Molecular characterization of the Salmonella enterica serovar Typhi Vi-typing bacteriophage E1

Some bacteriophages target potentially pathogenic bacteria by exploiting surface-associated virulence factors as receptors. For example, phage have been identified that exhibit specificity for Vi capsule producing Salmonella enterica serovar Typhi. Here we have characterized the Vi-associated E1-typing bacteriophage using a number of molecular approaches. The absolute requirement for Vi capsule expression for infectivity was demonstrated using different Vi-negative S. enterica derivatives. The phage particles were shown to have an icosahedral head and a long noncontractile tail structure. The genome is 45,362 bp in length with defined capsid and tail regions that exhibit significant homology to the S. enterica transducing phage ES18. Mass spectrometry was used to confirm the presence of a number of hypothetical proteins in the Vi phage E1 particle and demonstrate that a number of phage proteins are modified posttranslationally. The genome of the Vi phage E1 is significantly related to other bacteriophages belonging to the same serovar Typhi phage-typing set, and we demonstrate a role for phage DNA modification in determining host specificity.     

An AIL family protein promotes type three secretion system‐1‐independent invasion and pathogenesis of Salmonella enterica serovar Typhi

Adhesion and invasion of Intestinal Epithelial Cells (IECs) are critical for the pathogenesis of Salmonella Typhi, the aetiological agent of human typhoid fever. While type three secretion system‐1 (T3SS‐1) is a major invasion apparatus of Salmonella, independent invasion mechanisms were described for non‐typhoidal Salmonellae. Here, we show that T2942, an AIL‐like protein of S. Typhi Ty2 strain, is required for adhesion and invasion of cultured IECs. That invasion was T3SS‐1 independent was proved by ectopic expression of T2942 in the non‐invasive E. coli BL21 and double‐mutant Ty2 (Ty2Δt2942ΔinvG) strains. Laminin and fibronectin were identified as the host‐binding partners of T2942 with higher affinity for laminin. Standalone function of T2942 was confirmed by cell adhesion of the recombinant protein, while the protein or anti‐T2942 antiserum blocked adhesion/invasion of S. Typhi, indicating specificity. A 20‐amino acid extracellular loop was required for invasion, while several loop regions of T2942 contributed to adhesion. Further, T2942 cooperates with laminin‐binding T2544 for adhesion and T3SS‐1 for invasion. Finally, T2942 was required and synergistically worked with T3SS‐1 for pathogenesis of S. Typhi in mice. Considering wide distribution of T2942 among clinical strains, the protein or the 20‐mer peptide may be suitable for vaccine development.     

The Skp1‐like protein SSK1 is required for cross‐pollen compatibility in S‐RNase‐based self‐incompatibility

The self‐incompatibility (SI) response occurs widely in flowering plants as a means of preventing self‐fertilization. In these self/non‐self discrimination systems, plant pistils reject self or genetically related pollen. In the Solanaceae, Plantaginaceae and Rosaceae, pistil‐secreted S‐RNases enter the pollen tube and function as cytotoxins to specifically arrest self‐pollen tube growth. Recent studies have revealed that the S‐locus F‐box (SLF) protein controls the pollen expression of SI in these families. However, the precise role of SLF remains largely unknown. Here we report that PhSSK1 (Petunia hybrida SLF‐interacting Skp1‐like1), an equivalent of AhSSK1 of Antirrhinum hispanicum, is expressed specifically in pollen and acts as an adaptor in an SCF(Skp1‐Cullin1‐F‐box)^SLF complex, indicating that this pollen‐specific SSK1‐SLF interaction occurs in both Petunia and Antirrhinum, two species from the Solanaceae and Plantaginaceae, respectively. Substantial reduction of PhSSK1 in pollen reduced cross‐pollen compatibility (CPC) in the S‐RNase‐based SI response, suggesting that the pollen S determinant contributes to inhibiting rather than protecting the S‐RNase activity, at least in solanaceous plants. Furthermore, our results provide an example that a specific Skp1‐like protein other than the known conserved ones can be recruited into a canonical SCF complex as an adaptor.     

