GtgE is an effector protein from Salmonella Typhimurium that modulates trafficking of the Salmonella-containing vacuole. It exerts its function by cleaving the Rab-family GTPases Rab29, Rab32 and Rab38, thereby preventing the delivery of antimicrobial factors to the bacteria-containing vacuole. Here, the crystal structure of GtgE at 1.65?Å resolution is presented, and structure-based mutagenesis and in vivo infection assays are used to identify its catalytic triad. A panel of cysteine protease inhibitors were examined and it was determined that N-ethylmaleimide, antipain and chymostatin inhibit GtgE activity in vitro. These findings provide the basis for the development of novel therapeutic strategies to combat Salmonella infections.
The Cytotoxin associated gene A (CagA) protein of Helicobacter pylori is associated with increased virulence and risk of cancer. Recent proteomic studies have demonstrated an association of CagA with the human tumor suppressor Apoptosis-stimulating Protein of p53-2 (ASPP2). We present here a genetic, biochemical, and structural analysis of CagA with ASPP2. Domain delineation of the 120-kDa CagA protein revealed a stable N-terminal subdomain that was used in a yeast two-hybrid screen that identified the proline-rich domain of ASPP2 as a host cellular target. Biochemical experiments confirm this interaction. The cocrystal structure to 2.0-Å resolution of this N-terminal subdomain of CagA with a 7-kDa proline-rich sequence of ASPP2 reveals that this domain of CagA forms a highly specialized three-helix bundle, with large insertions in the loops connecting the helices. These insertions come together to form a deep binding cleft for a highly conserved 20-aa peptide of ASPP2. ASPP2 forms an extended helix in this groove of CagA, burying more than 1,000 Å(2) of surface area. This interaction is disrupted in vitro and in vivo by structure-based, loss-of-contact point mutations of key residues in either CagA or ASPP2. Disruption of CagA and ASPP2 binding alters the function of ASPP2 and leads to the decreased survival of H. pylori-infected cells.
Yersinia pestis injects numerous bacterial proteins into host cells through an organic nanomachine called the type 3 secretion system. One such substrate is the tyrosine phosphatase YopH, which requires an interaction with a cognate chaperone in order to be effectively injected. Here, the first crystal structure of a SycH-YopH complex is reported, determined to 1.9 Å resolution. The structure reveals the presence of (i) a nonglobular polypeptide in YopH, (ii) a so-called ?-motif in YopH and (iii) a conserved hydrophobic patch in SycH that recognizes the ?-motif. Biochemical studies establish that the ?-motif is critical to the stability of this complex. Finally, since previous work has shown that the N-terminal portion of YopH adopts a globular fold that is functional in the host cell, aspects of how this polypeptide adopts radically different folds in the host and in the bacterial environments are analysed.
Protein-protein interactions are typically identified by either biochemical purification coupled to mass spectrometry or genetic approaches exemplified by the yeast two-hybrid assay; however, neither assay works well for the identification of cofactors for poorly soluble proteins. Solubility of a poorly soluble protein is thought to increase upon cofactor binding, possibly by masking otherwise exposed hydrophobic domains. We have exploited this notion to develop a high-throughput genetic screen to identify interacting partners of an insoluble protein fused to chloramphenicol acetyltransferase by monitoring the survival of bacteria in the presence of a drug. In addition to presenting proof-of-principle experiments, we apply this screen to activation-induced cytidine deaminase (AID), a poorly soluble protein that is essential for antibody diversification. We identify a unique cofactor, RING finger protein 126 (RNF126), verify its interaction by traditional techniques, and show that it has functional consequences as RNF126 is able to ubiquitylate AID. Our results underpin the value of this screening technique and suggest a unique form of AID regulation involving RNF126 and ubiquitylation.
We identified an essential cell wall biosynthetic enzyme in Bacillus anthracis and an inhibitor thereof to which the organism did not spontaneously evolve measurable resistance. This work is based on the exquisite binding specificity of bacteriophage-encoded cell wall-hydrolytic lysins, which have evolved to recognize critical receptors within the bacterial cell wall. Focusing on the B. anthracis-specific PlyG lysin, we first identified its unique cell wall receptor and cognate biosynthetic pathway. Within this pathway, one biosynthetic enzyme, 2-epimerase, was required for both PlyG receptor expression and bacterial growth. The 2-epimerase was used to design a small-molecule inhibitor, epimerox. Epimerox prevented growth of several Gram-positive pathogens and rescued mice challenged with lethal doses of B. anthracis. Importantly, resistance to epimerox was not detected (<10(-11) frequency) in B. anthracis and S. aureus. These results describe the use of phage lysins to identify promising lead molecules with reduced resistance potential for antimicrobial development.
The cycle inhibiting factors (Cif), produced by pathogenic bacteria isolated from vertebrates and invertebrates, belong to a family of molecules called cyclomodulins that interfere with the eukaryotic cell cycle. Cif blocks the cell cycle at both the G?/S and G?/M transitions by inducing the stabilization of cyclin-dependent kinase inhibitors p21(waf1) and p27(kip1). Using yeast two-hybrid screens, we identified the ubiquitin-like protein NEDD8 as a target of Cif. Cif co-compartmentalized with NEDD8 in the host cell nucleus and induced accumulation of NEDD8-conjugated cullins. This accumulation occurred early after cell infection and correlated with that of p21 and p27. Co-immunoprecipitation revealed that Cif interacted with cullin-RING ubiquitin ligase complexes (CRLs) through binding with the neddylated forms of cullins 1, 2, 3, 4A and 4B subunits of CRL. Using an in vitro ubiquitylation assay, we demonstrate that Cif directly inhibits the neddylated CUL1-associated ubiquitin ligase activity. Consistent with this inhibition and the interaction of Cif with several neddylated cullins, we further observed that Cif modulates the cellular half-lives of various CRL targets, which might contribute to the pathogenic potential of diverse bacteria.
