Microbiology And Immunology

212 W.-P. Fung-Leung,VA Wallace, D. Gray et al. ... Wai-Ping Fung-LeungOO, Valerie A. Wallaceo, Dawn Grayo, William C. Sha*, Hanspeter Pircher A , Hung-Sia Teh+, DennisY. Lohn and Tak W. MakO ... Ontario Cancer Instituteo, Toronto, The RW Johnson Pharmaceutical ...

Naive T cells require costimulation for robust Ag-driven differentiation and survival. Members of the TNFR family have been shown to provide costimulatory signals conferring survival at distinct phases of the T cell response. In this study, we show that CD4 and CD8 T cells depend on TNFR type 2 (p75) for survival during clonal expansion, allowing larger accumulation of effector cells and conferring protection from apoptosis for a robust memory pool in vivo. We demonstrate using the MHC class I-restricted 2C TCR and MHC class II-restricted AND TCR transgenic systems that TNFR2 regulates the threshold for clonal expansion of CD4 and CD8 T cell subsets in response to cognate Ag. Using a novel recombinant Listeria monocytogenes (rLM) expressing a secreted form of the 2C agonist peptide (SIY) to investigate the role of TNFR2 for T cell immunity in vivo, we found that TNFR2 controls the survival and accumulation of effector cells during the primary response. TNFR2 ؊/؊ CD8 T cells exhibit loss of protection from apoptosis that is correlated with diminished survivin and Bcl-2 expression. Null mutant mice were more susceptible to rLM-SIY challenge at high doses of primary infection, correlating with impaired LM-specific T cell response in the absence of TNFR2-mediated costimulation. Moreover, the resulting memory pools specific for SIY and listeriolysin O epitopes derived from rLM-SIY were diminished in TNFR2 ؊/؊ mice. Thus, examination of Ag-driven T cell responses revealed a hitherto unknown costimulatory function for TNFR2 in regulating T cell survival during the differentiation program elicited by intracellular pathogen in vivo.

Ag encounter by naive CD8 T cells initiates a developmental program consisting of cellular proliferation, changes in gene expression, and the formation of effector and memory T cells. The strength and duration of TCR signaling are known to be important parameters regulating the differentiation of naive CD8 T cells, although the molecular signals arbitrating these processes remain poorly defined. The Ras-guanyl nucleotide exchange factor RasGRP1 has been shown to transduce TCR-mediated signals critically required for the maturation of developing thymocytes. To elucidate the role of RasGRP1 in CD8 T cell differentiation, in vitro and in vivo experiments were performed with 2C TCR transgenic CD8 T cells lacking RasGRP1. In this study, we report that RasGRP1 regulates the threshold of T cell activation and Ag-induced expansion, at least in part, through the regulation of IL-2 production. Moreover, RasGRP1−/− 2C CD8 T cells exhibit an anergic phenotype in response to cognate Ag stimulati...

erate signaling results in positive selection and differentiation into either mature CD4 ϩ CD8 Ϫ or CD4 Ϫ CD8 ϩ (SP) T cells. However, the molecular mechanism for distinguishing signals that lead to positive and negative selection remains to be defined.

Caulobacters are biofilm-forming members of the natural flora of soil and aquatic environments, which exhibit several characteristics that make them attractive for development of high surface area microbial bioreactors or biosensors. Although caulobacters are well characterized genetically, little is known about their biofilm-forming characteristics as a monoculture, or their tolerance of bioreactor-like conditions. Here we investigated the ability of caulobacters to spontaneously form high-density monolayers on artificial surfaces under a variety of environmental conditions, using phase contrast image analysis to assess biofilm density, and epifluorescence with the vital stain DiBAC(tm) to assess viability. With adequate nutrition, extremely dense monolayers formed within 24-48 h, and maintained near 100% viability in experiments ranging up to 22 days. When areas were abraded to remove cells, repopulation occurred rapidly with characteristics similar to the population of a clean su...

