Differences in the electrostatic surfaces of the type III secretion needle proteins PrgI, BsaL, and MxiH

Gram-negative bacteria use a needle-like protein assembly, the type III secretion apparatus, to inject virulence factors into target cells to initiate human disease. The needle is formed by the polymerization of approximately 120 copies of a small acidic protein that is conserved among diverse pathogens. We previously reported the structure of the BsaL needle monomer from Burkholderia pseudomallei by nuclear magnetic resonance (NMR) spectroscopy and others have determined the crystal structure of the Shigella flexneri MxiH needle. Here, we report the NMR structure of the PrgI needle protein of Salmonella typhimurium, a human pathogen associated with food poisoning. PrgI, BsaL, and MxiH form similar two helix bundles, however, the electrostatic surfaces of PrgI differ radically from those of BsaL or MxiH. In BsaL and MxiH, a large negative area is on a face formed by the helix alpha1-alpha2 interface. In PrgI, the major negatively charged surface is not on the "face" but instead is on the "side" of the two-helix bundle, and only residues from helix alpha1 contribute to this negative region. Despite being highly acidic proteins, these molecules contain large basic regions, suggesting that electrostatic contacts are important in needle assembly. Our results also suggest that needle-packing interactions may be different among these bacteria and provide the structural basis for why PrgI and MxiH, despite 63% sequence identity, are not interchangeable in S. typhimurium and S. flexneri.     

Synthesis and localization of the Salmonella SPI-1 type III secretion needle complex proteins PrgI and PrgJ

An essential component of type III secretion systems (TTSS) is a supramolecular structure termed the needle complex. In Salmonella enterica, at least four proteins make up this structure: InvG, PrgH, PrgK, and PrgI. Another protein, PrgJ, is thought to play a role in the assembly of this structure, but its function is poorly understood. We have analyzed the expression and localization of PrgJ and the needle protein PrgI in different S. enterica serovar Typhimurium mutant strains. We found that the levels of PrgI and PrgJ were significantly reduced in a TTSS-deficient invA mutant strain and that the decreased levels were due to protein instability. In addition, we found that PrgJ, although associated with the needle complex in wild-type S. enterica serovar Typhimurium, was absent from needle complexes obtained from an invJ mutant strain, which exhibits very long needle substructures. We suggest that PrgJ is involved in capping the needle substructure of the needle complex.     

Role of the Salmonella pathogenicity island 1 (SPI-1) protein InvB in type III secretion of SopE and SopE2, two Salmonella effector proteins encoded outside of SPI-1

Salmonella enterica subspecies 1 serovar Typhimurium encodes a type III secretion system (TTSS) within Salmonella pathogenicity island 1 (SPI-1). This TTSS injects effector proteins into host cells to trigger invasion and inflammatory responses. Effector proteins are recognized by the TTSS via signals encoded in their N termini. Specific chaperones can be involved in this process. The chaperones InvB, SicA, and SicP are encoded in SPI-1 and are required for transport of SPI-1-encoded effectors. Several key effector proteins, like SopE and SopE2, are located outside of SPI-1 but are secreted in an SPI-1-dependent manner. It has not been clear how these effector proteins are recognized by the SPI-1 TTSS. Using pull-down and coimmunoprecipitation assays, we found that SopE is copurified with InvB, the known chaperone for the SPI-1-encoded effector protein Sip/SspA. We also found that InvB is required for secretion and translocation of SopE and SopE2 and for stabilization of SopE2 in the bacterial cytosol. Our data demonstrate that effector proteins encoded within and outside of SPI-1 use the same chaperone for secretion via the SPI-1 TTSS.     

Analysis of the expression, secretion and translocation of the Salmonella enterica type III secretion system effector SteA

Many Gram-negative pathogens possess virulence-related type III secretion systems. Salmonella enterica uses two of these systems, encoded on the pathogenicity islands SPI-1 and SPI-2, respectively, to translocate more than 30 effector proteins into eukaryotic host cells. SteA is one of the few effectors that can be translocated by both systems. We investigated the conditions affecting the synthesis of this effector, its secretion to culture media and its translocation into host cells. Whereas steA was expressed under a wide range of conditions, some factors, including low and high osmolarity, and presence of butyrate, decreased expression. SteA was efficiently secreted to the culture media under both SPI-1 and SPI-2 inducing conditions. The kinetics of translocation into murine macrophages and human epithelial cells was studied using fusions with the 3xFLAG tag, and fusions with CyaA from Bordetella pertussis. Translocation into macrophages under non-invasive conditions was mainly dependent on the SPI-2-encoded type III secretion system but some participation of the SPI-1 system was also detected 6 hours post-infection. Interestingly, both type III secretion systems had a relevant role in the translocation of SteA into epithelial cells. Finally, a deletion approach allowed the identification of the N-terminal signal necessary for translocation of this effector. The amino acid residues 1-10 were sufficient to direct translocation into host cells through both type III secretion systems. Our results provide new examples of functional overlapping between the two type III secretion systems of Salmonella.     

