Ultrasound-controllable engineered bacteria for cancer immunotherapy

Abedi, Mohamad; Yao, Michael; Mittelstein, David R.; Bar‐Zion, Avinoam; Swift, Margaret; Lee‐Gosselin, Audrey; Barturen‐Larrea, Pierina; Buss, Marjorie T.; Shapiro, Mikhail G.

doi:10.1038/s41467-022-29065-2

Cited by 117 publications

(95 citation statements)

References 83 publications

(93 reference statements)

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“…On bacterial division, if a daughter cell does not receive any plasmid copies, the antitoxin rapidly degrades, and the active toxin kills the cell. While several natural TA systems have been studied (56) and some were investigated for plasmid retention in E. coli with promising results (41,57,58), their use in lactobacilli has not yet been explored. We first investigated the type II TA system, Txe/Axe, derived from Enterococcus faecium that was shown to ensure long-term plasmid retention in E. coli (41) .In this system, Txe is an endoribonuclease and Axe is its corresponding inhibitory protein.…”

Section: Resultsmentioning

confidence: 99%

Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli

Dey

Asensio

Kuttae

et al. 2022

Preprint

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Lactobacilli are ubiquitous in nature, often beneficially associated with animals as commensals and probiotics, and are extensively used in food fermentation. Due to this close-knit association, there is considerable interest to engineer them for healthcare applications in both humans and animals, for which high-performance and versatile genetic parts are greatly desired. For the first time, we describe two genetic modules in Lactiplantibacillus plantarum that achieve high-level gene expression using plasmids that can be retained without antibiotics, bacteriocins or genomic manipulations. These include (i) a promoter, PtlpA, from a phylogenetically distant bacterium, Salmonella typhimurium, that drives up to 5-fold higher level of gene expression compared to previously reported promoters and (ii) multiple toxin-antitoxin systems as a self-contained and easy-to-implement plasmid retention strategy that facilitates the engineering of tunable transient Genetically Modified Organisms. These modules and the fundamental factors underlying their functionality that are described in this work will greatly contribute to expanding the genetic programmability of lactobacilli for healthcare applications.

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Section: Resultsmentioning

confidence: 99%

Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli

Dey

Asensio

Kuttae

et al. 2022

Preprint

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“…86 Additionally, other heating mechanisms could be used for magnetic hyperthermia such as ultrasound which was shown previously. 42, 52, 92…”

Section: Resultsmentioning

confidence: 99%

Magnetothermal control of temperature-sensitive repressors in superparamagnetic iron nanoparticle-coatedBacillus subtilis

Greeson

Madsen

Makela

et al. 2022

Preprint

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Superparamagnetic iron oxide nanoparticles (SPIONs) are used as contrast agents in magnetic resonance imaging (MRI) and magnetic particle imaging (MPI) and resulting images can be used to guide magnetothermal heating. Alternating magnetic fields (AMF) cause local temperature increases in regions with SPIONs, and we investigated the ability of magnetic hyperthermia to regulate temperature-sensitive repressors (TSRs) of bacterial transcription. The TSR, TlpA39, was derived from a Gram-negative bacterium, and used here for thermal control of reporter gene expression in Gram-positive, Bacillus subtilis. In vitro heating of B. subtilis with TlpA39 controlling bacterial luciferase expression, resulted in a 14.6-fold (12-hour; h) and 1.8-fold (1-h) increase in reporter transcripts with a 9.0-fold (12-h) and 11.1-fold (1-h) increase in bioluminescence. To develop magnetothermal control, B. subtilis cells were coated with three SPION variations. Electron microscopy coupled with energy dispersive X-ray spectroscopy revealed an external association with, and retention of, SPIONs on B. subtilis. Furthermore, using long duration AMF we demonstrated magnetothermal induction of the TSRs in SPION-coated B. subtilis with a maximum of 4.6-fold increases in bioluminescence. After intramuscular injections of SPION-coated B. subtilis, histology revealed that SPIONs remained in the same locations as the bacteria. For in vivo studies, 1-h of AMF is the maximum exposure due to anesthesia constraints. Both in vitro and in vivo, there was no change in bioluminescence after 1-h of AMF treatment. Pairing TSRs with magnetothermal energy using SPIONs for localized heating with AMF can lead to transcriptional control that expands options for targeted bacteriotherapies.

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“…[63][64][65][66] In addition, the spatial distribution is important in guiding focused energy procedures applied to the cells, such as focused ultrasound. [67][68][69] Other potential applications include the study of the gastrointestinal (GI) microbiome and the tracking of probiotics designed to diagnose or treat GI conditions. 70,71 In these applications, luminescence imaging would provide limited in vivo resolution due to light scattering, and would be difficult to scale to larger animals or human patients.…”

Section: Discussionmentioning

confidence: 99%

Genomically mined acoustic reporter genes enable real-timein vivomonitoring of tumors and tumor-homing probiotics

Hurt

Buss

Duan

et al. 2021

Preprint

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A major outstanding challenge in the fields of biological research, synthetic biology and cell-based medicine is the difficulty of visualizing the function of natural and engineered cells noninvasively inside opaque organisms. Ultrasound imaging has the potential to address this challenge as a widely available technique with a tissue penetration of several centimeters and a spatial resolution below 100 um. Recently, the first genetically encoded reporter molecules were developed based on bacterial gas vesicles to link ultrasound signals to molecular and cellular function. However, the properties of these first-generation acoustic reporter genes (ARGs) resulted in limited sensitivity and specificity for imaging in the in vivo context. Here, we describe second-generation ARGs with greatly improved acoustic properties and expression characteristics. We identified these ARGs through a systematic phylogenetic screen of candidate gas vesicle gene clusters from diverse bacteria and archaea. The resulting constructs offer major qualitative and quantitative improvements, including the ability to produce nonlinear ultrasound contrast to distinguish their signals from those of background tissues, and a reduced burden of expression in probiotic hosts. We demonstrate the utility of these next-generation ARGs by imaging the in situ gene expression of tumor-homing probiotic bacteria, revealing the unique spatial distribution of tumor colonization by these cells noninvasively in living subjects. *equal contribution

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Ultrasound-controllable engineered bacteria for cancer immunotherapy

Cited by 117 publications

References 83 publications

Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli

Novel genetic modules encoding high-level antibiotic-free protein expression in probiotic lactobacilli

Magnetothermal control of temperature-sensitive repressors in superparamagnetic iron nanoparticle-coatedBacillus subtilis

Genomically mined acoustic reporter genes enable real-timein vivomonitoring of tumors and tumor-homing probiotics

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