The type III secretion system needle, tip, and translocon.

Many Gram-negative bacteria pathogenic to plants and animals deploy the type III secretion system (T3SS) to inject virulence factors into their hosts. All bacteria that rely on the T3SS to cause infectious diseases in humans have developed antibiotic resistance. The T3SS is an attractive target for developing new antibiotics because it is essential in virulence, and part of its structural component is exposed on the bacterial surface. The structural component of the T3SS is the needle apparatus, which is assembled from over 20 different proteins and consists of a base, an extracellular needle, a tip, and a translocon. This review summarizes the current knowledge on the structure and assembly of the needle, tip, and translocon.

Natural product derivative Gossypolone inhibits Musashi family of RNA-binding proteins.

BACKGROUND: The Musashi (MSI) family of RNA-binding proteins is best known for the role in post-transcriptional regulation of target mRNAs. Elevated MSI1 levels in a variety of human cancer are associated with up-regulation of Notch/Wnt signaling. MSI1 binds to and negatively regulates translation of Numb and APC (adenomatous polyposis coli), negative regulators of Notch and Wnt signaling respectively. METHODS: Previously, we have shown that the natural product (-)-gossypol as the first known small molecule inhibitor of MSI1 that down-regulates Notch/Wnt signaling and inhibits tumor xenograft growth in vivo. Using a fluorescence polarization (FP) competition assay, we identified gossypolone (Gn) with a > 20-fold increase in Ki value compared to (-)-gossypol. We validated Gn binding to MSI1 using surface plasmon resonance, nuclear magnetic resonance, and cellular thermal shift assay, and tested the effects of Gn on colon cancer cells and colon cancer DLD-1 xenografts in nude mice. RESULTS: In colon cancer cells, Gn reduced Notch/Wnt signaling and induced apoptosis. Compared to (-)-gossypol, the same concentration of Gn is less active in all the cell assays tested. To increase Gn bioavailability, we used PEGylated liposomes in our in vivo studies. Gn-lip via tail vein injection inhibited the growth of human colon cancer DLD-1 xenografts in nude mice, as compared to the untreated control (P < 0.01, n = 10). CONCLUSION: Our data suggest that PEGylation improved the bioavailability of Gn as well as achieved tumor-targeted delivery and controlled release of Gn, which enhanced its overall biocompatibility and drug efficacy in vivo. This provides proof of concept for the development of Gn-lip as a molecular therapy for colon cancer with MSI1/MSI2 overexpression.

A protein secreted by the Salmonella type III secretion system controls needle filament assembly.

Type III protein secretion systems (T3SS) are encoded by several pathogenic or symbiotic bacteria. The central component of this nanomachine is the needle complex. Here we show in a Salmonella Typhimurium T3SS that assembly of the needle filament of this structure requires OrgC, a protein encoded within the T3SS gene cluster. Absence of OrgC results in significantly reduced number of needle substructures but does not affect needle length. We show that OrgC is secreted by the T3SS and that exogenous addition of OrgC can complement a ∆orgC mutation. We also show that OrgC interacts with the needle filament subunit PrgI and accelerates its polymerization into filaments in vitro. The structure of OrgC shows a novel fold with a shared topology with a domain from flagellar capping proteins. These findings identify a novel component of T3SS and provide new insight into the assembly of the type III secretion machine.

Characterization of Small-Molecule Scaffolds That Bind to the Shigella Type†III Secretion System Protein IpaD.

Many pathogens such as Shigella and other bacteria assemble the type III secretion system (T3SS) nanoinjector to inject virulence proteins into their target cells to cause infectious diseases in humans. The rise of drug resistance among pathogens that rely on the T3SS for infectivity, plus the dearth of new antibiotics require alternative strategies in developing new antibiotics. The Shigella T3SS tip protein IpaD is an attractive target for developing anti-infectives because of its essential role in virulence and its exposure on the bacterial surface. Currently, the only known small molecules that bind to IpaD are bile salt sterols. In this study we identified four new small-molecule scaffolds that bind to IpaD, based on the methylquinoline, pyrrolidine-aniline, hydroxyindole, and morpholinoaniline scaffolds. NMR mapping revealed potential hotspots in IpaD for binding small molecules. These scaffolds can be used as building blocks in developing small-molecule inhibitors of IpaD that could lead to new anti-infectives.

