biological context. OBJECTIVE: To investigate if HAMLET can be used for colon cancer treatment and prevention. Apc(Min)(/+) mice, which carry mutations relevant to hereditary and sporadic human colorectal tumours, were used as a model for human disease. METHOD: HAMLET was given perorally in therapeutic and prophylactic regimens. Tumour burden and animal survival of HAMLET-treated and sham-fed mice were compared. Tissue analysis focused on Wnt/ß-catenin signalling, proliferation markers and gene expression, using microarrays, immunoblotting, immunohistochemistry and ELISA. Confocal microscopy, reporter assay, immunoprecipitation, immunoblotting, ion flux assays and holographic imaging were used to determine effects on colon cancer cells. RESULTS: Peroral HAMLET administration reduced tumour progression and mortality in Apc(Min)(/+) mice. HAMLET accumulated specifically in tumour tissue, reduced ß-catenin and related tumour markers. Gene expression analysis detected inhibition of Wnt signalling and a shift to a more differentiated phenotype. In colon cancer cells with APC mutations, HAMLET altered ß-catenin integrity and localisation through an ion channel-dependent pathway, defining a new mechanism for controlling ß-catenin signalling. Remarkably, supplying HAMLET to the drinking water from the time of weaning also significantly prevented tumour development. CONCLUSIONS: These data identify HAMLET as a new, peroral agent for colon cancer prevention and treatment, especially needed in people carrying APC mutations, where colon cancer remains a leading cause of death.

Colony phase variation switch modulates antimicrobial tolerance and biofilm formation in Acinetobacter baumannii.

Carbapenem-resistant Acinetobacter baumannii causes one of the most difficult-to-treat nosocomial infections. Polycationic drugs like polymyxin B or colistin and tetracycline drugs such as doxycycline or minocycline are commonly used to treat infections caused by carbapenem-resistant A. baumannii. Here, we show that a subpopulation of cells associated with the opaque/translucent colony phase variation by A. baumannii AB5075 displays differential tolerance to subinhibitory concentrations of colistin and tetracycline. Using a variety of microscopic techniques, we demonstrate that extracellular polysaccharide moieties mediate colistin tolerance to opaque A. baumannii at single-cell level and that mushroom-shaped biofilm structures protect opaque bacteria at the community level. The colony switch phenotype is found to alter several traits of A. baumannii, including long-term survival under desiccation, tolerance to ethanol, competition with Escherichia coli, and intracellular survival in the environmental model host Acanthamoeba castellanii. Additionally, our findings suggest that extracellular DNA associated with membrane vesicles can promote colony switching in a DNA recombinase-dependent manner.IMPORTANCEAs a WHO top-priority drug-resistant microbe, Acinetobacter baumannii significantly contributes to hospital-associated infections worldwide. One particularly intriguing aspect is its ability to reversibly switch its colony morphotype on agar plates, which has been remarkably underexplored. In this study, we employed various microscopic techniques and phenotypic assays to investigate the colony phase variation switch under different clinically and environmentally relevant conditions. Our findings reveal that the presence of a poly N-acetylglucosamine-positive extracellular matrix layer contributes to the protection of bacteria from the bactericidal effects of colistin. Furthermore, we provide intriguing insights into the multicellular lifestyle of A. baumannii, specifically in the context of colony switch variation within its predatory host, Acanthamoeba castellanii.

Macrophage innate immune responses delineate between defective translocon assemblies produced by Yersinia pseudotuberculosis YopD mutants.

Translocon pores formed in the eukaryotic cell membrane by a type III secretion system facilitate the translocation of immune-modulatory effector proteins into the host cell interior. The YopB and YopD proteins produced and secreted by pathogenic Yersinia spp. harboring a virulence plasmid-encoded type III secretion system perform this pore-forming translocator function. We had previously characterized in vitro T3SS function and in vivo pathogenicity of a number of strains encoding sited-directed point mutations in yopD. This resulted in the classification of mutants into three different classes based upon the severity of the phenotypic defects. To investigate the molecular and functional basis for these defects, we explored the effectiveness of RAW 264.7 cell line to respond to infection by representative YopD mutants of all three classes. Signature cytokine profiles could separate the different YopD mutants into distinct categories. The activation and suppression of certain cytokines that function as central innate immune response modulators correlated well with the ability of mutant bacteria to alter anti-phagocytosis and programmed cell death pathways. These analyses demonstrated that sub-optimal translocon pores impact the extent and magnitude of host cell responsiveness, and this limits the capacity of pathogenic Yersinia spp. to fortify against attack by both early and late arms of the host innate immune response.

