A Caulobacter crescentus Microbicide Protects from Vaginal Infection with HIV-1 in Humanized Bone Marrow-Liver-Thymus Mice.

Over 2 million people are infected with HIV-1 annually. Approximately half of these new infections occur in women residing in low-income countries, where their access to and control over HIV-1 preventative measures are often limited, indicating that female-controlled prevention options for HIV-1 are urgently needed. Microbicides that can be topically applied to the vaginal tract in advance of sexual activity represent a promising female-controlled prevention option for HIV-1.

Topologically-guided continuous protein crystallization controls bacterial surface layer self-assembly.

Many bacteria and most archaea possess a crystalline protein surface layer (S-layer), which surrounds their growing and topologically complicated outer surface. Constructing a macromolecular structure of this scale generally requires localized enzymatic machinery, but a regulatory framework for S-layer assembly has not been identified. By labeling, superresolution imaging, and tracking the S-layer protein (SLP) from C.

Generation of a Dual-Target, Safe, Inexpensive Microbicide that Protects Against HIV-1 and HSV-2 Disease.

HSV-2 infection is a significant health problem and a major co-morbidity factor for HIV-1 acquisition, increasing risk of infection 2-4 fold. Condom based prevention strategies for HSV-2 and HIV-1 have not been effective at stopping the HIV-1 pandemic, indicating that alternative prevention strategies need to be investigated. We have previously developed an inexpensive HIV-1 specific microbicide that utilizes the S-layer mediated display capabilities of Caulobacter crescentus, and have shown that recombinant C.

Environmental Calcium Controls Alternate Physical States of the Caulobacter Surface Layer.

Surface layers (S-layers) are paracrystalline, proteinaceous structures found in most archaea and many bacteria. Often the outermost cell envelope component, S-layers serve diverse functions including aiding pathogenicity and protecting against predators. We report that the S-layer of Caulobacter crescentus exhibits calcium-mediated structural plasticity, switching irreversibly between an amorphous aggregate state and the crystalline state.

Surface-layer protein from Caulobacter crescentus: expression, purification and X-ray crystallographic analysis.

Protein surface layers are self-assembling, paracrystalline lattices on the surface of many prokaryotes. Surface-layer proteins have not benefited from widespread structural analysis owing to their resistance to crystallization. Here, the successful expression of a truncated version of RsaA, the surface-layer protein from Caulobacter crescentus, from a Caulobacter protein-expression system is reported.

The core and O-polysaccharide structure of the Caulobacter crescentus lipopolysaccharide.

Here we describe the analysis of the structure of the lipopolysaccharide (LPS) from Caulobacter crescentus strain JS1025, a derivative of C. crescentus CB15 NA1000 with an engineered amber mutation in rsaA, leading to the loss of the protein S-layer and gene CCNA_00471 encoding a putative GDP-L-fucose synthase. LPS was isolated using an aqueous membrane disruption method.

Development of an HIV-1 Microbicide Based on Caulobacter crescentus: Blocking Infection by High-Density Display of Virus Entry Inhibitors.

The HIV/AIDS pandemic remains an enormous global health concern. Despite effective prevention options, 2.6 million new infections occur annually, with women in developing countries accounting for more than half of these infections.

Evaluating secretion and surface attachment of SapA, an S-layer-associated metalloprotease of Caulobacter crescentus.

Caulobacter crescentus is used to display foreign peptides at high density as insertions into the surface (S)-layer protein (RsaA). Many recombinant RsaA proteins, however, are cleaved by SapA, a 71-kDa metalloprotease, suggesting a role in maintaining S-layer integrity. When overexpressed on a multicopy plasmid SapA was detected on the surface by fluorescent antibody only if RsaA and the O-side chain of LPS that mediates S-layer attachment were removed by mutation, indicating an outer membrane location beneath the S-layer.

Enhanced neutralization of HIV by antibodies displayed on the S-layer of Caulobacter crescentus.

Innovative methods of prevention are needed to stop the more than two million new HIV-1 infections annually, particularly in women. Local application of anti-HIV antibodies has been shown to be effective at preventing infection in nonhuman primates; however, the concentrations needed are cost prohibitive. Display of antibodies on a particulate platform will likely prolong effectiveness of these anti-HIV agents and lower the cost of goods.

Analysis of the intact surface layer of Caulobacter crescentus by cryo-electron tomography.

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.

Analysis of high-level S-layer protein secretion in Caulobacter crescentus.

Caulobacter crescentus exhibits a hexagonally arranged protein layer on its outermost surface. RsaA, the sole protein of this "S-layer", is secreted by a type I (ABC) transporter. Few type I transporters show high-level secretion, and few bacterial S-layers have been carefully examined for the amount of protein synthesis capacity needed to maintain cell coverage.

Development of an HIV-1 specific microbicide using Caulobacter crescentus S-layer mediated display of CD4 and MIP1alpha.

The development of alternative strategies to prevent HIV infection is a global public health priority. Initial efforts in anti-HIV microbicide development have met with poor success as the strategies have relied on a non-specific mechanism of action. Here, we report the development of a microbicide aimed at specifically blocking HIV entry by displaying molecular components of the HIV/host cell attachment complex on the surface of Caulobacter crescentus, a harmless aquatic bacterium.

