Anti-tumor immunotherapy via antigen delivery from a live attenuated genetically engineered Pseudomonas aeruginosa type III secretion system-based vector.

Immunotherapy requiring an efficient T lymphocyte response is initiated by antigen delivery to antigen-presenting cells. Several studies have assessed the efficiency of various antigen loading procedures, including microbial vectors. Here a live strain of Pseudomonas aeruginosa was engineered to translocate a recombinant antigenic protein into mammalian cells via the type III secretion system, a bacterial device translocating effector proteins into host cells. Optimization of the vector included virulence attenuation and determination of the N-terminal sequence allowing translocation of fused antigens into cells. In vitro delivery of an ovalbumin fragment by the bacterial vector into dendritic cells induced the activation of ovalbumin-specific CD8(+) T lymphocytes. Mice injected with the ovalbumin-delivering vector developed ovalbumin-specific CD8(+) T lymphocytes and were resistant to a subsequent challenge with an ovalbumin-expressing melanoma. Moreover, in a curative assay, injection of the vaccine vector 5 and 12 days after tumor implantation led to a complete cure in five of six animals. These results highlight the utility of type III secretion system-based vectors for anti-tumor immunotherapy.     

Vaccine Protection against Bacillus cereus-Mediated Respiratory Anthrax-Like Disease in Mice

Bacillus cereus strains harboring a pXO1-like virulence plasmid cause respiratory anthrax-like disease in humans, particularly in welders. We developed mouse models for intraperitoneal as well as aerosol challenge with spores of B. cereus G9241, harboring pBCXO1 and pBC218 virulence plasmids. Compared to wild-type B. cereus G9241, spores with a deletion of the pBCXO1-carried protective antigen gene (pagA1) were severely attenuated, whereas spores with a deletion of the pBC218-carried protective antigen homologue (pagA2) were not. Anthrax vaccine adsorbed (AVA) immunization raised antibodies that bound and neutralized the pagA1-encoded protective antigen (PA1) but not the PA2 orthologue encoded by pagA2. AVA immunization protected mice against a lethal challenge with spores from B. cereus G9241 or B. cereus Elc4, a strain that had been isolated from a fatal case of anthrax-like disease. As the pathogenesis of B. cereus anthrax-like disease in mice is dependent on pagA1 and PA-neutralizing antibodies provide protection, AVA immunization may also protect humans from respiratory anthrax-like death.     

Hereditary hemochromatosis restores the virulence of plague vaccine strains

Nonpigmented Yersinia pestis (pgm) strains are defective in scavenging host iron and have been used in live-attenuated vaccines to combat plague epidemics. Recently, a Y. pestis pgm strain was isolated from a researcher with hereditary hemochromatosis who died from laboratory-acquired plague. We used hemojuvelin-knockout (Hjv(-/-)) mice to examine whether iron-storage disease restores the virulence defects of nonpigmented Y. pestis. Unlike wild-type mice, Hjv(-/-) mice developed lethal plague when challenged with Y. pestis pgm strains. Immunization of Hjv(-/-) mice with a subunit vaccine that blocks Y. pestis type III secretion generated protection against plague. Thus, individuals with hereditary hemochromatosis may be protected with subunit vaccines but should not be exposed to live-attenuated plague vaccines.     

Collateral sensitivity of resistant MRP1-overexpressing cells to flavonoids and derivatives through GSH efflux

The multidrug resistance protein 1 (MRP1) is involved in multidrug resistance of cancer cells by mediating drug efflux out of cells, often in co-transport with glutathione (GSH). GSH efflux mediated by MRP1 can be stimulated by verapamil. In cells overexpressing MRP1, we have previously shown that verapamil induced a huge intracellular GSH depletion which triggered apoptosis of the cells. That phenomenon takes place in the more global anticancer strategy called “collateral sensitivity” and could be exploited to eradicate some chemoresistant cancer cells. Seeking alternative compounds to verapamil, we screened a library of natural flavonoids and synthetic derivatives. A large number of these compounds stimulate MRP1-mediated GSH efflux and the most active ones have been evaluated for their cytotoxic effect on MRP1-overexpressing cells versus parental cells. Interestingly, some are highly and selectively cytotoxic for MRP1-cells, leading them to apoptosis. However, some others do not exhibit any cytotoxicity while promoting a strong GSH efflux, indicating that GSH efflux is necessary but not sufficient for MRP1-cells apoptosis. In support to this hypothesis, structure activity relationships show that the absence of a hydroxyl group at position 3 of the flavonoid C ring is an absolute requirement for induction of MRP1-cells death, but is not for GSH efflux stimulation. Chrysin (compound 8) and its derivatives, compounds 11 and 22, exhibit a high selectivity toward MRP1-cells with a IC₅₀ value of 4.1 μM for compound 11 and 4.9 μM for chrysin and compound 22, making them among the best described selective killer compounds of multidrug ABC transporter-overexpressing cells.     

Motivation to Consent and Adhere to the FORT Randomized Controlled Trial

The aim of this qualitative study was to identify the motivational factors that influence cancer survivors to participate and adhere to the fear of cancer recurrence (FCR) FORT randomized controlled trial (RCT). Fifteen women diagnosed with breast and gynecological cancer who took part in the FORT RCT were interviewed about their experience to consent and adhere to the trial. The transcribed interviews were content analyzed within a relational autonomy framework. The analysis revealed that the participants’ motivation to consent and adhere to the FORT RCT was structured around thirteen subthemes grouped into four overarching themes: (1) Personal Influential Factors; (2) Societal Motivations; (3) Structural Influences; and (4) Gains in Emotional Support. The unique structures of the trial such as the group format, the friendships formed with other participants in their group and with the group leaders, and the right timing of the trial within their cancer survivorship trajectory all contributed to their motivation to consent and adhere to the FORT RCT. While their initial motivation to participate was mostly altruistic, it was their personal gains obtained over the course of the trial that contributed to their adherence. Potential gains in emotional and social support from psycho-oncology trials should be capitalized when approaching future participants as a mean to improve on motivations to consent and adhere.     