ACTH decreases the expression and secretion of apolipoprotein B in HepG2 cell cultures

Administration of adrenocorticotropic hormone (ACTH) has been shown to decrease plasma concentrations of apolipoprotein B (apoB) containing lipoproteins, including lipoprotein(a), in man. However, the mechanism behind this hypolipidemic effect is unknown. This study aimed at distinguishing between the main possibilities (increased elimination or decreased production of lipoproteins) using HepG2 cell cultures. Addition of ACTH to the cell culture medium selectively down-regulated apoB mRNA expression and apoB secretion in a dose-dependent manner. At 100 pmol/liter ACTH, the apoB mRNA level was about 40% lower than in the untreated cells, and the secretion of apoB into the medium was decreased to a similar extent. The expression and secretion of other apolipoproteins (apoA-I, apoE, and apoM), however, were not affected by ACTH. Under normal culture conditions the level of secretion of apoB from HepG2 cells is quite low. In the presence of 0.4 mmol/liter oleic acid secretion of apoB increased 3-fold, but this phenomenon was not seen in ACTH-treated cells. Binding and internalization of radiolabeled low density lipoprotein (LDL) by HepG2 cell, as well as LDL-receptor mRNA and scavenger receptor B-I mRNA levels, were not influenced by ACTH. In conclusion, ACTH directly and selectively down-regulated the production and secretion of apoB in HepG2 cell cultures, suggesting that a principal mechanism behind the cholesterol-lowering effect of ACTH in vivo may be a decreased production rate of apoB-containing lipoproteins from the liver.     

Characterization of soluble complexes of the Shigella flexneri type III secretion system ATPase

The conserved ATPase of type III secretion systems is critical to the export of substrates through the apparatus. We present a characterization of the native T3SS ATPase, Spa47, from the cytoplasm of Shigella flexneri, demonstrating it to be in two distinct high-molecular-weight complexes with Spa33: MxiN and MxiK.     

Characterization of Nops,nodulation outer proteins,secreted via the type III secretion system of NGR234

The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secretion system (TTSS), proteins called Nops (nodulation outer proteins). Abolition of TTSS-dependent protein secretion has either no effect or leads to a change in the number of nodules on selected plants. More dramatically, Nops impair nodule development on Crotalaria juncea roots, resulting in the formation of nonfixing pseudonodules. A double mutation of nopX and nopL, which code for two previously identified secreted proteins, leads to a phenotype on Pachyrhizus tuberosus differing from that of a mutant in which the TTSS is not functional. Use of antibodies and a modification of the purification protocol revealed that NGR234 secretes additional proteins in a TTSS-dependent manner. One of them was identified as NopA, a small 7-kDa protein. Single mutations in nopX and nopL were also generated to assess the involvement of each Nop in protein secretion and nodule formation. Mutation of nopX had little effect on NopL and NopA secretion but greatly affected the interaction of NGR234 with many plant hosts tested. NopL was not necessary for the secretion of any Nops but was required for efficient nodulation of some plant species. NopL may thus act as an effector protein whose recognition is dependent upon the hosts' genetic background.     

pH-dependent secretion of SseB, a product of the SPI-2 type III secretion system of Salmonella typhimurium

The type III secretion system of Salmonella pathogenicity island 2 (SPI-2) is required for bacterial replication inside macrophages. SseB has been considered a putative target of the secretion system on the basis of its similarity with EspA, a protein secreted by the type III secretion system of enteropathogenic Escherichia coli (EPEC). EspA forms a filamentous structure on the bacterial cell surface and is involved in translocation of proteins into the eukaryotic cytosol. In this paper, we show that SseB is a secreted protein that associates with the surface of the bacterial cell and might, therefore, also be required for delivery of SPI-2 effector proteins to the eukaryotic cell cytosol. SseB begins to accumulate inside the bacterial cell when the culture enters early stationary phase. However, SseB is only secreted if the bacteria are grown at low pH or if the pH is shifted after growth from 7.0 to below pH 5.0. The secretion occurs within minutes of acidification and is totally dependent on a functional SPI-2 type III secretion system. As the pH of the Salmonella-containing vacuole inside host cells has been shown to acidify to between pH 4.0 and 5.0, and as SPI-2 gene expression occurs inside host cells, low pH might be a physiological stimulus for SPI-2-mediated secretion in vivo.     