Identification of a new small ubiquitin-like modifier (SUMO)-interacting motif in the E3 ligase PIASy.

Small ubiquitin-like modifier (SUMO) conjugation is a reversible post-translational modification process implicated in the regulation of gene transcription, DNA repair, and cell cycle. SUMOylation depends on the sequential activities of E1 activating, E2 conjugating, and E3 ligating enzymes. SUMO E3 ligases enhance transfer of SUMO from the charged E2 enzyme to the substrate. We have previously identified PIASy, a member of the Siz/protein inhibitor of activated STAT (PIAS) RING family of SUMO E3 ligases, as essential for mitotic chromosomal SUMOylation in frog egg extracts and demonstrated that it can mediate effective SUMOylation. To address how PIASy catalyzes SUMOylation, we examined various truncations of PIASy for their ability to mediate SUMOylation. Using NMR chemical shift mapping and mutagenesis, we identified a new SUMO-interacting motif (SIM) in PIASy. The new SIM and the currently known SIM are both located at the C terminus of PIASy, and both are required for the full ligase activity of PIASy. Our results provide novel insights into the mechanism of PIASy-mediated SUMOylation. PIASy adds to the growing list of SUMO E3 ligases containing multiple SIMs that play important roles in the E3 ligase activity.

The fungal natural product azaphilone-9 binds to HuR and inhibits HuR-RNA interaction in vitro.

The RNA-binding protein Hu antigen R (HuR) binds to AU-rich elements (ARE) in the 3'-untranslated region (UTR) of target mRNAs. The HuR-ARE interactions stabilize many oncogenic mRNAs that play important roles in tumorigenesis. Thus, small molecules that interfere with the HuR-ARE interaction could potentially inhibit cancer cell growth and progression. Using a fluorescence polarization (FP) competition assay, we identified the compound azaphilone-9 (AZA-9) derived from the fungal natural product asperbenzaldehyde, binds to HuR and inhibits HuR-ARE interaction (IC50 ~1.2 µM). Results from surface plasmon resonance (SPR) verified the direct binding of AZA-9 to HuR. NMR methods mapped the RNA-binding interface of HuR and identified the involvement of critical RNA-binding residues in binding of AZA-9. Computational docking was then used to propose a likely binding site for AZA-9 in the RNA-binding cleft of HuR. Our results show that AZA-9 blocks key RNA-binding residues of HuR and disrupts HuR-RNA interactions in vitro. This knowledge is needed in developing more potent AZA-9 derivatives that could lead to new cancer therapy.

NMR identification of the binding surfaces involved in the Salmonella and Shigella Type III secretion tip-translocon protein-protein interactions.

The type III secretion system (T3SS) is essential for the pathogenesis of many bacteria including Salmonella and Shigella, which together are responsible for millions of deaths worldwide each year. The structural component of the T3SS consists of the needle apparatus, which is assembled in part by the protein-protein interaction between the tip and the translocon. The atomic detail of the interaction between the tip and the translocon proteins is currently unknown. Here, we used NMR methods to identify that the N-terminal domain of the Salmonella SipB translocon protein interacts with the SipD tip protein at a surface at the distal region of the tip formed by the mixed α/ß domain and a portion of its coiled-coil domain. Likewise, the Shigella IpaB translocon protein and the IpaD tip protein interact with each other using similar surfaces identified for the Salmonella homologs. Furthermore, removal of the extreme N-terminal residues of the translocon protein, previously thought to be important for the interaction, had little change on the binding surface. Finally, mutations at the binding surface of SipD reduced invasion of Salmonella into human intestinal epithelial cells. Together, these results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and advance our understanding of the assembly of the T3SS needle apparatus. Proteins 2016; 84:1097-1107. © 2016 Wiley Periodicals, Inc.