The GmFWL1 (FW2‐2‐like) nodulation gene encodes a plasma membrane microdomain‐associated protein

The soybean gene GmFWL1 (FW2‐2‐like1) belongs to a plant‐specific family that includes the tomato FW2‐2 and the maize CNR1 genes, two regulators of plant development. In soybean, GmFWL1 is specifically expressed in root hair cells in response to rhizobia and in nodules. Silencing of GmFWL1 expression significantly reduced nodule numbers supporting its role during soybean nodulation. While the biological role of GmFWL1 has been described, its molecular function and, more generally, the molecular function of plant FW2‐2‐like proteins is unknown. In this study, we characterized the role of GmFWL1 as a membrane microdomain‐associated protein. Specifically, using biochemical, molecular and cellular methods, our data show that GmFWL1 interacts with various proteins associated with membrane microdomains such as remorin, prohibitins and flotillins. Additionally, comparative genomics revealed that GmFWL1 interacts with GmFLOT2/4 (FLOTILLIN2/4), the soybean ortholog to Medicago truncatula FLOTILLIN4, a major regulator of the M. truncatula nodulation process. We also observed that, similarly to MtFLOT4 and GmFLOT2/4, GmFWL1 was localized at the tip of the soybean root hair cells in response to rhizobial inoculation supporting the early function of GmFWL1 in the rhizobium infection process.     

Multi‐isotype antibody responses against the multimeric Salmonella Typhi recombinant hemolysin E antigen

The detection and measurement of different antibody isotypes in the serum provide valuable indicators of the different stages of typhoid infection. Here, the ability of S. Typhi recombinant hemolysin E (HlyE) to detect multi‐isotype antibody responses in sera of patients with typhoid and paratyphoid A was investigated using an indirect antibody immunoassay. Nanogram amounts of HlyE were found to be sufficient for detection of IgG and IgA isotypes and, in a study of individuals' sera (n = 100), the immunoassay was able to distinguish between typhoid and non‐typhoid sera. The overall sensitivity, specificity and efficiency of the ELISA were 70% (39/56), 100% (44/44) and 83% respectively.     

The Salmonella enterica giant adhesin SiiE binds to polarized epithelial cells in a lectin‐like manner

The invasion of polarized epithelial cells by Salmonella enterica requires the cooperative activity of the Salmonella pathogenicity island (SPI) 1‐encoded type III secretion system (T3SS) and the SPI4‐encoded giant non‐fimbrial adhesin SiiE. SiiE is a highly repetitive protein composed of 53 bacterial Ig (BIg) domains and mediates binding to the apical side of polarized epithelial cells. We analysed the binding properties of SiiE and observed lectin‐like activity. SiiE‐dependent cell invasion can be ablated by chemical or enzymatic deglycosylation. Lectin blockade experiments revealed that SiiE binding is specific for glycostructures with terminal N‐acetyl‐glucosamine (GlcNAc) and/or α 2,3‐linked sialic acid. In line with these data, we found that SiiE‐expressing Salmonella bind to the GlcNAc polymer chitin. Various recombinant SiiE fragments were analysed for host cell binding. We observed that C‐terminal portions of SiiE bind to the apical side of polarized cells and the intensity of binding increases with the number of BIg domains present in the recombinant proteins. Based on these results, we propose that SiiE mediates multiple interactions per molecule with glycoproteins and/or glycosylated phospholipids present in the apical membrane of polarized epithelial cells. Thisintimate binding enables the subsequent function of the SPI1‐T3SS, resulting in host cell invasion.     