Csu pili dependent biofilm formation and virulence of Acinetobacter baumannii.

Acinetobacter baumannii has emerged as one of the most common extensive drug-resistant nosocomial bacterial pathogens. Not only can the bacteria survive in hospital settings for long periods, but they are also able to resist adverse conditions. However, underlying regulatory mechanisms that allow A. baumannii to cope with these conditions and mediate its virulence are poorly understood. Here, we show that bi-stable expression of the Csu pili, along with the production of poly-N-acetyl glucosamine, regulates the formation of Mountain-like biofilm-patches on glass surfaces to protect bacteria from the bactericidal effect of colistin. Csu pilus assembly is found to be an essential component of mature biofilms formed on glass surfaces and of pellicles. By using several microscopic techniques, we show that clinical isolates of A. baumannii carrying abundant Csu pili mediate adherence to epithelial cells. In addition, Csu pili suppressed surface-associated motility but enhanced colonization of bacteria into the lungs, spleen, and liver in a mouse model of systemic infection. The screening of c-di-GMP metabolizing protein mutants of A. baumannii 17978 for the capability to adhere to epithelial cells led us to identify GGDEF/EAL protein AIS_2337, here denoted PdeB, as a major regulator of Csu pili-mediated virulence and biofilm formation. Moreover, PdeB was found to be involved in the type IV pili-regulated robustness of surface-associated motility. Our findings suggest that the Csu pilus is not only a functional component of mature A. baumannii biofilms but also a major virulence factor promoting the initiation of disease progression by mediating bacterial adherence to epithelial cells.

HAMLET: functional properties and therapeutic potential.

Human α-lactalbumin made lethal to tumor cells (HAMLET) is the first member in a new family of protein-lipid complexes that kills tumor cells with high selectivity. The protein component of HAMLET is α-lactalbumin, which in its native state acts as a substrate specifier in the lactose synthase complex, thereby defining a function essential for the survival of lactating mammals. In addition, α-lactalbumin acquires tumoricidal activity after partial unfolding and binding to oleic acid. The lipid cofactor serves the dual role as a stabilizer of the altered fold of the protein and a coactivator of specific steps in tumor cell death. HAMLET is broadly tumoricidal, suggesting that the complex identifies conserved death pathways suitable for targeting by novel therapies. Sensitivity to HAMLET is defined by oncogene expression including Ras and c-Myc and by glycolytic enzymes. Cellular targets are located in the cytoplasmic membrane, cytoskeleton, mitochondria, proteasomes, lysosomes and nuclei, and specific signaling pathways are rapidly activated, first by interactions of HAMLET with the cell membrane and subsequently after HAMLET internalization. Therapeutic effects of HAMLET have been demonstrated in human skin papillomas and bladder cancers, and HAMLET limits the progression of human glioblastomas, with no evidence of toxicity for normal brain or bladder tissue. These findings open up new avenues for cancer therapy and the understanding of conserved death responses in tumor cells.

Bacterial protein MakA causes suppression of tumour cell proliferation via inhibition of PIP5K1α/Akt signalling.

Recently, we demonstrated that a novel bacterial cytotoxin, the protein MakA which is released by Vibrio cholerae, is a virulence factor, causing killing of Caenorhabditis elegans when the worms are grazing on the bacteria. Studies with mammalian cell cultures in vitro indicated that MakA could affect eukaryotic cell signalling pathways involved in lipid biosynthesis. MakA treatment of colon cancer cells in vitro caused inhibition of growth and loss of cell viability. These findings prompted us to investigate possible signalling pathways that could be targets of the MakA-mediated inhibition of tumour cell proliferation. Initial in vivo studies with MakA producing V. cholerae and C. elegans suggested that the MakA protein might target the PIP5K1α phospholipid-signalling pathway in the worms. Intriguingly, MakA was then found to inhibit the PIP5K1α lipid-signalling pathway in cancer cells, resulting in a decrease in PIP5K1α and pAkt expression. Further analyses revealed that MakA inhibited cyclin-dependent kinase 1 (CDK1) and induced p27 expression, resulting in G2/M cell cycle arrest. Moreover, MakA induced downregulation of Ki67 and cyclin D1, which led to inhibition of cell proliferation. This is the first report about a bacterial protein that may target signalling involving the cancer cell lipid modulator PIP5K1α in colon cancer cells, implying an anti-cancer effect.