Characterization of the Binding of Hydroxyindole, Indoleacetic acid, and Morpholinoaniline to the Salmonella Type†III Secretion System Proteins SipD and SipB.

Many Gram-negative bacteria require the type III secretion system (T3SS) to cause infectious diseases in humans. A looming public health problem is that all bacterial pathogens that require the T3SS to cause infectious diseases in humans have developed multidrug resistance to current antibiotics. The T3SS is an attractive target for the development of new antibiotics because of its critical role in virulence. An initial step in developing anti-T3SS-based therapeutics is the identification of small molecules that can bind to T3SS proteins. Currently, the only small molecules that are known to bind to the Salmonella T3SS proteins SipD and SipB are bile salts (to SipD) and sphingolipids and cholesterol (to SipB). Herein we report the results of a surface plasmon resonance screen of 288 compounds wherein the binding of 4-morpholinoaniline to SipD, 3-indoleacetic acid to SipB, and 5-hydroxyindole to both SipD and SipB were identified. We also identified by NMR the SipD surfaces involved in binding. These three compounds represent a new class of molecules that can bind to T3SS tip (SipD) and translocon (SipB) proteins that could find use in future drug design.

Characterization of the Shigella and Salmonella Type†III Secretion System Tip-Translocon Protein-Protein Interaction by Paramagnetic Relaxation Enhancement.

Many Gram-negative pathogens, such as Shigella and Salmonella, assemble the type III secretion system (T3SS) to inject virulence proteins directly into eukaryotic cells to initiate infectious diseases. The needle apparatus of the T3SS consists of a base, an extracellular needle, a tip protein complex, and a translocon. The atomic structure of the assembled tip complex and the translocon is unknown. Here, we show by NMR paramagnetic relaxation enhancement (PRE) that the mixed α-ß domain at the distal region of the Shigella and Salmonella tip proteins interacts with the N-terminal ectodomain of their major translocon proteins. Our results reveal the binding surfaces involved in the tip-translocon protein-protein interaction and provide insights about the assembly of the needle apparatus of the T3SS.

Nuclear Magnetic Resonance Characterization of the Type III Secretion System Tip Chaperone Protein PcrG of Pseudomonas aeruginosa.

Lung infection with Pseudomonas aeruginosa is the leading cause of death among cystic fibrosis patients. To initiate infection, P. aeruginosa assembles a protein nanomachine, the type III secretion system (T3SS), to inject bacterial proteins directly into target host cells. An important regulator of the P. aeruginosa T3SS is the chaperone protein PcrG, which forms a complex with the tip protein, PcrV. In addition to its role as a chaperone to the tip protein, PcrG also regulates protein secretion. PcrG homologues are also important in the T3SS of other pathogens such as Yersinia pestis, the causative agent of bubonic plague. The atomic structure of PcrG or any member of the family of tip protein chaperones is currently unknown. Here, we show by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy that PcrG lacks a tertiary structure. However, it is not completely disordered but contains secondary structures dominated by two long α-helices from residue 16 to 41 and from residue 55 to 76. The helices of PcrG are partially formed, have similar backbone dynamics, and are flexible. NMR titrations show that the entire length of PcrG residues from position 9 to 76 is involved in binding to PcrV. PcrG adds to the growing list of partially folded or unstructured proteins with important roles in type III secretion.

The LcrG Tip Chaperone Protein of the Yersinia pestis Type III Secretion System Is Partially Folded.

