Alp7R Regulates Expression of the Actin-Like Protein Alp7A in Bacillus subtilis

Derman, Alan I.; Nonejuie, Poochit; Michel, Brittany C.; Truong, Bao Dinh; Fujioka, Akina; Erb, Marcella L.; Pogliano, Joe

doi:10.1128/jb.06550-11

Cited by 16 publications

(19 citation statements)

References 52 publications

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“…The pSC02 therapeutic plasmid was constructed by cloning a gBlock (IDT) encoding a tac promoter and an E. coli codon-optimized sequence for the A4 anti-CD47 nanobody 9 with an C-terminal hemagglutinin tag into the multiple cloning site of a pAH162 plasmid 32 . Additionally, two stabilizing elements, the hok/sok system 33 and alp7 partitioning system 34 were introduced into pSC02 to minimize plasmid loss in vivo. pSC01 and pSC02 were transformed into chemically competent E. coli Pir1 + (Invitrogen).…”

Section: Strains and Plasmidsmentioning

confidence: 99%

Programmable bacteria induce durable tumor regression and systemic antitumor immunity

Chowdhury

Hinchliffe

Castro

et al. 2019

Preprint

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Co-corresponding authors SUMMARY PARAGRAPHSynthetic biology is driving a new era of medicine through the genetic programming of living cells 1,2 . This transformative approach allows for the creation of engineered systems that intelligently sense and respond to diverse environments, ultimately adding specificity and efficacy that extends beyond the capabilities of molecular-based therapeutics 3-5 . One particular focus area has been the engineering of bacteria as therapeutic delivery systems to selectively release therapeutic payloads in vivo 6-8 . Here, we engineered a nonpathogenic E. coli to specifically lyse within the tumor microenvironment and release an encoded nanobody antagonist of CD47 (CD47nb) 9 , an anti-phagocytic receptor commonly overexpressed in several human cancers 10,11 . We show that intratumoral delivery of CD47nb by tumor-colonizing bacteria increases activation of tumor-infiltrating T cells, stimulates rapid tumor regression, prevents metastasis, and leads to long-term survival in a syngeneic tumor model. Moreover, we report that local injection of CD47nb bacteria stimulates systemic antitumor immune responses that reduce the growth of untreated tumorsproviding, to the best of our knowledge, the first demonstration of an abscopal effect induced by a bacteria cancer therapy. Thus, engineered bacteria may be used for safe and local delivery of immunotherapeutic payloads leading to systemic antitumor immunity.

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Section: Strains and Plasmidsmentioning

confidence: 99%

Programmable bacteria induce durable tumor regression and systemic antitumor immunity

Chowdhury

Hinchliffe

Castro

et al. 2019

Preprint

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“…The plasmids used in this study are listed in Supplementary Table 1. The construction of plasmid pAID3219 (pP xyl alp7R-gfp), where gfp encodes green fluorescent protein, was described previously (Derman et al, 2012). Plasmid pAID3604 (pP xyl alp7RΔ36-gfp) was constructed by amplification of pAID3129 with oligonucleotide primers P1 and P2, restriction of the amplicon with AvrII and SphI, and ligation to pAID3129 restricted with AvrII and SphI.…”

Section: Right-angle Light Scatteringmentioning

confidence: 99%

“…Only background green fluorescence could be detected from control strains JP3349 [PY79 amyE::P hyspank divIVA-alp7R TAA -gfp] and JP3567 [PY79 amyE::P hyspank divIVA-alp7RΔ36 TAA -gfp] at all induction levels surveyed, indicating that there was no read-though into gfp when the alp7R stop codon was present. Media and antibiotic supplementation was as described in Derman et al, 2012. Live-Cell Microscopy. Agarose pads for microscopy were prepared and imaged as described in Derman et al, 2012.…”

Section: Right-angle Light Scatteringmentioning

confidence: 99%

“…In addition to the actin-like protein Alp7A, this operon encodes Alp7R, a member of the Ribbon-Helix-Helix (RHH) family of dimeric DNA-binding proteins 14 . The Alp7R dimers bind to nine nearby DNA sequence repeats (the alp7C locus), to form a DNA-protein complex that is required for plasmid partitioning 13,15 . A previous study suggested that in vivo Alp7A forms dynamically unstable filaments that interact with the Alp7R/C complex to create a DNA-segregating spindle.…”

Section: Introductionmentioning

confidence: 99%

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Polymer dynamics of Alp7A reveals two ‘critical’ concentrations that govern dynamically unstable actin-like proteins

Petek

Derman

Royal

et al. 2017

Preprint

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ABSTRACT:Dynamically unstable polymers capture and move cellular cargos in both bacteria and eukaryotes, but the regulation of their assembly remains poorly understood. Here we describe polymerization of Alp7A, a bacterial Actin-Like Protein (ALP) that segregates the low copynumber plasmid pLS20 in Bacillus subtilis. Purified Alp7A forms dynamically unstable polymers with two critical points: an intrinsic critical concentration (0.6 µM), observed when ATP hydrolysis is blocked, and a dynamic critical concentration (10.3 µM), observed when ATP hydrolysis occurs. From biochemical and kinetic analysis, the intrinsic critical concentration reflects a balance between filament elongation and shortening, while the dynamic critical concentration reflects a balance between filament nucleation and catastrophic disassembly. Although Alp7A does not form stable polymers at physiological concentrations, rapid nucleation by an accessory factor, Alp7R, decreases the dynamic critical concentration into the physiological range. Intrinsic and dynamic critical concentrations are fundamental parameters that can be used to describe the behavior of all dynamically unstable polymers.

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“…The bacterial actin AlfA is encoded on the B. subtilis plasmid pLS32 and is found in an operon with AlfB (ParR-like) and a parC sequence 20, 26 . The bacterial actin Alp7A, in conjunction with Alp7R (ParR-like) and a lp7C (parC- like), is responsible for the segregation of plasmid pLS20 in B. subtilis 27, 60 . Although the basic architecture of all these systems is similar there appears to be substantial differences between their bacterial actins, as we will discuss.…”

Section: Parm and Other Plasmid Segregating Bacterial Actinsmentioning

confidence: 99%

Bacterial Actins and Their Diversity

2013

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For many years bacteria were considered rather simple organisms, but the dogmatic notion that subcellular organization is a eukaryotic trait has been overthrown for more than a decade. The discovery of homologs of the eukaryotic cytoskeletal proteins actin, tubulin, and intermediate filaments in bacteria has been instrumental in changing this view. Over the recent years we gained an incredible level of insight into the diverse family of bacterial actins and their molecular workings. Here we review the functional, biochemical and structural features of the most well-studied bacterial actins.

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Alp7R Regulates Expression of the Actin-Like Protein Alp7A in Bacillus subtilis

Cited by 16 publications

References 52 publications

Programmable bacteria induce durable tumor regression and systemic antitumor immunity

Programmable bacteria induce durable tumor regression and systemic antitumor immunity

Polymer dynamics of Alp7A reveals two ‘critical’ concentrations that govern dynamically unstable actin-like proteins

Bacterial Actins and Their Diversity

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