The protein interacting with C‐kinase (PICK1) interacts with and attenuates parkin‐associated endothelial‐like (PAEL) receptor‐mediated cell death

The parkin‐associated endothelial‐like receptor (PAELR, GPR37) is an orphan G protein‐coupled receptor that interacts with and is degraded by parkin‐mediated ubiquitination. Mutations in parkin are thought to result in PAELR accumulation and increase neuronal cell death in Parkinson's disease. In this study, we find that the protein interacting with C‐kinase (PICK1) interacts with PAELR. Specifically, the Postsynaptic density protein‐95/Discs large/ZO‐1 (PDZ) domain of PICK1 interacted with the last three residues of the c‐terminal (ct) located PDZ motif of PAELR. Pull‐down assays indicated that recombinant and native PICK1, obtained from heterologous cells and rat brain tissue, respectively, were retained by a glutathione S‐transferase fusion of ct‐PAELR. Furthermore, coimmunoprecipitation studies isolated a PAELR‐PICK1 complex from transiently transfected cells. PICK1 interacts with parkin and our data showed that PICK1 reduces PAELR expression levels in transiently transfected heterologous cells compared to a PICK1 mutant that does not interact with PAELR. Finally, PICK1 over‐expression in HEK293 cells reduced cell death induced by PAEALR over‐expression during rotenone treatment and these effects of PICK1 were attenuated during inhibition of the proteasome. These results suggest a role for PICK1 in preventing PAELR‐induced cell toxicity.

     

The H‐NS‐like protein StpA represses the RpoS (σ38) regulon during exponential growth of Salmonella Typhimurium

StpA is a paralogue of the nucleoid‐associated protein H‐NS that is conserved in a range of enteric bacteria and had no known function in Salmonella Typhimurium. We show that 5% of the Salmonella genome is regulated by StpA, which contrasts with the situation in Escherichia coli where deletion of stpA only had minor effects on gene expression. The StpA‐dependent genes of S. Typhimurium are a specific subset of the H‐NS regulon that are predominantly under the positive control of σ³⁸ (RpoS), CRP‐cAMP and PhoP. Regulation by StpA varied with growth phase; StpA controlled σ³⁸ levels at mid‐exponential phase by preventing inappropriate activation of σ³⁸ during rapid bacterial growth. In contrast, StpA only activated the CRP‐cAMP regulon during late exponential phase. ChIP‐chip analysis revealed that StpA binds to PhoP‐dependent genes but not to most genes of the CRP‐cAMP and σ³⁸ regulons. In fact, StpA indirectly regulates σ³⁸‐dependent genes by enhancing σ³⁸ turnover by repressing the anti‐adaptor protein rssC. We discovered that StpA is essential for the dynamic regulation of σ³⁸ in response to increased glucose levels. Our findings identify StpA as a novel growth phase‐specific regulator that plays an important physiological role by linking σ³⁸ levels to nutrient availability.     

Expression of a recombinant elastin‐like protein in pichia pastoris

The translation of highly repetitive gene sequences is often associated with reduced levels of protein expression and may be prone to mutational events. In this report, we describe a modified concatemerization strategy to construct a gene with enhanced sequence diversity that encodes a highly repetitive elastin‐like protein polymer for expression in Pichia pastoris. Specifically, degenerate oligonucleotides were used to create a monomer library, which after concatemerization yielded a genetically nonrepetitive DNA sequence that encoded identical pentapeptide repeat sequences. By limiting genetic repetition, the risk of genetic deletions, rearrangements, or premature termination errors during protein synthesis is minimized. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Intrinsically disordered inhibitor of glutamine synthetase is a functional protein with random‐coil‐like pKa values