The type III secretion system (T3SS) is essential in the pathogenesis of Yersinia pestis, the causative agent of plague. A small protein, LcrG, functions as a chaperone to the tip protein LcrV, and the LcrG-LcrV interaction is important in regulating protein secretion through the T3SS. The atomic structure of the LcrG family is currently unknown. However, because of its predicted helical propensity, many have suggested that the LcrG family forms a coiled-coil structure. Here, we show by NMR and CD spectroscopy that LcrG lacks a tertiary structure and it consists of three partially folded α-helices spanning residues 7-38, 41-46, and 58-73. NMR titrations of LcrG with LcrV show that the entire length of a truncated LcrG (residues 7-73) is involved in binding to LcrV. However, there is regional variation in how LcrG binds to LcrV. The C-terminal region of a truncated LcrG (residues 52-73) shows tight binding interaction with LcrV while the N-terminal region (residues 7-51) shows weaker interaction with LcrV. This suggests that there are at least two binding events when LcrG binds to LcrV. Biological assays and mutagenesis indicate that the C-terminal region of LcrG (residues 52-73) is important in blocking protein secretion through the T3SS. Our results reveal structural and mechanistic insights into the atomic conformation of LcrG and how it binds to LcrV.

Natural product (-)-gossypol inhibits colon cancer cell growth by targeting RNA-binding protein Musashi-1.

Musashi-1 (MSI1) is an RNA-binding protein that acts as a translation activator or repressor of target mRNAs. The best-characterized MSI1 target is Numb mRNA, whose encoded protein negatively regulates Notch signaling. Additional MSI1 targets include the mRNAs for the tumor suppressor protein APC that regulates Wnt signaling and the cyclin-dependent kinase inhibitor P21(WAF-1). We hypothesized that increased expression of NUMB, P21 and APC, through inhibition of MSI1 RNA-binding activity might be an effective way to simultaneously downregulate Wnt and Notch signaling, thus blocking the growth of a broad range of cancer cells. We used a fluorescence polarization assay to screen for small molecules that disrupt the binding of MSI1 to its consensus RNA binding site. One of the top hits was (-)-gossypol (Ki = 476 ± 273 nM), a natural product from cottonseed, known to have potent anti-tumor activity and which has recently completed Phase IIb clinical trials for prostate cancer. Surface plasmon resonance and nuclear magnetic resonance studies demonstrate a direct interaction of (-)-gossypol with the RNA binding pocket of MSI1. We further showed that (-)-gossypol reduces Notch/Wnt signaling in several colon cancer cell lines having high levels of MSI1, with reduced SURVIVIN expression and increased apoptosis/autophagy. Finally, we showed that orally administered (-)-gossypol inhibits colon cancer growth in a mouse xenograft model. Our study identifies (-)-gossypol as a potential small molecule inhibitor of MSI1-RNA interaction, and suggests that inhibition of MSI1's RNA binding activity may be an effective anti-cancer strategy.

The bacterial type III secretion system as a target for developing new antibiotics.

Antibiotic resistance in pathogens requires new targets for developing novel antibacterials. The bacterial type III secretion system (T3SS) is an attractive target for developing antibacterials as it is essential in the pathogenesis of many Gram-negative bacteria. The T3SS consists of structural proteins, effectors, and chaperones. Over 20 different structural proteins assemble into a complex nanoinjector that punctures a hole on the eukaryotic cell membrane to allow the delivery of effectors directly into the host cell cytoplasm. Defects in the assembly and function of the T3SS render bacteria non-infective. Two major classes of small molecules, salicylidene acylhydrazides and thiazolidinones, have been shown to inhibit multiple genera of bacteria through the T3SS. Many additional chemically and structurally diverse classes of small molecule inhibitors of the T3SS have been identified as well. While specific targets within the T3SS of a few inhibitors have been suggested, the vast majority of specific protein targets within the T3SS remain to be identified or characterized. Other T3SS inhibitors include polymers, proteins, and polypeptides mimics. In addition, T3SS activity is regulated by its interaction with biologically relevant molecules, such as bile salts and sterols, which could serve as scaffolds for drug design.