Structure of the mature kinetoplastids mitoribosome and insights into its large subunit biogenesis

Kinetoplastids are unicellular eukaryotic parasites responsible for such human pathologies as Chagas disease, sleeping sickness, and leishmaniasis. They have a single large mitochondrion, essential for the parasite survival. In kinetoplastid mitochondria, most of the molecular machineries and gene expression processes have significantly diverged and specialized, with an extreme example being their mitochondrial ribosomes. These large complexes are in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Structural studies performed in Trypanosoma brucei already highlighted the numerous peculiarities of these mitoribosomes and the maturation of their small subunit. However, several important aspects mainly related to the large subunit (LSU) remain elusive, such as the structure and maturation of its ribosomal RNA. Here we present a cryo-electron microscopy study of the protozoans Leishmania tarentolae and Trypanosoma cruzi mitoribosomes. For both species, we obtained the structure of their mature mitoribosomes, complete rRNA of the LSU, as well as previously unidentified ribosomal proteins. In addition, we introduce the structure of an LSU assembly intermediate in the presence of 16 identified maturation factors. These maturation factors act on both the intersubunit and the solvent sides of the LSU, where they refold and chemically modify the rRNA and prevent early translation before full maturation of the LSU.

Kinetoplastids are unicellular eukaryotic parasites, causative agents of several human and livestock pathologies (1). They are potentially lethal, affecting more than 20 million people worldwide (1). Owing in part to their parasitic nature, they strongly diverged from other eukaryotic model species. Kinetoplastids evolved to live in and infect a large variety of eukaryotic organisms in very different molecular environments. Consequently, beyond the general similarities, kinetoplastid species have diverged evolutionarily from one another, and their protein sequence identity can be relatively low. They have a single large mitochondrion, a crucial component of their cellular architecture (2), where gene expression machineries have also largely diverged, notably their mitochondrial ribosomes (mitoribosomes) (3–10). These sophisticated RNA-protein complexes translate the few mRNAs still encoded by mitochondrial genomes. The mitoribosomes’ composition and structure diverged greatly from their bacterial ancestor, with the kinetoplastid mitoribosomes the most extreme case described to date, with highly reduced rRNAs and more than 80 supernumerary ribosomal proteins (r-proteins) compared with bacteria, completely reshaping the overall ribosome structure.Recent structural studies performed in Trypanosoma brucei have highlighted the particularities of this mitoribosome structure and composition, as well as the assembly processes of the small subunit (SSU) (3, 6). However, despite the very comprehensive structural characterization of the full T. brucei SSU and its maturation, several pivotal aspects related to the large subunit (LSU) have remained uncharacterized. For instance, a large portion of the LSU at the intersubunit side, including the whole rRNA peptidyl-transfer center (PTC) along with several r-proteins, were unresolved (3). Moreover, in contrast to the SSU, nearly nothing is known about LSU maturation and assembly. More generally, the maturation of the mitoribosomes in all eukaryotic species remains largely underexplored. Here we present a cryo-electron microscopy (EM) investigation of the full mature mitoribosomes from two different kinetoplastids, Leishmania tarentolae and Trypanosoma cruzi. In addition, we reveal the structure of an assembly intermediate of the LSU displaying unprecedented details on rRNA maturation in these very singular mitoribosomes, some of which likely can be generalized to the maturation of all rRNA.To obtain high-resolution reconstructions of the full kinetoplastid mitoribosomes, we purified mitochondrial vesicles from both L. tarentolae and T. cruzi and directly purified the mitoribosomes from the sucrose gradient (Methods) (SI Appendix, Fig. S1). All of our collected fractions were analyzed by nano-liquid chromatography tandem mass spectrometry (LC-MS/MS) (SI Appendix, Tables S1 and S2) to determine their proteomic composition. We collected micrographs from multiple vitrified samples corresponding to different sucrose gradient density peaks for both species, and following image processing, we obtained cryo-EM reconstructions of the full mitoribosomes, as well as of the dissociated SSU. Moreover, we also derived reconstructions of what appeared to be an assembly intermediate of L. tarentolae LSU. After extensive rounds of two-dimensional (2D) and three-dimensional (3D) classification and refinement we obtained the structure of L. tarentolae and T. cruzi complete and mature mitoribosomes at 3.9 Å and 6 Å, respectively (SI Appendix, Figs. S2–S4). Other notable features include the intersubunit contacts and two distinct rotational states in T. cruzi. Similarly to T. brucei (3), our cryo-EM analysis revealed a reconstruction of an early initiation complex from T. cruzi at 3.1 Å for the body and 3.2 Å for the head of the SSU (SI Appendix, Fig. S5). Further focused refinement on the LSU, SSU head, and SSU body generated reconstructions at 3.6, 3.8, and 4 Å, respectively, for L. tarentolae (SI Appendix, Figs. S2 and S3), and 3.7 and 4.5 Å for T. cruzi LSU and SSU, respectively (SI Appendix, Fig. S4). Combined, these reconstructions, along with the MS data allowed us to build nearly complete atomic models, with only few protein densities remaining unidentified.