The sequential action of glutamine synthetase (GS) and glutamate synthase (GOGAT) in cyanobacteria allows the incorporation of ammonium into carbon skeletons. In the cyanobacterium Synechocystis sp. PCC 6803, the activity of GS is modulated by the interaction with proteins, which include a 65‐residue‐long intrinsically disordered protein (IDP), the inactivating factor IF7. This interaction is regulated by the presence of charged residues in both IF7 and GS. To understand how charged amino acids can affect the binding of an IDP with its target and to provide clues on electrostatic interactions in disordered states of proteins, we measured the pK_a values of all IF7 acidic groups (Glu32, Glu36, Glu38, Asp40, Asp58, and Ser65, the backbone C‐terminus) at 100 mM NaCl concentration, by using NMR spectroscopy. We also obtained solution structures of IF7 through molecular dynamics simulation, validated them on the basis of previous experiments, and used them to obtain theoretical estimates of the pK_a values. Titration values for the two Asp and three Glu residues of IF7 were similar to those reported for random‐coil models, suggesting the lack of electrostatic interactions around these residues. Furthermore, our results suggest the presence of helical structure at the N‐terminus of the protein and of conformational changes at acidic pH values. The overall experimental and in silico findings suggest that local interactions and conformational equilibria do not play a role in determining the electrostatic features of the acidic residues of IF7.     

Proteome targets of ubiquitin‐like samp1ylation are associated with sulfur metabolism and oxidative stress in Haloferax volcanii

Small archeal modifier proteins (SAMPs) are related to ubiquitin in tertiary structure and in their isopeptide linkage to substrate proteins. SAMPs also function in sulfur mobilization to form biomolecules such as molybdopterin and thiolated tRNA. While SAMP1 is essential for anaerobic growth and covalently attached to lysine residues of its molybdopterin synthase partner MoaE (K240 and K247), the full diversity of proteins modified by samp1ylation is not known. Here, we expand the knowledge of proteins isopeptide linked to SAMP1. LC‐MS/MS analysis of ‐Gly‐Gly signatures derived from SAMP1 S85R conjugates cleaved with trypsin was used to detect sites of sampylation (23 lysine residues) that mapped to 11 target proteins. Many of the identified target proteins were associated with sulfur metabolism and oxidative stress including MoaE, SAMP‐activating E1 enzyme (UbaA), methionine sulfoxide reductase homologs (MsrA and MsrB), and the Fe‐S assembly protein SufB. Several proteins were found to have multiple sites of samp1ylation, and the isopeptide linkage at SAMP3 lysines (K18, K55, and K62) revealed hetero‐SAMP chain topologies. Follow‐up affinity purification of selected protein targets (UbaA and MoaE) confirmed the LC‐MS/MS results. 3D homology modeling suggested sampy1ylation is autoregulatory in inhibiting the activity of its protein partners (UbaA and MoaE), while occurring on the surface of some protein targets, such as SufB and MsrA/B. Overall, we provide evidence that SAMP1 is a ubiquitin‐like protein modifier that is relatively specific in tagging its protein partners as well as proteins associated with oxidative stress response.     

Ubiquitin‐specific protease‐like 1 (USPL1) is a SUMO isopeptidase with essential,non‐catalytic functions

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity‐based search with the suicide inhibitor haemagglutinin (HA)‐SUMO‐vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin‐specific protease‐like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l—an essential but distant zebrafish homologue of USPL1—also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low‐abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.     

Microtubule‐binding protein doublecortin‐like kinase 1 (DCLK1) guides kinesin‐3‐mediated cargo transport to dendrites

In neurons, the polarized distribution of vesicles and other cellular materials is established through molecular motors that steer selective transport between axons and dendrites. It is currently unclear whether interactions between kinesin motors and microtubule‐binding proteins can steer polarized transport. By screening all 45 kinesin family members, we systematically addressed which kinesin motors can translocate cargo in living cells and drive polarized transport in hippocampal neurons. While the majority of kinesin motors transport cargo selectively into axons, we identified five members of the kinesin‐3 (KIF1) and kinesin‐4 (KIF21) subfamily that can also target dendrites. We found that microtubule‐binding protein doublecortin‐like kinase 1 (DCLK1) labels a subset of dendritic microtubules and is required for KIF1‐dependent dense‐core vesicles (DCVs) trafficking into dendrites and dendrite development. Our study demonstrates that microtubule‐binding proteins can provide local signals for specific kinesin motors to drive polarized cargo transport.     