Clinical practice guidelines on the diagnosis and treatment of gastroesophageal reflux disease (GERD)

In the last two decades gastroesophageal reflux disease (GERD), initially thought to be a disease only common  in the West, is described  increasingly in Asia, including the Philippines. A recent local report indicated that the prevalence of erosive esophagitis (EE), a common complication of GERD, has more than doubled, i.e., 2.9% to  6.3%,  between the two time periods of 1994-1997 and 2000-2003, respectively. GERD causes recurrent annoying symptoms which are common  reasons  for  clinic  visits  and consultations thus, it is the objective of these guidelines to provide both primary care physicians  (PCPs) and specialists a current, evidence-based, country-specific recommendations for the optimal management  of  GERD.  These  guidelines  are  intended   to   empower   PCPs   to   make   a   clinic-based diagnosis of GERD, to start an empiric acid-suppressive therapy in the appropriate patient,and direct them to select which GERD patient may need to undergo investigations to ascertain further the diagnosis of GERD or to assess outcomes of therapy. We acknowledge that studies published in the future may influence the impact on our confidence on the recommendations enumerated in  these guidelines thus, we commit to update this document when it is deemed appropriate.

NMR model of PrgI-SipD interaction and its implications in the needle-tip assembly of the Salmonella type III secretion system.

Salmonella and other pathogenic bacteria use the type III secretion system (T3SS) to inject virulence proteins into human cells to initiate infections. The structural component of the T3SS contains a needle and a needle tip. The needle is assembled from PrgI needle protomers and the needle tip is capped with several copies of the SipD tip protein. How a tip protein docks on the needle is unclear. A crystal structure of a PrgI-SipD fusion protein docked on the PrgI needle results in steric clash of SipD at the needle tip when modeled on the recent atomic structure of the needle. Thus, there is currently no good model of how SipD is docked on the PrgI needle tip. Previously, we showed by NMR paramagnetic relaxation enhancement (PRE) methods that a specific region in the SipD coiled coil is the binding site for PrgI. Others have hypothesized that a domain of the tip protein-the N-terminal α-helical hairpin-has to swing away during the assembly of the needle apparatus. Here, we show by PRE methods that a truncated form of SipD lacking the α-helical hairpin domain binds more tightly to PrgI. Further, PRE-based structure calculations revealed multiple PrgI binding sites on the SipD coiled coil. Our PRE results together with the recent NMR-derived atomic structure of the Salmonella needle suggest a possible model of how SipD might dock at the PrgI needle tip. SipD and PrgI are conserved in other bacterial T3SSs; thus, our results have wider implication in understanding other needle-tip complexes.

Structure of the Yersinia pestis tip protein LcrV refined to 1.65†Šresolution.

The human pathogen Yersinia pestis requires the assembly of the type III secretion system (T3SS) for virulence. The structural component of the T3SS contains an external needle and a tip complex, which is formed by LcrV in Y. pestis. The structure of an LcrV triple mutant (K40A/D41A/K42A) in a C273S background has previously been reported to 2.2 Šresolution. Here, the crystal structure of LcrV without the triple mutation in a C273S background is reported at a higher resolution of 1.65 Å. Overall the two structures are similar, but there are also notable differences, particularly near the site of the triple mutation. The refined structure revealed a slight shift in the backbone positions of residues Gly28-Asn43 and displayed electron density in the loop region consisting of residues Ile46-Val63, which was disordered in the original structure. In addition, the helical turn region spanning residues Tyr77-Gln95 adopts a different orientation.

Structure and biophysics of type III secretion in bacteria.

Many plant and animal bacterial pathogens assemble a needle-like nanomachine, the type III secretion system (T3SS), to inject virulence proteins directly into eukaryotic cells to initiate infection. The ability of bacteria to inject effectors into host cells is essential for infection, survival, and pathogenesis for many Gram-negative bacteria, including Salmonella, Escherichia, Shigella, Yersinia, Pseudomonas, and Chlamydia spp. These pathogens are responsible for a wide variety of diseases, such as typhoid fever, large-scale food-borne illnesses, dysentery, bubonic plague, secondary hospital infections, and sexually transmitted diseases. The T3SS consists of structural and nonstructural proteins. The structural proteins assemble the needle apparatus, which consists of a membrane-embedded basal structure, an external needle that protrudes from the bacterial surface, and a tip complex that caps the needle. Upon host cell contact, a translocon is assembled between the needle tip complex and the host cell, serving as a gateway for translocation of effector proteins by creating a pore in the host cell membrane. Following delivery into the host cytoplasm, effectors initiate and maintain infection by manipulating host cell biology, such as cell signaling, secretory trafficking, cytoskeletal dynamics, and the inflammatory response. Finally, chaperones serve as regulators of secretion by sequestering effectors and some structural proteins within the bacterial cytoplasm. This review will focus on the latest developments and future challenges concerning the structure and biophysics of the needle apparatus.

