Anti-tumor effects of the bacterium caulobacter crescentus in murine tumor models

Bhatnagar, Prerak; Awasthi, Amit; Nomellini, John F.; Smit, John; Suresh, Mavanur R.

doi:10.4161/cbt.5.5.2553

Cited by 42 publications

(37 citation statements)

References 33 publications

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“…To further explore the possible contributions of 14-3-3␥ among seven isoforms to reveal the hematopoietic cell neoplasms (35,36), we selected a human erythroleukemia cells line, K562 (37), a mouse lymphocytic leukemia cell line, L1210 (38), and a mouse T cell lymphoma cell line, EL4, for the study (39). The cells were transfected to achieve overexpression of 14-3-3␥ or its specific siRNA.…”

Section: Forced Expression Of Sirna Specific For 14-3-3␥ Suppressed Smentioning

confidence: 99%

14-3-3γ Is Stimulated by IL-3 and Promotes Cell Proliferation

Ajjappala

Kim

et al. 2009

The Journal of Immunology

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*IL-3 plays important roles in the growth and survival of hematopoietic progenitor cells, processes modeled in studies of the IL-3-dependent cell line Ba/F3. To gain insights into molecular mechanisms governing cell fate, we examined the patterns of proteins up-regulated following stimulation of Ba/F3 cells with IL-3. Through two-dimensional electrophoresis and proteomicsbased approaches, we identified 11 proteins. Of these, expression of 14-3-3␥ was significantly increased by IL-3 stimulation at both the transcriptional and translational levels. 14-3-3␥ overexpression in Ba/F3 cells abrogated dependence on IL-3 and was associated with activation of PI3K and MAPK signaling cascades, suggesting that the functions of 14-3-3␥ in normal hematopoietic progenitors are to promote survival and growth through the activation of distinct signaling pathways. Additionally, the upregulation of Bax and Bad was seen with the ablation of 14-3-3␥, resulting in cell death. These results indicate that deregulated expression of 14-3-3␥ may contribute to malignant transformation, possibly providing a new target for therapeutic intervention in hematopoietic neoplasms.

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Section: Forced Expression Of Sirna Specific For 14-3-3␥ Suppressed Smentioning

confidence: 99%

14-3-3γ Is Stimulated by IL-3 and Promotes Cell Proliferation

Ajjappala

Kim

et al. 2009

The Journal of Immunology

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“…One interest in understanding S layers comes from their potential applications in nanotechnology (46) and therapeutic applications, such as anti-HIV microbicide development (37) and cancer therapy (9). The concept is to display heterologous proteins from within the S-layer structure in order to create dense arrays of foreign insertions.…”

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confidence: 99%

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

et al. 2010

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The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.Surface layers (S layers) are the outermost cell wall component in many archaea and bacteria (6, 44). Most S layers are composed of a single protein or glycoprotein species that selforganizes into two-dimensional (2D) lattices of various sizes, usually with square or hexagonal symmetry (7,14,43). This geometrical arrangement is almost the only commonality among species, since sequence homology between S-layer proteins is low and functionality differs in many cases. In many archaea, the S layer is the only cell wall component, so it may have a role in shape determination. However, in bacteria such as Caulobacter crescentus, the role is more likely related to protection against a variety of predatorial assaults (8).One interest in understanding S layers comes from their potential applications in nanotechnology (46) and therapeutic applications, such as anti-HIV microbicide development (37) and cancer therapy (9). The concept is to display heterologous proteins from within the S-layer structure in order to create dense arrays of foreign insertions. Resolving the S-layer organization and structure at high resolution in cells as close to a native state as possible is crucial to understand or predict where proteins are displayed in the array, particularly when more than one foreign peptide is being displayed simultaneously.A significant limitation has been the dif...

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“…This presentation system has many potential applications, such as the development of whole-cell vaccines, tumor suppressors, cellular adsorbents, and peptide display libraries and the screening of antibody libraries (3,13,23). We have identified sites where insertions of nucleotides that maintain reading frames into the full-length rsaA gene can be made without impeding the synthesis, folding, secretion, and self-assembly of the corresponding protein on the bacterial cell surface (5,6,35).…”

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confidence: 99%

S-Layer-Mediated Display of the Immunoglobulin G-Binding Domain of Streptococcal Protein G on the Surface ofCaulobacter crescentus: Development of an Immunoactive Reagent

Nomellini

Duncan

Dorocicz

et al. 2007

Appl Environ Microbiol

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The immunoglobulin G (IgG)-binding streptococcal protein G is often used for immunoprecipitation or immunoadsorption-based assays, as it exhibits binding to a broader spectrum of host species IgG and IgG subclasses than the alternative, Staphylococcus aureus protein A. Caulobacter crescentus produces a hexagonally arranged paracrystalline protein surface layer (S-layer) composed of a single secreted protein, RsaA, that is notably tolerant of heterologous peptide insertions while maintaining the surface-attached crystalline character. Here, a protein G IgG-binding domain, GB1, was expressed as an insertion into full-length RsaA on the cell surface to produce densely packed immunoreactive particles. GB1 insertions at five separate sites were expressed, and all bound rabbit and goat IgG, but expression levels were reduced compared to those of wild-type RsaA and poor binding to mouse IgG was noted. To remedy this, we used the 20-amino-acid Muc1 peptide derived from human mucins as a spacer, since insertions of multiple tandem repeats were well tolerated for RsaA secretion and assembly. This strategy worked remarkably well, and recombinant RsaA proteins, containing up to three GB1 domains, surrounded by Muc1 peptides, not only were secreted and assembled but did so at wild-type levels. The ability to bind IgG (including mouse IgG) increased as GB1 units were added, and those with three GB1 domains bound twice as much rabbit IgG per cell as S. aureus cells (Pansorbin). The ability of recombinant protein G-Caulobacter cells to function as immunoactive reagents was assessed in an immunoprecipitation assay using a FLAG-tagged protein and anti-FLAG mouse monoclonal antibody; their performance was comparable to that of protein G-Sepharose beads. This work demonstrates the potential for using cells expressing recombinant RsaA/GB1 in immunoassays, especially considering that protein G-Caulobacter cells are more cost-effective than protein G beads and exhibit a broader species and IgG isotype binding range than protein A.

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Anti-tumor effects of the bacterium caulobacter crescentus in murine tumor models

Cited by 42 publications

References 33 publications

14-3-3γ Is Stimulated by IL-3 and Promotes Cell Proliferation

14-3-3γ Is Stimulated by IL-3 and Promotes Cell Proliferation

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

S-Layer-Mediated Display of the Immunoglobulin G-Binding Domain of Streptococcal Protein G on the Surface ofCaulobacter crescentus: Development of an Immunoactive Reagent

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