S-layer-mediated display of the immunoglobulin G-binding domain of streptococcal protein G on the surface of Caulobacter crescentus: development of an immunoactive reagent.

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.

S-layer anchoring and localization of an S-layer-associated protease in Caulobacter crescentus.

The S-layer of the gram-negative bacterium Caulobacter crescentus is composed of a single protein, RsaA, that is secreted and assembled into a hexagonal crystalline array that covers the organism. Despite the widespread occurrence of comparable bacterial S-layers, little is known about S-layer attachment to cell surfaces, especially for gram-negative organisms. Having preliminary indications that the N terminus of RsaA anchors the monomer to the cell surface, we developed an assay to distinguish direct surface attachment from subunit-subunit interactions where small RsaA fragments are incubated with S-layer-negative cells to assess the ability of the fragments to reattach.

Anti-tumor effects of the bacterium Caulobacter crescentus in murine tumor models.

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.

Evaluation of a new system for developing particulate enzymes based on the surface (S)-layer protein (RsaA) of Caulobacter crescentus: fusion with the beta-1,4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi yields a robust, catalytically active product.

Immobilized biocatalysts, including particulate enzymes, represent an attractive tool for research and industrial applications because they combine the specificity of native enzymes with the advantage that they can be readily separated from end product and reused. We demonstrated the use of the Caulobacter crescentus surface (S)-layer protein (RsaA) secretion apparatus for the generation of particulate enzymes. Specifically, a candidate protein made previously by fusion of the beta-1,4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi with the C-terminus of RsaA was evaluated.

Transcriptional regulation of the S-layer protein type I secretion system in Caulobacter crescentus.

The Gram-negative Caulobacter crescentus exports RsaA, the crystalline S-layer subunit protein using a dedicated type I secretion system. The protein and two transporter genes (rsaADE) are located together, comparable to the Escherichia coli type I hemolysin hlyCABD operon, where read through of a stem loop following hlyCA results in reduced transcription of the hlyBD. Using two genetic approaches and a direct assessment of transcription from regions 5' to the genes we learned that rsaD and rsaE were transcribed together as a separate transcript from rsaA.

Comparison of S-layer secretion genes in freshwater caulobacters.

Our freshwater caulobacter collection contains about 40 strains that are morphologically similar to Caulobacter crescentus. All elaborate a crystalline protein surface (S) layer made up of protein monomers 100-193 kDa in size. We conducted a comparative study of S-layer secretion in 6 strains representing 3 size groups of S-layer proteins: small (100-108 kDa), medium (122-151 kDa), and large (181-193 kDa).

Two outer membrane proteins are required for maximal type I secretion of the Caulobacter crescentus S-layer protein.

Transport of RsaA, the crystalline S-layer subunit protein of Caulobacter crescentus, is mediated by a type I secretion mechanism. Two proteins have been identified that play the role of the outer membrane protein (OMP) component in the RsaA secretion machinery. The genes rsaF(a) and rsaF(b) were identified by similarity to the Escherichia coli hemolysin secretion OMP TolC by using the C.

Caulobacter crescentus synthesizes an S-layer-editing metalloprotease possessing a domain sharing sequence similarity with its paracrystalline S-layer protein.

Strains of Caulobacter crescentus elaborate an S-layer, a two-dimensional protein latticework which covers the cell surface. The S-layer protein (RsaA) is secreted by a type I mechanism (relying on a C-terminal signal) and is unusual among type I secreted proteins because high levels of protein are produced continuously. In efforts to adapt the S-layer for display of foreign peptides and proteins, we noted a proteolytic activity that affected S-layer monomers with foreign inserts.

Development of small high-copy-number plasmid vectors for gene expression in Caulobacter crescentus.

Caulobacter crescentus is a bacterium with a distinctive life cycle and so it is studied as a cell development model. In addition, we have adapted this bacterium for recombinant protein production and display based on the crystalline surface protein (S)-layer and its C-terminal secretion signal. We report here the development of small, high-copy-number plasmid vectors and methods for producing an obligate expression host.

Recombinant vaccines against infectious hematopoietic necrosis virus: production by the Caulobacter crescentus S-layer protein secretion system and evaluation in laboratory trials.

We report the development of an IHNV vaccine produced by a new protein production system based on the bacterium Caulobacter crescentus. The subunit vaccines that were tested contain a 184 amino acid segment of the IHNV glycoprotein in different fusion arrangements with the C. crescentus S-layer protein.

Expression and testing of Pseudomonas aeruginosa vaccine candidate proteins prepared with the Caulobacter crescentus S-layer protein expression system.

A novel bacterial protein secretion system was used to produce vaccine candidates against Pseudomonas aeruginosa. The surface protein (RsaA) of Caulobacter crescentus was adapted to produce recombinant vaccine proteins based on the pilus tip epitope ('adhesintope') of P. aeruginosa.

Secretion of the Caulobacter crescentus S-layer protein: further localization of the C-terminal secretion signal and its use for secretion of recombinant proteins.

The secretion signal of the Caulobacter crescentus S-layer protein (RsaA) was localized to the C-terminal 82 amino acids of the molecule. Protein yield studies showed that 336 or 242 C-terminal residues of RsaA mediated secretion of >50 mg of a cellulase passenger protein per liter to the culture fluids.

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

The 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.