Differential Internalization of Thrombin-Derived Host Defense Peptides into Monocytes and Macrophages.

Proteolytic cleavage of thrombin generates C-terminal host defense peptides exerting multiple immunomodulatory effects in response to bacterial stimuli. Previously, we reported that thrombin-derived C-terminal peptides (TCPs) are internalized in monocytes and macrophages in a time- and temperature-dependent manner. In this study, we investigated which endocytosis pathways are responsible for the internalization of TCPs. Using confocal microscopy and flow cytometry, we show that both clathrin-dependent and clathrin-independent pathways are involved in the internalization of the prototypic TCP GKY25 in RAW264.7 and human monocyte-derived M1 macrophages, whereas the uptake of GKY25 in monocytic THP-1 cells is mainly dynamin-dependent. Internalized GKY25 was transported to endosomes and finally lysosomes, where it remained detectable for up to 10 h. Comparison of GKY25 uptake with that of the natural occurring TCPs HVF18 and FYT21 indicates that the pathway of TCP endocytosis is not only cell type-dependent but also depends on the length and composition of the peptide as well as the presence of LPS and bacteria. Finally, using neutron reflectometry, we show that the observed differences between HVF18 and the other 2 TCPs may be explained partially by differences in membrane insertion. Taken together, we show that TCPs are differentially internalized into monocytes and macrophages.

Bone mineral: A trojan horse for bone cancers. Efficient mitochondria targeted delivery and tumor eradication with nano hydroxyapatite containing doxorubicin.

Efficient systemic pharmacological treatment of solid tumors is hampered by inadequate tumor concentration of cytostatics necessitating development of smart local drug delivery systems. To overcome this, we demonstrate that doxorubicin (DOX), a cornerstone drug used for osteosarcoma treatment, shows reversible accretion to hydroxyapatite (HA) of both nano (nHA) and micro (mHA) size. nHA particles functionalized with DOX get engulfed in the lysosome of osteosarcoma cells where the acidic microenvironment causes a disruption of the binding between DOX and HA. The released DOX then accumulates in the mitochondria causing cell starvation, reduced migration and apoptosis. The HA+DOX delivery system was also tested in-vivo on osteosarcoma bearing mice. Locally delivered DOX via the HA particles had a stronger tumor eradication effect compared to the controls as seen by PET-CT and immunohistochemical staining of proliferation and apoptosis markers. These results indicate that in addition to systemic chemotherapy, an adjuvant nHA could be used as a carrier for intracellular delivery of DOX for prevention of tumor recurrence after surgical resection in an osteosarcoma. Furthermore, we demonstrate that nHA particles are pivotal in this approach but a combination of nHA with mHA could increase the safety associated with particulate nanomaterials while maintaining similar therapeutic potential.

Protein-lipid interaction at low pH induces oligomerization of the MakA cytotoxin from Vibrio cholerae.

The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae. As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.

Eco-evolutionary feedbacks mediated by bacterial membrane vesicles.

Bacterial membrane vesicles (BMVs) are spherical extracellular organelles whose cargo is enclosed by a biological membrane. The cargo can be delivered to distant parts of a given habitat in a protected and concentrated manner. This review presents current knowledge about BMVs in the context of bacterial eco-evolutionary dynamics among different environments and hosts. BMVs may play an important role in establishing and stabilizing bacterial communities in such environments; for example, bacterial populations may benefit from BMVs to delay the negative effect of certain evolutionary trade-offs that can result in deleterious phenotypes. BMVs can also perform ecosystem engineering by serving as detergents, mediators in biochemical cycles, components of different biofilms, substrates for cross-feeding, defense systems against different dangers and enzyme-delivery mechanisms that can change substrate availability. BMVs further contribute to bacteria as mediators in different interactions, with either other bacterial species or their hosts. In short, BMVs extend and deliver phenotypic traits that can have ecological and evolutionary value to both their producers and the ecosystem as a whole.

Ecotin and LamB in Escherichia coli influence the susceptibility to Type VI secretion-mediated interbacterial competition and killing by Vibrio cholerae.