Biogenesis, regulation, and targeting of the type III secretion system

The type III secretion system (T3SS) is employed by a number of Gram-negative bacterial pathogens to inject toxins into eukaryotic cells. The biogenesis of this complex machinery requires the regulated interaction between over 20 cytosolic, periplasmic, and membrane-imbedded proteins, many of which undergo processes such as polymerization, partner recognition, and partial unfolding. Elements of this intricate macromolecular system have been characterized through electron microscopy, crystallography, and NMR techniques, allowing for an initial understanding of the spatiotemporal regulation of T3SS-related events. Here, we report recent advances in the structural characterization of T3SS proteins from a number of bacteria, and provide an overview of recently identified small molecule T3SS inhibitors that could potentially be explored for novel antibacterial development.     

solution structure, backbone dynamics, and interaction with Cdc42 of Salmonella guanine nucleotide exchange factor SopE2

SopE and SopE2 are delivered by the Salmonella type III secretion system into eukaryotic cells to promote cell invasion. SopE and SopE2 are potent guanine nucleotide exchange factors (GEFs) for Rho GTPases Cdc42 and Rac1 and constitute a novel class of Rho GEFs. Although the sequence of SopE-like GEFs is not at all homologous to those of the Dbl homology domain-containing eukaryotic GEFs, the mechanism of nucleotide release seems to have significant similarities. We have determined the solution structure of the catalytic domain (residues 69-240) of SopE2, showing that SopE2(69-240) comprises two three-helix bundles (alpha1alpha4alpha5 and alpha2alpha3alpha6) arranged in a Lambda shape. Compared to the crystal structure of SopE(78-240) in complex with Cdc42, SopE2(69-240) exhibits a less open Lambda shape due to movement of SopE(78-240) helices alpha2 and alpha5 to accommodate binding to the Cdc42 switch regions. In an NMR titration to investigate the SopE2(69-240)-Cdc42 interaction, the SopE2(69-240) residues affected by binding Cdc42 were very similar to the SopE(78-240) residues that contact Cdc42 in the SopE(78-240)-Cdc42 complex. Analysis of the backbone (15)N dynamics of SopE2(69-240) revealed flexibility in residues that link the two three-helix bundles, including the alpha3-alpha4 linker that incorporates a beta-hairpin and the catalytic loop, and the alpha5-alpha6 loop, and flexibility in residues involved in interaction with Cdc42. Together, these observations provide experimental evidence of a previously proposed mechanism of GEF-mediated nucleotide exchange based on the Rac1-Tiam1 complex structure, with SopE/E2 flexibility, particularly in the interbundle loops, enabling conformational rearrangements of the nucleotide binding region of Cdc42 through an induced fit type of binding. Such flexibility in SopE/E2 may also facilitate interaction through adaptive binding with alternative target proteins such as Rab5, allograft inflammatory factor 1, and apolipoprotein A-1.     

Apoptosis and expression of vasopressin, insulin, and Bcl-2 in the neurosecretory system of aged mice

Despite a great many works dealing with apoptosis, the occurrence of this process at later stages of ontogenesis still is far from clear. The goal of the present study was to elucidate role of insulin and antiapoptotic protein Bcl-2 in initiation of apoptosis and preservation of functional status of hypothalamic neurosecretory cells of mice in aging. Neurosecretory cells of aged mice were shown to be eliminated intensively via apoptosis; however, functional activity (synthesis of vasopressin) of remaining cells is comparable with that in young animals. Hypothalamic neurosecretory cells were revealed to synthesize insulin, but its content in neurons of old animals decreases in correlation with initiation of apoptosis. It was shown that protein Bcl-2 in neurons of aged mice did not prevent initiation of apoptosis. It was also established that α-interferon had no apoptosis-protective effect to hypothalamic neurons in aging and suppressed synthesis of vasopressin, insulin, and Bcl-2 in cells of young and old mice.     

Characterization of the Yersinia enterocolitica type III secretion ATPase YscN and its regulator, YscL

Type III secretion is a mechanism used by a broad range of gram-negative bacteria to neutralize eukaryotic defenses by enabling translocation of bacterial proteins directly into the cytoplasm of host cells. The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. By studying the biochemical properties of YscN and YscL of Yersinia enterocolitica, we have characterized them as the ATPase and ATPase regulator, respectively, of the type III secretion system of this organism. In vivo, YscL and YscN interact with each other, and the overexpression of glutathione S-transferase-YscL abolishes secretion and down-regulates the expression of secretion apparatus components.     

Cell individuality: the bistable gene expression of the type III secretion system in Dickeya dadantii 3937

Dickeya dadantii 3937 is a gram-negative phytopathogenic bacterium that expresses genes encoding a type III secretion system (T3SS) in a bistable pattern when cultured in a homogeneous minimal media. In this work, we further characterized the bistable gene expression of T3SS at the single-cell level. We demonstrated that bistable expression of the HrpL-regulon genes, such as hrpA and hrpN, is controlled by the same regulatory mechanism. We also showed that the expression level of the T3SS master regulatory gene hrpL plays an important role in the development of the bistable expression of hrpA. A high expression level of hrpL is required but unable to guarantee the high-state expression of hrpA in a cell. In addition, bistable expression patterns of T3SS genes in other gram-negative pathogens of the Enterobacteriaceae and Pseudomonadaceae families were also described in this study. This suggests that the T3SS bistability might be a conserved population behavior in several gram-negative bacterial pathogens.     