The Salmonella type III secretion system inner rod protein PrgJ is partially folded.

The type III secretion system (T3SS) is essential in the pathogenesis of many bacteria. The inner rod is important in the assembly of the T3SS needle complex. However, the atomic structure of the inner rod protein is currently unknown. Based on computational methods, others have suggested that the Salmonella inner rod protein PrgJ is highly helical, forming a folded 3 helix structure. Here we show by CD and NMR spectroscopy that the monomeric form of PrgJ lacks a tertiary structure, and the only well-structured part of PrgJ is a short α-helix at the C-terminal region from residues 65-82. Disruption of this helix by glycine or proline mutation resulted in defective assembly of the needle complex, rendering bacteria incapable of secreting effector proteins. Likewise, CD and NMR data for the Shigella inner rod protein MxiI indicate this protein lacks a tertiary structure as well. Our results reveal that the monomeric forms of the T3SS inner rod proteins are partially folded.

Structural characterization of the Crimean-Congo hemorrhagic fever virus Gn tail provides insight into virus assembly.

The RNA virus that causes the Crimean Congo Hemorrhagic Fever (CCHF) is a tick-borne pathogen of the Nairovirus genus, family Bunyaviridae. Unlike many zoonotic viruses that are only passed between animals and humans, the CCHF virus can also be transmitted from human to human with an overall mortality rate approaching 30%. Currently, there are no atomic structures for any CCHF virus proteins or for any Nairovirus proteins. A critical component of the virus is the envelope Gn glycoprotein, which contains a C-terminal cytoplasmic tail. In other Bunyaviridae viruses, the Gn tail has been implicated in host-pathogen interaction and viral assembly. Here we report the NMR structure of the CCHF virus Gn cytoplasmic tail, residues 729-805. The structure contains a pair of tightly arranged dual ßßα zinc fingers similar to those found in the Hantavirus genus, with which it shares about 12% sequence identity. Unlike Hantavirus zinc fingers, however, the CCHF virus zinc fingers bind viral RNA and contain contiguous clusters of conserved surface electrostatics. Our results provide insight into a likely role of the CCHF virus Gn zinc fingers in Nairovirus assembly.

The crystal structures of the Salmonella type III secretion system tip protein SipD in complex with deoxycholate and chenodeoxycholate.

The type III secretion system (T3SS) is a protein injection nanomachinery required for virulence by many human pathogenic bacteria including Salmonella and Shigella. An essential component of the T3SS is the tip protein and the Salmonella SipD and the Shigella IpaD tip proteins interact with bile salts, which serve as environmental sensors for these enteric pathogens. SipD and IpaD have long central coiled coils and their N-terminal regions form α-helical hairpins and a short helix α3 that pack against the coiled coil. Using AutoDock, others have predicted that the bile salt deoxycholate binds IpaD in a cleft formed by the α-helical hairpin and its long central coiled coil. NMR chemical shift mapping, however, indicated that the SipD residues most affected by bile salts are located in a disordered region near helix α3. Thus, how bile salts interact with SipD and IpaD is unclear. Here, we report the crystal structures of SipD in complex with the bile salts deoxycholate and chenodeoxycholate. Bile salts bind SipD in a region different from what was predicted for IpaD. In SipD, bile salts bind part of helix α3 and the C-terminus of the long central coiled coil, towards the C-terminus of the protein. We discuss the biological implication of the differences in how bile salts interact with SipD and IpaD.