Protein type III secretion systems (T3SSs) are organic nanosyringes that achieve an energy-dependent translocation of bacterial proteins through the two membranes of Gram-negative organisms. Examples include the pathogenic systems of animals, plants and symbiotic bacteria that inject factors into eukaryotic cells, and the flagellar export system that secretes flagellin. T3SSs possess a core of several membrane-associated proteins that are conserved across all known bacterial species that use this system. The Salmonella protein InvA is one of the most highly conserved proteins of this core of critical T3SS components. The crystal structure of a C-terminal domain of InvA reveals an unexpected homology to domains that have been repeatedly found as building blocks of other elements of the T3SS apparatus. This suggests the surprising hypothesis that evolution has produced a significant component of the apparatus structure through a series of gene-duplication and gene-rearrangement events.
Virulence associated protein type III secretion systems (T3SSs) are intricately structured organic nanosyringes that achieve the translocation of bacterial proteins from the prokaryotic cytoplasm across three membranes into the host cytosol. The substrates for these systems number in the hundreds, with remarkably diverse biological activities, modulating host cell biology for the benefit of the pathogen. Although there has been tremendous progress on the structure and function of the T3SS substrates, there has been comparatively little progress on the much more highly conserved secretion apparatus itself. This review summarizes recent advances in the field of structural microbiology that have begun to address this shortcoming, finally bringing to bear the power of structural biology to this central virulence system of Gram-negative bacterial pathogens.
The CagA protein of Helicobacter pylori interacts with numerous cellular factors and is associated with increased virulence and risk of gastric carcinoma. We present here the cocrystal structure of a subdomain of CagA with the human kinase PAR1b/MARK2, revealing that a CagA peptide mimics substrates of this kinase family, resembling eukaryotic protein kinase inhibitors. Mutagenesis of conserved residues central to this interaction renders CagA inactive as an inhibitor of MARK2.
Processes as diverse as receptor binding and signaling, cytoskeletal dynamics, and programmed cell death are manipulated by mimics of host proteins encoded by pathogenic bacteria. We show here that the Salmonella virulence factor SspH2 belongs to a growing class of bacterial effector proteins that harness and subvert the eukaryotic ubiquitination pathway. This virulence protein possesses ubiquitination activity that depends on a conserved cysteine residue. A crystal structure of SspH2 reveals a canonical leucine-rich repeat (LRR) domain that interacts with a unique E3 ligase [which we have termed NEL for Novel E3 Ligase] C-terminal fold unrelated to previously observed HECT or RING-finger E3 ligases. Moreover, the LRR domain sequesters the catalytic cysteine residue contained in the NEL domain, and we suggest a mechanism for activation of the ligase requiring a substantial conformational change to release the catalytic domain for function. We also show that the N-terminal domain targets SspH2 to the apical plasma membrane of polarized epithelial cells and propose a model whereby binding of the LRR to proteins at the target site releases the ligase domain for site-specific function.
The bacterial protein tyrosine phosphatase YopH is an essential virulence determinant in Yersinia pestis and a potential antibacterial drug target. Here we report our studies of screening for small molecule inhibitors of YopH using both high throughput and in silico approaches. The identified inhibitors represent a diversity of chemotypes and novel pTyr mimetics, providing a starting point for further development and fragment-based design of multi-site binding inhibitors. We demonstrate that the applications of high throughput and virtual screening, when guided by structural binding mode analysis, is an effective approach for identifying potent and selective inhibitors of YopH and other protein phosphatases for rational drug design.
Pseudomonas syringae injects numerous bacterial proteins into host plant cells through a type 3 secretion system (T3SS). One of the first such bacterial effectors discovered, HopA1, is a protein that has unknown functions in the host cell but possesses close homologs that trigger the plant hypersensitive response in resistant strains. Like the virulence factors in many bacterial pathogens of animals, HopA1 depends upon a cognate chaperone in order to be effectively translocated by the P. syringae T3SS. Herein, we report the crystal structure of a complex of HopA1(21-102) with its chaperone, ShcA, determined to 1.56-Å resolution. The structure reveals that three key features of the chaperone-effector interactions found in animal pathogens are preserved in the Gram-negative pathogens of plants, namely, (i) the interaction of the chaperone with a nonglobular polypeptide of the effector, (ii) an interaction centered on the so-called ?-motif, and (iii) the presence of a conserved hydrophobic patch in the chaperone that recognizes the ?-motif. Structure-based mutagenesis and biochemical studies have established that the ?-motif is critical for the stability of this complex. Overall, these results show that the ?-motif interactions are broadly conserved in bacterial pathogens utilizing T3SSs, spanning an interkingdom host range.
SseI is secreted into host cells by Salmonella and contributes to the establishment of systemic infections. The crystal structure of the C-terminal domain of SseI has been solved to 1.70 Å resolution, revealing it to be a member of the cysteine protease superfamily with a catalytic triad consisting of Cys178, His216 and Asp231 that is critical to its virulence activities. Structure-based analysis revealed that SseI is likely to possess either acyl hydrolase or acyltransferase activity, placing this virulence factor in the rapidly growing class of enzymes of this family utilized by bacterial pathogens inside eukaryotic cells.
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