David N. Cook

Genes acrA and acrB encode a stress‐induced efflux system of Escherichia coli

Defined mutations of acrA or acrB (formerly acrE) genes increased the susceptibility of Escherichia coli to a range of small inhibitor molecules. Deletion of acrAB increased susceptibility to cephalothin and cephaloridine, but the permeability of these β‐lactams across the outer membrane was not increased. This finding is inconsistent with the earlier hypothesis that acrAB mutations increase drug susceptibility by increasing the permeability of the outer membrane, and supports our model that acrAB codes for a multi‐drug efflux pump. The natural environment of an enteric bacterium such as E. coli is enriched in bile salts and fatty acids. An acrAB deletion mutant was found to be hypersusceptible to bile salts and to decanoate. In addition, acrAB expression was elevated by growth in 5mM decanoate. These results suggest that one major physiological function of AcrAB is to protect E. coli against these and other hydrophobic inhibitors. Transcription of acrAB is increased by other stress conditions including 4% ethanol, 0.5 M NaCl, and stationary phase in Luria‐Bertani medium. Finally, acrAB expression was shown to be increased in mar (multiple‐antibiotic‐resistant) mutants.

Photochemical inactivation of viruses and bacteria in platelet concentrates by use of a novel psoralen and long-wavelength ultraviolet light

BACKGROUND: A photochemical treatment process has been developed for the inactivation of viruses and bacteria in platelet concentrates. This process is based on the photochemical reaction of a novel psoralen, S‐ 59, with nucleic acids upon illumination with long‐wavelength ultraviolet light (UVA, 320–400 nm). STUDY DESIGN AND METHODS: High levels of pathogens were added to single‐donor platelet concentrates containing 3 to 5 × 10(11) platelets in 300 mL of 35‐percent autologous plasma and 65‐percent platelet additive solution. After treatment with S‐59 (150 microM) and UVA (0–3 J/cm2), the infectivity of each pathogen was measured with established biologic assays. In vitro platelet function after photochemical treatment was evaluated during 7 days of storage by using a panel of 14 assays. The in vivo recovery and life span of photochemically treated platelets were evaluated after 24 hours of storage in a primate transfusion model. RESULTS: The following levels of pathogen inactivation were achieved:>10(6.7) plaque‐forming units (PFU) per mL of cell‐free human immunodeficiency virus (HIV),>10(6.6) PFU per mL of cell‐associated HIV,>10(6.8) infectious dose (ID50) per mL of duck hepatitis B virus (a model for hepatitis B virus),>10(6.5) PFU per mL of bovine viral diarrhea virus (a model for hepatitis C virus),>10(6.6) colony‐forming units of Staphylococcus epidermidis, and>10(5.6) colony‐forming units of Klebsiella pneumoniae. Expression of integrated HIV was inhibited by 0.1 microM S‐ 59 and 1 J per cm2 of UVA. In vitro and in vivo platelet function were adequately maintained after antiviral and antibacterial treatment. CONCLUSION: Photochemical treatment of platelet concentrates offers the potential for reducing transfusion‐related viral and bacterial diseases.

Efflux pumps and drug resistance in gram-negative bacteria.

The outer membrane of Gram-negative bacteria can only slow down the influx of lipophilic inhibitors, and so these bacteria need active efflux pumps of broad specificity to survive. Pumps such as the Escherichia coli Acr system and its homologs make Gram-negative bacteria resistant to dyes, detergents and antibiotics.

Selective Targeting of Antitumor Immune Responses with Engineered Live-Attenuated Listeria monocytogenes

Improved immunization and ex vivo T-cell culture strategies can generate larger numbers and more potent tumor-specific effector cells than previously possible. Nonetheless, the capacity of these cells to eliminate established tumors is limited by their ability to efficiently enter tumor-bearing organs and mediate their effector function. In the current study, we show that the administration of an engineered organ-homing microbe selectively targets tumor-specific immune responses to metastases within that organ. Specifically, an attenuated Listeria monocytogenes strain, which preferentially infects the liver following systemic administration, dramatically enhances the activity of a cancer vaccine against liver metastases but not metastases in the lung. This enhanced activity results from both local recruitment of innate immune effectors as well as concentration and increased activation of vaccine-induced antitumor T cells within the liver. These findings show a general approach to focus systemic cancer immunotherapies to specific organs bearing tumor metastases by taking advantage of differential tropisms and the proinflammatory nature of microbes.

Vesicular stomatitis virus: An exciting new therapeutic oncolytic virus candidate for cancer or just another chapter from Field's Virology?

Selected mutant strains of vesicular stomatitis virus (VSV) are described that are unable to combat endogenous IFN-beta signaling within infected normal cells and as a result are dramatically more selective for productive growth in tumor cells having a defective antiviral response. The VSV mutants may have the potential to be used clinically as a systemic oncolytic agent for the treatment of distal and metastatic cancers.

Regulation of carotenoid and bacteriochlorophyll biosynthesis genes and identification of an evolutionarily conserved gene required for bacteriochlorophyll accumulation.

