The Caulobacter crescentus Paracrystalline S-Layer Protein Is Secreted by an ABC Transporter (Type I) Secretion Apparatus

Caulobacter crescentus is a gram-negative bacterium that produces a two-dimensional crystalline array on its surface composed of a single 98-kDa protein, RsaA. Secretion of RsaA to the cell surface relies on an uncleaved C-terminal secretion signal. In this report, we identify two genes encoding components of the RsaA secretion apparatus. These components are part of a type I secretion system involving an ABC transporter protein. These genes, lying immediately 3′ of rsaA, were found by screening a Tn5 transposon library for the loss of RsaA transport and characterizing the transposon-interrupted genes. The two proteins presumably encoded by these genes were found to have significant sequence similarity to ABC transporter and membrane fusion proteins of other type I secretion systems. The greatest sequence similarity was found to the alkaline protease (AprA) transport system of Pseudomonas aeruginosa and the metalloprotease (PrtB) transport system of Erwinia chrysanthemi. The prtB and aprA genes were introduced into C. crescentus, and their products were secreted by the RsaA transport system. Further, defects in the S-layer protein transport system led to the loss of this heterologous secretion. This is the first report of an S-layer protein secreted by a type I secretion apparatus. Unlike other type I secretion systems, the RsaA transport system secretes large amounts of its substrate protein (it is estimated that RsaA accounts for 10 to 12% of the total cell protein). Such levels are expected for bacterial S-layer proteins but are higher than for any other known type I secretion system.     

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. 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.     

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). 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 secrete the S-layer protein C-terminus of 1 strain examined. Inactivation of the ATP-binding cassette transporter abolished S-layer protein secretion, indicating that the type I transporter is necessary for S-layer protein secretion. Finally, while all of the S-layer proteins of this subset of strains were secreted by type I mechanisms, there were significant differences in genome positions of the transporter genes that correlated with S-layer protein size.     

新月柄杆菌RsaA分泌系统用于大肠杆菌胞外递送重组蛋白的初步研究

目的：构建基于新月柄杆菌RsaA外运机制的以大肠杆菌为宿主的原核胞外分泌表达载体系统。方法：利用分子克隆手段,按RsaA分泌系统操纵子组织方式,将RsaA系统外运功能基因配合以异源调控序列克隆至pQE30骨架质粒。以绿色荧光蛋白（GFP）为报告分子、大肠杆菌M15为宿主茵,诱导表达后通过Western Blotting检测培养上清中GFP的表达。结果：获得了与设计完全一致的pQABPS载体,利用该载体系统,在培养上清中报告分子GFP的表达明显增加,且是通过特异的RsaA外运机制被分泌至胞外的,而非渗漏表达或简单的信号肽引导。结论：在大肠杆菌中重现了RsaA分泌系统的外运功能,为该系统在基因工程领域的应用研究打下了良好基础。     

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. In doing so, we found that the RsaA anchoring region lies in the first approximately 225 amino acids and that this RsaA anchoring region requires a smooth lipopolysaccharide species found in the outer membrane. By making mutations at six semirandom sites, we learned that relatively minor perturbations within the first approximately 225 amino acids of RsaA caused loss of anchoring. In other studies, we confirmed that only this N-terminal region has a direct role in S-layer anchoring. As a by-product of the anchoring studies, we discovered that Sap, the C. crescentus S-layer-associated protease, recognized a cleavage site in the truncated RsaA fragments that is not detected by Sap in full-length RsaA. This, in turn, led to the discovery that Sap was an extracellular membrane-bound protease, rather than intracellular, as previously proposed. Moreover, Sap was secreted to the cell surface primarily by the S-layer type I secretion apparatus.     

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. The cleavage was precise, resulting in fragments with an unambiguous N-terminal sequence. We developed an assay to screen for loss of this activity (i.e., presentation of foreign peptides without degradation), using transposon and traditional mutagenesis. A metalloprotease gene designated sap (S-layer-associated protease) was identified which could complement the protease-negative mutants. The N-terminal half of Sap possessed significant similarity to other type I secreted proteases (e.g., alkaline protease of Pseudomonas aeruginosa), including the characteristic RTX repeat sequences, but the C-terminal half which normally includes the type I secretion signal exhibited no such similarity. Instead, there was a region of significant similarity to the N-terminal region of RsaA. We hypothesize that Sap evolved by combining the catalytic portion of a type I secreted protease with an S-layer-like protein, perhaps to associate with nascent S-layer monomers to "scan" for modifications.     

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.     

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. 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.     

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

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, with a predicted pI of 3.46. As with most other sequenced S-layer proteins, RsaA contained no cysteine residues. A homology scan of the Swiss Protein Bank 17 produced no close matches to the predicted RsaA sequence. However, RsaA protein shared measurable homology with some exported proteins of other bacteria, including the hemolysins. Of particular interest was a specific region of the RsaA protein that was homologous to the repeat regions of glycine and aspartate residues found in several proteases and hemolysins. These repeats are implicated in the binding of calcium for proper structure and biological activity of these proteins. Those present in the RsaA protein may perform a similar function, since S-layer assembly and surface attachment requires calcium. RsaA protein also shared some homology with 10 other S-layer proteins, with the Campylobacter fetus S-layer protein scoring highest.     