ABSTRACTInnovative 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. Here, we demonstrate that the bacteriumCaulobacter crescentusand its highly expressed surface-layer (S-layer) protein can provide this antibody display platform. Caulobacters displaying protein G, alone or with CD4 codisplay, successfully captured HIV-1-specific antibodies and demonstrated functional neutralization. Compared to soluble antibodies, a neutralizing anti-HIV antibody displayed onCaulobacterwas as effective or more effective at neutralizing diverse HIV-1 isolates. Moreover, when an antibody reactive with an epitope induced by CD...

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.

ABSTRACTThis report describes a previously unrecognized role for bacterial surface layers as barriers that confer protection against antimicrobial peptides. As antimicrobial peptides exist in natural environments, S-layers may provide a bacterial survival mechanism that has been selected for through evolution.

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 β-1,4-glycanase (Cex) from the cellulolytic bacterium Cellulomonas fimi with the C-terminus of RsaA was evaluated. Cex/RsaA cleaved the glycosidic linkage in the artificial substrate p-nitrophenyl-β-D-cellobioside with a K M similar to that of native Cex (1.1 mM for Cex/RsaA vs 0.60 mM for Cex), indicating that the particulate Cex enzyme was able to bind substrate with wild-type affinity. By contrast, the k cat value was significantly reduced (0.08 s -1 for Cex/RsaA vs 15.8 s -1 for Cex), likely owing to the fact that the RsaA C-terminus induces spontaneous unstructured aggregation of the recombinant protein. Here, we demonstrated that not only can an RsaA fusion protein be cheaply produced and purified to a 96 Duncan et al.

There is a growing class of elementary particle detectors, large-water Cerenkov detectors, that have a body of water (thousands of tons) as a sensitive medium. Particles are detected when they interact with the water and produce Cerenkov light, so detection efficiency relies on the transparency of the water. These detectors are active typically for many years, so biological activity (primarily bacterial growth) is one of the means by which the transparency of the water may be reduced. We present the results of a measurement of light scattering and absorption from a population of Escherichia coli in water, which is used as a model for bacteria in general. One can separate the scattering and absorption by varying the refractive index of the medium by using a solute of high molecular weight. We show that the results can be understood simply in terms of light scattering from small spheres (radius wavelength) with an effective refractive index, nb, plus a small amount of absorption in the ultraviolet. We compare this scattering with Rayleigh scattering in pure water.

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. Secretion was mediated by the RsaA type 1 transporter since secretion was eliminated in transporter deficient strains or by C-terminal deletions in SapA (the presumed location of type 1 secretion signals). Secretion was required to become an active protease; mass spectrometry suggested this might be due to N-terminal processing during secretion, a feature shared with other type 1-secreted proteases. Overexpression leads to additional processing C-terminal to the protease domain, producing a 45-kDa protein. This was demonstrated to be self-processing. Deletion analysis revealed the C-terminal 100 amino acids were sufficient for anchoring and secretion. When protein G was fused to the last 238 amino acids of SapA it was secreted, surface attached and bound immunoglobulin, indicating potential for foreign protein display.

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). All contained genes predicted to encode ATP-binding cassette transporters and membrane fusion proteins highly similar to those of C. crescentus, indicating that the S-layer proteins were all secreted by a type I system. The S-layer proteins' C-termini showed unexpectedly low sequence similarity but contained conserved residues and predicted secondary structure features typical of type I secretion signals. Cross-expression studies showed that the 6 strains recognized secretion signals from C. crescentus and Pseudomonas aeruginosa and similarly that C. crescentus was able to sec...

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. Here we determined RsaA levels by quantitative immunoblotting methods, learned that very stable mRNA is a key factor in high-level secretion, and found that the transporter was capable of still higher secretion. A propensity for RsaA to aggregate was a barrier to quantitation, but with the use of S-layer shedding mutants and methods to keep RsaA soluble, we learned that ~31% of cell protein is RsaA. When multiple copies of rsaA were introduced, the level increased to ~51% of cell protein, a higher level than we are aware of for any protein in any bacterium. Unexpectedly, in comparing normal and S-layer shedding strains, an ...

