Nucleotide sequence analysis of the gene encoding the <i>Caulobacter crescentus</i> paracrystalline surface layer protein

Gilchrist, Angus; Fisher, James; Smit, John

doi:10.1139/m92-033

Cited by 64 publications

(63 citation statements)

References 44 publications

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“…[27][28][29] It is possible that the smooth lipopolysaccharide anchoring the S-layer to the cell surface may have some role in this anti-tumor effects. 16,17 This could be due to nonspecific stimulation of the host immune system. In support of this idea, Caulobacter LPS is known to have adjuvanation properties, stimulating a stronger immune response to an E. coli flagellar antigen.…”

Section: Discussionmentioning

confidence: 99%

“…Since S layer protein assembles on the outside of the cell, it is a secreted protein that is non-covalently anchored to the cell surface via a specific smooth lipopolysaccharide. 16,17 The gene (rsaA) encoding the C. crescentus S layer subunit protein (RsaA) has been characterized and used to display foreign peptides on the cell surface in a dense, highly ordered manner. 18 Our ongoing studies are directed towards the development of cancer vaccine with Caulobacter-S-layer expressing tandemly repeated copies of the human MUC1 antigen along with additional immune modulating segments.…”

Section: Introductionmentioning

confidence: 99%

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

Bhatnagar

Awasthi²,

Nomellini³

et al. 2006

Cancer Biology & Therapy

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Caulobacter crescentus is a gram negative, non-pathogenic bacterium, common in aquatic and soil environments. One feature of note is a protein surface layer (S-layer) composed of a single protein, organized as a self-assembled crystalline array that coats the bacterium. In the course of efforts to express cancer-associated peptides as genetic insertions into the S-layer, we noted a tumor suppressive effect of the unmodified bacterium. C. crescentus was examined for anti-tumor activity against three transplantable tumor mouse models: Lewis lung carcinoma cells transfected with the MUC1 gene in C57BL/6, murine mammary carcinoma (EMT-6) in BALB/c (both in prophylactic and therapeutic mode) and murine leukemia cells (L1210) in DBA2. Mice were immunized three times i.p. with C. crescentus (2 x 10 7 cells/mouse). In prophylactic mode, the mice were challenged with tumor cells two weeks after the last immunization. Immunization with live C. crescentus resulted in anti-tumor activity in all three transplantable tumor models, as measured by prolonged survival, reduced tumor mass or reduced number of lung nodules, compared to saline control groups. In the Lewis lung and the EMT-6 mammary carcinoma murine models the number of lung nodules as well as the tumor weight was lower in mice treated with C. crescentus, compared to the control group; for EMT-6, this was observed in prophylactic and therapeutic modes. In the murine leukemia and Lewis lung carcinoma models prolonged survival was observed in the groups of mice immunized with Caulobacters. In most cases the live C. crescentus cells were markedly more efficacious than heat killed or formalin fixed cells, despite the fact that they do not grow or persist in mice. The results suggest that C. crescentus may be a safe, bacterial immunomodulator for the treatment of tumors.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Bhatnagar

Awasthi²,

Nomellini³

et al. 2006

Cancer Biology & Therapy

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“…It is estimated that 60 000 RsaA monomers are interlinked to form the S-layer. The correct crystallization of the structure into its native hexagonal pattern requires calcium ions (Gilchrist et al, 1992;Smit et al, 1992;Walker et al, 1992Walker et al, , 1994Bingle et al, 1997). Secretion of the protein relies on a stable C-terminal secretion signal (Bingle et al, 1997) and a three-component bacterial ABC-transporter system, two components of which have been cloned and sequenced (P. Awram and J.…”