Engineering a recyclable elastin‐like polypeptide capturing scaffold for non‐chromatographic protein purification

Previously, we reported a non‐chromatographic protein purification method exploiting the highly specific interaction between the dockerin and cohesin domains from Clostridium thermocellum and the reversible aggregation property of elastin‐like polypeptide (ELP) to provide fast and cost‐effective protein purification. However, the bound dockerin‐intein tag cannot be completely dissociated from the ELP‐cohesin capturing scaffold due to the high binding affinity, resulting in a single‐use approach. In order to further reduce the purification cost by recycling the ELP capturing scaffold, a truncated dockerin domain with the calcium‐coordinating function partially impaired was employed. We demonstrated that the truncated dockerin domain was sufficient to function as an effective affinity tag, and the target protein was purified directly from cell extracts in a single binding step followed by intein cleavage. The efficient EDTA‐mediated dissociation of the bound dockerin‐intein tag from the ELP‐cohesin capturing scaffold was realized, and the regenerated ELP capturing scaffold was reused in another purification cycle without any decrease in the purification efficiency. This recyclable non‐chromatographic based affinity method provides an attractive approach for efficient and cost‐effective protein purification. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:968–971, 2013     

The microtubule‐associated kinase‐like protein RUNKEL functions in somatic and syncytial cytokinesis

The microtubule (MT)‐associated putative kinase RUNKEL (RUK) is an important component of the phragmoplast machinery involved in cell plate formation in Arabidopsis somatic cytokinesis. Since loss‐of‐function ruk mutants display seedling lethality, it was previously not known whether RUK functions in mature sporophytes or during gametophyte development. In this study we utilized RUK proteins that lack the N‐terminal kinase domain to further examine biological processes related to RUK function. Truncated RUK proteins when expressed in wild‐type Arabidopsis plants cause cellularization defects not only in seedlings and adult tissues but also during male meiocyte development, resulting in abnormal pollen and reduced fertility. Ultrastructural analysis of male tetrads revealed irregular and incomplete or absent intersporal cell walls, caused by disorganized radial MT arrays. Moreover, in ruk mutants endosperm cellularization defects were also caused by disorganized radial MT arrays. Intriguingly, in seedlings expressing truncated RUK proteins, the kinesin HINKEL, which is required for the activation of a mitogen‐activated protein kinase signaling pathway regulating phragmoplast expansion, was mislocalized. Together, these observations support a common role for RUK in both phragmoplast‐based cytokinesis in somatic cells and syncytial cytokinesis in reproductive cells.     

Follistatin‐like 1 in vertebrate development

Follistatin‐like 1 (Fstl1) is a member of the secreted protein acidic rich in cysteins (SPARC) family and has been implicated in many different signaling pathways, including bone morphogenetic protein (BMP) signaling. In many different developmental processes like, dorso‐ventral axis establishment, skeletal, lung and ureter development, loss of function experiments have unveiled an important role for Fstl1. Fstl1 largely functions through inhibiting interactions with the BMP signaling pathway, although, in various disease models, different signaling pathways, like activation of pAKT, pAMPK, Na/K‐ATPase, or innate immune responses, are linked to Fstl1. How Fstl1 inhibits BMP signaling remains unclear, although it is known that Fstl1 does not function through a scavenging mechanism, like the other known extracellular BMP inhibitors such as noggin. It has been proposed that Fstl1 interferes with BMP receptor complex formation and as such inhibits propagation of the BMP signal into the cell. Future challenges will encompass the identification of the factors that determine the mechanisms that underlie the fact that Fstl1 acts by interfering with BMP signaling during development, but through other signaling pathways during disease. Birth Defects Research (Part C) 99:61–69, 2013. © 2013 Wiley Periodicals, Inc.