The secreted endoglucanase (CenA) from the Gram-positive bacterium Cellulomonas fimi and a deletion derivative (ΔCenA) lacking the N-terminal leader peptide of native CenA were used to explore the potential of ΔCenA as a reporter molecule in Caulobacter crescentus. Expression of cenA in C. crescentus yielded extracellular endoglucanase activity, suggesting that the N-terminal leader peptide of CenA could direct the enzyme to the periplasm where it subsequently leaked into the medium. In contrast, expression of ΔcenA yielded only cell-associated endoglucanase activity; this suggested that the enzyme retained activity in the C. crescentus cytoplasm. Using the putative cytoplasmic and periplasmic forms of ΔCenA as markers, a simple assay for periplasmic ΔCenA hybrids was developed. This assay indicated that ΔCenA activity was largely independent of cellular location. To facilitate the use of ΔCenA as a reporter, a broad host range translational fusion vector (pEC215) incorporating Δcen...

Using a gene fusion approach, hybrid proteins were created by linking alkaline phosphatase (PhoA) or a cellulase reporter (ΔCenA) to four large N-terminal portions of the Caulobacter crescentus surface (S)-layer protein (RsaA; 1026 amino acids). Three of the sites (amino acids 189, 220, 315) were selected on the basis of TnphoA experiments that suggested the first 250–350 amino acids of RsaA could mediate export of PhoA from the cytoplasm while the fourth lay only 21 amino acids from the C-terminus. Expression of all fusions except rsaA(315):ΔcenA and rsaA(315):phoA was toxic to C. crescentus. None of the gene fusions were toxic when expressed by Escherichia coli DH5α, where all the hybrid proteins accumulated as inclusion bodies. The toxicity of hybrid proteins encoding 189, 220, and 1005 RsaA-derived amino acids was related to the nature of the hybrid protein itself because truncated RsaA peptides lacking their reporter domains were nontoxic. Further study of RsaA(ΔC21) showed tha...

The entire nucleotide sequence of the rsaA gene, encoding the paracrystalline surface (S) layer protein (RsaA) of Caulobacter crescentus CB15A, was determined. The rsaA gene encoded a protein of 1026 amino acids, with a predicted molecular weight of 98 132. Protease cleavage of mature RsaA protein and amino acid sequencing of retrievable peptides yielded two peptides: one aligned with a region approximately two-thirds the way into the predicted amino acid sequence and the second peptide corresponded to the predicted carboxy terminus. Thus, no cleavage processing of the carboxy portion of the RsaA protein occurred during export, and with the exception of the removal of the initial methionine residue, the protein was not processed by cleavage to produce the mature protein. The predicted RsaA amino acid profile was unusual, with small neutral residues predominating. Excepting aspartate, charged amino acids were in relatively low proportion, resulting in an especially acidic protein, wi...

The paracrystalline surface layer (S-layer) of Caulobacter crescentus is composed of a single protein (RsaA, 1026 amino acids) that associates noncovalently with the lipopolysaccharide of the outer membrane. Like many other extracellular proteins of Gram-negative bacteria, the S-layer protein is not processed during transport to the cell surface. To study the secretion of RsaA, several N-terminal deletions of the protein were made by modifying the 5′-region of the rsaA gene. This analysis showed that portions of the N-terminus totalling the first 775 N-terminal amino acids (75% of the protein) could be removed from RsaA without abolishing secretion of the remainder of the protein. Although the RsaA N-terminus was not required for secretion, an N-terminal domain consisting of either 34 or 52 RsaA-derived amino acids promoted export of the alkaline phosphatase reporter (PhoA) and a cellulase reporter (ΔCenA) from the cytoplasm; using the cellulase reporter, the efficiency of hybrid pr...

Abstract: This project was focussed on issues related to tne adhesion device used by marine and freshwater biofouling Caulobacter bacteria. The longterm goal of these studies was to improve our understanding of how fouling bacteria attach to surfaces and to consider ...