Section: Introductionmentioning

confidence: 99%

Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus

Bingle

Nomellini

Smit

1997

Molecular Microbiology

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SummaryThe paracrystalline surface (S)-layer of Caulobacter crescentus is composed of a single secreted protein (RsaA) that interlocks in a hexagonal pattern to completely envelop the bacterium. Using a genetic approach, we inserted a 12 amino acid peptide from Pseudomonas aeruginosa strain K pilin at numerous semirandom positions in RsaA. We then used an immunological screen to identify those sites that presented the inserted pilin peptide on the C. crescentus cell surface as a part of the S-layer. Eleven such sites (widely separated in the primary sequence) were identified, demonstrating for the first time that S-layers can be readily exploited as carrier proteins to display 'epitope-size' heterologous peptides on bacterial cell surfaces. Whereas intact RsaA molecules carrying a pilin peptide could always be found on the surface of C. crescentus regardless of the particular insertion site, introduction of the pilin peptide at 9 of the 11 sites resulted in some proteolytic cleavage of RsaA. Two types of proteolytic phenomena were observed. The first was characterized by a single cleavage within the pilin peptide insert with both fragments of the S-layer protein remaining anchored to the outer membrane. The other proteolytic phenomenon was characterized by cleavage of the S-layer protein at a point distant from the site of the pilin peptide insertion. This cleavage always occurred at the same location in RsaA regardless of the particular insertion site, yielding a surface-anchored 26 kDa proteolytic fragment bearing the RsaA N-terminus; the C-terminal cleavage product carrying the pilin peptide was released into the growth medium. When the results of this work were combined with the results of a previous study, the RsaA primary sequence could be divided into three regions with respect to the location of a peptide insertion and its effect on S-layer biogenesis: (i) insertions in the extreme N-terminus of RsaA either produce no apparent effect on S-layer biogenesis or disrupt surface-anchoring of the protein; (ii) insertions in the extreme C-terminus either produce no apparent effect on S-layer biogenesis or disrupt protein secretion; and (iii) insertions more centrally located in the protein either have no apparent effect on S-layer biogenesis or result in proteolytic cleavage of RsaA. These data are discussed in relation to our previous assignment of the RsaA N-and C-terminus as regions that are important for surface anchoring and secretion respectively.

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“…In past studies, the S-layer protein of C. crescentus CB1SA (39) (formerly called 130K, but now called RsaA [14]) was purified from an extracellular milieu of proteins by solubilization in sodium dodecyl sulfate (SDS) and gel filtration chromatography (38). Antibody to this preparation, however, does not label the intact S-layer structure, nor have we been able to achieve in vitro crystallization of an S layer with such preparations.…”

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confidence: 99%

Isolation and comparison of the paracrystalline surface layer proteins of freshwater caulobacters

1992

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Several methods for isolation of the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A were evaluated. Treatment of cells with HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid) buffer at pH 2 was the most effective means of selectively removing RsaA from cells, and after neutralization, the protein was capable of reassembling into a paracrystalline structure. Ethylene glycol-bis(13-aminoethyl ether)-N,N,N',N'-tetraacetic acid treatment could also be used to extract RsaA and yielded protein capable of reassembly. The success of the methods was likely related to disruption of calcium-mediated bonding; calcium was required for recrystallization, while magnesium and strontium ions were ineffective. Antibody was raised against purified RsaA and, along with the S-layer extraction techniques, was used to evaluate 42 strains of caulobacters isolated from a variety of aquatic and wastewater treatment locations. A single characteristic protein could be isolated from the 35 strains that produced an S layer; with one exception, no proteins were extracted from strains that had no S layer. The presumed S-layer proteins ranged in size from 100 to 193 kDa.All of these proteins specifically reacted with anti-RsaA serum by Western immunoblot analysis. In strain CB15A, a specific S-layer-associated oligosaccharide has been proposed to be involved in a calcium-mediated attachment of the S layer to the cell surface. This molecule was detected by Western immunoblotting with a specific antiserum and on polyacrylamide gels stained for polysaccharides. A comparable band was found in all S-layer-producing strains and for most, S-layer-associated oligosaccharide-specific antibody reacted with them in Western analysis. Overall, in freshwater caulobacters at least portions of their S-layer structures appear to be strongly conserved entities, as well as the means of attachment to the cell surface.Caulobacters are one of many genera of bacteria that elaborate a paracrystalline surface (S) layer on their outermost surface (37). The S layer of one species, Caulobacter crescentus, has received the most attention, but in a survey of caulobacters isolated from wastewater treatment systems the most common type of caulobacter we encountered shares morphological similarities with C. crescentus strains, including a hexagonally packed S-layer structure (27).The cloned S-layer protein gene of C. crescentus CB15hybridized to specific regions of the genome for most of these S-layer-producing caulobacters, yet only under moderate-stringency conditions, and restriction length polymorphism analysis with the S-layer gene as the probe failed to reveal patterns of close relatedness between the strains (27).This was recently correlated with a 16S rRNA comparative analysis which showed that these caulobacters were a coherent group but still sufficiently different to have significant variation in their overall genomic DNA composition (41). We were interested in determining how similar their S-layer structures are from the s...

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Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein

Cited by 64 publications

References 44 publications

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

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

Cell‐surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S‐layer of Caulobacter crescentus

Isolation and comparison of the paracrystalline surface layer proteins of freshwater caulobacters

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