Type III secretion of the Salmonella effector protein SopE is mediated via an N-terminal amino acid signal and not an mRNA sequence

Type III secretion systems (TTSS) are virulence-associated components of many gram-negative bacteria that translocate bacterial proteins directly from the bacterial cytoplasm into the host cell. The Salmonella translocated effector protein SopE has no consensus cleavable amino-terminal secretion sequence, and the mechanism leading to its secretion through the Salmonella pathogenicity island 1 (SPI-1) TTSS is still not fully understood. There is evidence from other bacteria which suggests that the TTSS signal may reside within the 5' untranslated region (UTR) of the mRNA of secreted effectors. We investigated the role of the 5' UTR in the SPI-1 TTSS-mediated secretion of SopE using promoter fusions and obtained data indicating that the mRNA sequence is not involved in the secretion process. To clarify the proteinaceous versus RNA nature of the signal, we constructed frameshift mutations in the amino-terminal region of SopE of Salmonella enterica serovar Typhimurium SL1344. Only constructs with the native amino acid sequence were secreted, highlighting the importance of the amino acid sequence versus the mRNA sequence for secretion. Additionally, we obtained frameshift mutation data suggesting that the first 15 amino acids are important for secretion of SopE independent of the presence of the chaperone binding site. These data shed light on the nature of the signal for SopE secretion and highlight the importance of the amino-terminal amino acids for correct targeting and secretion of SopE via the SPI-1-encoded TTSS during host cell invasion.     

Yersinia enterocolitica type III secretion chaperone SycH. Recombinant expression, purification, characterisation, and crystallisation

All pathogenic Yersinia species (Y. enterocolitica, Y. pestis, and Y. pseudotuberculosis) share a type three secretion system (TTSS) that allows translocation of effector proteins into host cells. Yersinia enterocolitica SycH is a chaperone assisting the transport of the effector YopH and two regulatory components of the TTSS, YscM1 and YscM2. We have recombinantly expressed SycH in Escherichia coli. Purification of tag-free SycH to near homogeneity was achieved by combining ammonium sulfate precipitation, anion exchange chromatography, and gel filtration. Functionality of purified SycH was proven by demonstrating binding to YopH. SycH crystals were grown that diffracted to 2.94 Å resolution. Preliminary crystallographic data and biochemical findings suggest that SycH forms homotetramers. SycH may therefore represent a novel class of TTSS chaperones. In addition, we found that YopH was enzymatically active in the presence of SycH. This implies that the function of the secretion chaperone SycH is not to keep YopH in a globally unfolded state prior to secretion.     

Identification of SopE2, a Salmonella secreted protein which is highly homologous to SopE and involved in bacterial invasion of epithelial cells

Type III secreted Sop protein effectors are delivered into target eukaryotic cells and elicit cellular responses underlying Salmonella pathogenicity. In this work, we have identified another secreted protein, SopE2, and showed that SopE2 is an important invasion-associated effector. SopE2 is encoded by the sopE2 gene which is present and conserved in pathogenic strains of Salmonella. SopE2 is highly homologous to SopE, a protein encoded by a gene within a temperate bacteriophage and present in only some pathogenic strains.     

Regulation of the type III secretion system in phytopathogenic bacteria

The type III secretion system (TTSS) is a specialized protein secretion machinery used by numerous gram-negative bacterial pathogens of animals and plants to deliver effector proteins directly into the host cells. In plant-pathogenic bacteria, genes encoding the TTSS were discovered as hypersensitive response and pathogenicity (hrp) genes, because mutation of these genes typically disrupts the bacterial ability to cause diseases on host plants and to elicit hypersensitive response on nonhost plants. The hrp genes and the type III effector genes (collectively called TTSS genes hereafter) are repressed in nutrient-rich media but induced when bacteria are infiltrated into plants or incubated in nutrient-deficient inducing media. Multiple regulatory components have been identified in the plant-pathogenic bacteria regulating TTSS genes under various conditions. In Ralstonia solanacearum, several signal transduction components essential for the induction of TTSS genes in plants are dispensable for the induction in inducing medium. In addition to the inducing signals, recent studies indicated the presence of negative signals in the plant regulating the Pseudomonas syringae TTSS genes. Thus, the levels of TTSS gene expression in plants likely are determined by the interactions of multiple signal transduction pathways. Studies of the hrp regulons indicated that TTSS genes are coordinately regulated with a number of non-TTSS genes.