Secretory delivery of recombinant proteins in attenuated Salmonella strains: potential and limitations of Type I protein transporters

Live attenuated Salmonella strains have been extensively explored as oral delivery systems for recombinant vaccine antigens and effector proteins with immunoadjuvant and immunomodulatory potential. The feasibility of this approach was demonstrated in human vaccination trials for various antigens. However, immunization efficiencies with live vaccines are generally significantly lower compared to those monitored in parenteral immunizations with the same vaccine antigen. This is, at least partly, due to the lack of secretory expression systems, enabling large-scale extracellular delivery of vaccine and effector proteins by these strains. Because of their low complexity and the terminal location of the secretion signal in the secreted protein, Type I (ATP-binding cassette) secretion systems appear to be particularly suited for development of such recombinant extracellular expression systems. So far, the Escherichia coli hemolysin system is the only Type I secretion system, which has been adapted to recombinant protein secretion in Salmonella. However, this system has a number of disadvantages, including low secretion capacity, complex genetic regulation, and structural restriction to the secreted protein, which eventually hinder high-level in vivo delivery of recombinant vaccines and effector proteins. Thus, the development of more efficient recombinant protein secretion systems, based on Type I exporters can help to improve efficacies of live recombinant Salmonella vaccines. Type I secretion systems, mediating secretion of bacterial surface layer proteins, such as RsaA in Caulobacter crescentus, are discussed as promising candidates for improved secretory delivery systems.     

基于新月柄杆菌RsaA外运机制的EspA及EspA-IL-24融合蛋白胞外分泌表达研究

目的：实现大肠杆菌分泌蛋白（Esp）A及EspA与白细胞介素（IL）-24融合蛋白的胞外分泌表达,进一步验证基于新月柄杆菌RsaA外运机制的原核胞外分泌表达载体系统的有效性和通用性,并改造优化该系统。方法：利用分子克隆手段,按RsaA分泌系统操纵子组织方式,将获得的RsaA系统元件编码序列和异源调控序列克隆至pQE30骨架质粒,构建新的胞外分泌表达质粒pQABP2S;以大肠杆菌为宿主菌诱导表达EspA及EspA-IL-24融合蛋白,并通过Westernblot检测目标蛋白在培养上清中的表达。结果：获得了新的胞外分泌表达载体pQABP2S;与对照相比,该载体宿主系统培养上清中目标蛋白EspA及EspA-IL-24的表达量明显增加。结论：在大肠杆菌中通过RsaA分泌系统可实现分子大小不同的EspA及EspA-IL-24融合蛋白的特异性分泌表达,进一步证实该分泌表达策略的有效性和通用性;调整调控序列以优化分泌系统的尝试,为此类基因工程技术平台的开发提供了借鉴。     

The Caulobacter crescentus outer membrane protein Omp58 (RsaF) is not required for paracrystalline S-layer secretion

To identify the outer membrane protein component of the Caulobacter crescentus CB2 surface-layer export machinery we used the Serratia marcescens LipD protein to find homologs in the CB2 genome. From two homologous sequences found, one encodes a putative OMP with a predicted molecular mass of 57.5 kDa, termed Omp58 (formerly RsaF). Comparison of membrane protein profiles revealed a protein with an appropriate molecular mass present in wild-type, but not CB2 omp58::kanamycin, a mutant strain with an inactivated omp58 gene. Disruption of omp58 did not affect surface-layer production, suggesting that Omp58 is not involved in surface-layer protein secretion and, thus, may not be the outer membrane protein component of the C. crescentus surface-layer export system.     

A deficiency of microsomal triglyceride transfer protein reduces apolipoprotein B secretion

Microsomal triglyceride transfer protein (MTP) transfers lipids to apolipoprotein B (apoB) within the endoplasmic reticulum, a process that involves direct interactions between apoB and the large subunit of MTP. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. We hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of half-normal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the MTP:apoB ratio would be even lower). To test this hypothesis, we examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to our expectations, half-normal levels of MTP reduced the plasma apoB100 levels to the same extent ( approximately 25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum rather than the MTP:apoB ratio is the critical determinant of lipoprotein secretion. Finally, we found that heterozygosity for an apoB knockout mutation lowered plasma apoB100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice.     

Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels.

Aquaporin-5 (AQP5) is a water-selective transporting protein expressed in epithelial cells of serous acini in salivary gland. We generated AQP5 null mice by targeted gene disruption. The genotype distribution from intercross of founder AQP5 heterozygous mice was 70:69:29 wild-type:heterozygote:knockout, indicating impaired prenatal survival of the null mice. The knockout mice had grossly normal appearance, but grew approximately 20% slower than litter-matched wild-type mice when placed on solid food after weaning. Pilocarpine-stimulated saliva production was reduced by more than 60% in AQP5 knockout mice. Compared with the saliva from wild-type mice, the saliva from knockout mice was hypertonic (420 mosM) and dramatically more viscous. Amylase and protein secretion, functions of salivary mucous cells, were not affected by AQP5 deletion. Water channels AQP1 and AQP4 have also been localized to salivary gland; however, pilocarpine stimulation studies showed no defect in the volume or composition of saliva in AQP1 and AQP4 knockout mice. These results implicate a key role for AQP5 in saliva fluid secretion and provide direct evidence that high epithelial cell membrane water permeability is required for active, near-isosmolar fluid transport.