BACKGROUND: A prevailing action of the Type VI secretion system (T6SS) in several Gram-negative bacterial species is inter-bacterial competition. In the past several years, many effectors of T6SS were identified in different bacterial species and their involvement in inter-bacterial interactions were described. However, possible defence mechanisms against T6SS attack among prey bacteria were not well clarified yet. METHODS: Escherichia coli was assessed for susceptibility to T6SS-mediated killing by Vibrio cholerae. TheT6SS-mediated bacterial killing assays were performed in absence or presence of different protease inhibitors and with different mutant E. coli strains. Expression levels of selected proteins were monitored using SDS-PAGE and immunoblot analyses. RESULTS: The T6SS-mediated killing of E. coli by V. cholerae was partly blocked when the serine protease inhibitor Pefabloc was present. E. coli lacking the periplasmic protease inhibitor Ecotin showed enhanced susceptibility to killing by V. cholerae. Mutations affecting E. coli membrane stability also caused increased susceptibility to killing by V. cholerae. E. coli lacking the maltodextrin porin protein LamB showed reduced susceptibility to killing by V. cholerae whereas E. coli with induced high levels of LamB showed reduced survival in inter-bacterial competition. CONCLUSIONS: Our study identified two proteins in E. coli, the intrinsic protease inhibitor Ecotin and the outer membrane porin LamB, that influenced E. coli susceptibility to T6SS-mediated killing by V. cholerae. GENERAL SIGNIFICANCE: We envision that it is feasible to explore these findings to target and modulate their expression to obtain desired changes in inter-bacterial competition in vivo, e.g. in the gastrointestinal microbiome.

Bladder cancer therapy using a conformationally fluid tumoricidal peptide complex.

Partially unfolded alpha-lactalbumin forms the oleic acid complex HAMLET, with potent tumoricidal activity. Here we define a peptide-based molecular approach for targeting and killing tumor cells, and evidence of its clinical potential (ClinicalTrials.gov NCT03560479). A 39-residue alpha-helical peptide from alpha-lactalbumin is shown to gain lethality for tumor cells by forming oleic acid complexes (alpha1-oleate). Nuclear magnetic resonance measurements and computational simulations reveal a lipid core surrounded by conformationally fluid, alpha-helical peptide motifs. In a single center, placebo controlled, double blinded Phase I/II interventional clinical trial of non-muscle invasive bladder cancer, all primary end points of safety and efficacy of alpha1-oleate treatment are reached, as evaluated in an interim analysis. Intra-vesical instillations of alpha1-oleate triggers massive shedding of tumor cells and the tumor size is reduced but no drug-related side effects are detected (primary endpoints). Shed cells contain alpha1-oleate, treated tumors show evidence of apoptosis and the expression of cancer-related genes is inhibited (secondary endpoints). The results are especially encouraging for bladder cancer, where therapeutic failures and high recurrence rates create a great, unmet medical need.

Transcriptome Profiling of Staphylococcus aureus Associated Extracellular Vesicles Reveals Presence of Small RNA-Cargo.

Bacterial extracellular vesicles (EVs) have a vital role in bacterial pathogenesis. However, to date, the small RNA-cargo of EVs released by the opportunistic pathogen Staphylococcus aureus has not been characterized. Here, we shed light on the association of small RNAs with EVs secreted by S. aureus MSSA476 cultured in iron-depleted bacteriologic media supplemented with a subinhibitory dosage of vancomycin to mimic infection condition. Confocal microscopy analysis on intact RNase-treated EVs indicated that RNA is associated with EV particles. Transcriptomic followed by bioinformatics analysis of EV-associated RNA revealed the presence of potential gene regulatory small RNAs and high levels of tRNAs. Among the EV-associated enriched small RNAs were SsrA, RsaC and RNAIII. Our finding invites new insights into the potential role of EV-associated RNA as a modulator of host-pathogen interaction.

Analysis of colony phase variation switch in Acinetobacter baumannii clinical isolates.

Reversible switching between opaque and translucent colony formation is a novel feature of Acinetobacter baumannii that has been associated with variations in the cell morphology, surface motility, biofilm formation, antibiotic resistance and virulence. Here, we assessed a number of phenotypic alterations related to colony switching in A. baumannii clinical isolates belonging to different multi-locus sequence types. Our findings demonstrated that these phenotypic alterations were mostly strain-specific. In general, the translucent subpopulations of A. baumannii produced more dense biofilms, were more piliated, and released larger amounts of outer membrane vesicles (OMVs). In addition, the translucent subpopulations caused reduced fertility of Caenorhabditis elegans. When assessed for effects on the immune response in RAW 264.7 macrophages, the OMVs isolated from opaque subpopulations of A. baumannii appeared to be more immunogenic than the OMVs from the translucent form. However, also the OMVs from the translucent subpopulations had the potential to evoke an immune response. Therefore, we suggest that OMVs may be considered for development of new immunotherapeutic treatments against A. baumannii infections.