The temporal expression of ten clustered genes required for carotenoid (crt) and bacteriochlorophyll (bch) biosynthesis was examined during the transition from aerobic respiration to anaerobiosis requisite for the development of the photosynthetic membrane in the bacterium Rhodobacter capsulatus. Accumulation of crtA, crtC, crtD, crtE, crtF, crtK, bchC and bchD mRNAs increased transiently and coordinately, up to 12-fold following removal of oxygen from the growth medium, paralleling increases in mRNAs encoding pigment-binding polypeptides of the photosynthetic apparatus. The crtB and crtI genes, in contrast, were expressed similarly in the presence or absence of oxygen. The regulation patterns of promoters for the crtA and crtI genes and the bchCXYZ operon were characterized using lacZ transcriptional fusion and qualitatively reflected the corresponding mRNA accumulation patterns. We also report that the bchI gene product, encoded by a DNA sequence previously considered to be a portion of crtA, shares 49% sequence identity with the nuclear-encoded Arabidopsis thaliana Cs chloroplast protein required for normal pigmentation in plants.

Supercoiling Induced by Transcription

The biological implications of supercoiling by transcription are potentially significant (Pruss and Drlica 1989; Lilley and Higgins 1991), and a fundamental question is the extent to which transcription determines the level of DNA supercoiling in vivo. Transcription can induce supercoiling of the template by virtue of the topological relationship between DNA and elongating RNA polymerase (Liu and Wang 1987). In some models, transcription elongation requires that polymerase follows the helical screw of the DNA such that there one 360° rotation between the enzyme and DNA for each 10.5 bp transcribed (Gamper and Hearst 1982). Since RNA polymerase elongates at the rate of about 40 nucleotides/s, an efficiently anchored transcription complex should introduce approximately four negative super-turns upstream and four positive superturns downstream from an actively expressed gene each second. This would suggest extraordinarily fast rates of localized supercoiling after the onset of transcription. The goal of this chapter is to summarize and examine our understanding of the kinetics and mechanisms of supercoiling induced by transcription and to relate these insights to the mechanics of transcription elongation.

Abstract PR06: Drugging the human microbiome for combination with tumor immunotherapy

The human gut microbiome is a diverse, dynamic, and complex ecosystem that modulates host processes including metabolism, inflammation, and cellular and humoral immune responses. Recent studies have suggested that the microbiome may also influence the development of certain cancers such as colorectal cancer, and equally importantly, tumor response to systemic therapy, especially immunotherapy. Multiple groups are exploring the therapeutic utility of the microbiome to enhance clinical response through the use of defined oral therapeutics comprising living commensal bacteria, which would represent a new therapeutic modality. Exploiting the microbiome for therapeutic benefit is not without its challenges due to the heterogeneity of the gut microbiota across healthy donors and patients. In addition, many aspects of conventional small molecule and biologics drug discovery and development do not apply to this novel class of living drugs. We present an approach that leverages the concept of “reverse translation,” using genomic and immunologic characterization of patient samples from interventional studies to define and better understand the organisms and mechanisms that contribute to response or non-response to immunotherapy. We are investigating the relationship between the composition of the gut microbiome prior to therapy and the antitumor response in patients receiving checkpoint inhibitors (CPI), as well as how CPI treatment modulates the microbiome in both responders and nonresponders. Fecal and blood samples are collected before and during therapy from cancer patients who receive approved CPI; tumor types include renal, bladder, and NSCLC. Whole metagenomic shotgun sequencing of patient microbiomes is used to identify higher order (e.g., order- and family-level) “microbial signatures” that associate with response to CPI treatment. We then utilize proprietary algorithms that enable species- and strain-level resolution of microbial signatures. In addition, global and targeted metabolomics are used to identify functional pathways associated with outcome, and these pathways can be linked to species and strains identified by genomic analysis. Our discovery strategy iterates computational analyses and machine learning approaches with empirical in vitro and ex vivo screening of strains and consortia to inform selection and drive drug design. Data from such a comprehensive approach is invaluable for designing compositions of bacteria that form “functional ecological networks” that can impact response to CPI therapy. Finally, our microbial library of >14,000 isolates from healthy human subjects captures the phylogenetic diversity and functional breadth of the gastrointestinal microbiome, and provides a robust platform to build unique compositions. Such compositions, when tested in syngeneic tumor models in germ-free mice, can provide a preliminary readout of the contributions of members of the consortia and enable candidate identification. We present examples of reverse translation in patients with recurrent Clostridium difficile infection and ulcerative colitis, a form of inflammatory bowel disease, that have led to the translation of three drugs that are currently in clinical trials. This roadmap provides insight into how similar drugs can be discovered and developed in the setting of immunotherapy to augment the efficacy of CPIs by altering the cancer-immune set point. This abstract is also being presented as Poster A06. Citation Format: David N. Cook, Jonathan Peled, Marcel van den Brink, Lata Jayaraman. Drugging the human microbiome for combination with tumor immunotherapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr PR06.