Crystal Structure of the P Pilus Rod Subunit PapA

P pili are important adhesive fibres involved in kidney infection by uropathogenic Escherichia coli strains. P pili are assembled by the conserved chaperone-usher pathway, which involves the PapD chaperone and the PapC usher. During pilus assembly, subunits are incorporated into the growing fiber via the donor-strand exchange (DSE) mechanism, whereby the chaperone's G1 β-strand that complements the incomplete immunoglobulin-fold of each subunit is displaced by the N-terminal extension (Nte) of an incoming subunit. P pili comprise a helical rod, a tip fibrillum, and an adhesin at the distal end. PapA is the rod subunit and is assembled into a superhelical right-handed structure. Here, we have solved the structure of a ternary complex of PapD bound to PapA through donor-strand complementation, itself bound to another PapA subunit through DSE. This structure provides insight into the structural basis of the DSE reaction involving this important pilus subunit. Using gel filtration chromatography and electron microscopy on a number of PapA Nte mutants, we establish that PapA differs in its mode of assembly compared with other Pap subunits, involving a much larger Nte that encompasses not only the DSE region of the Nte but also the region N-terminal to it. Author Summary. Bacterial adhesion to a host is a crucial step that determines the onset of bacterial infection. It is mediated through recognition of a receptor on the host cell surface by a protein called an adhesin displayed on the surface of the bacterium. Many adhesins are displayed at the tip of specialized organelles called pili, some of which are assembled by the ubiquitous chaperone-usher pathway. In this pathway, each pilus subunit is assisted in folding by a chaperone. The resulting chaperone-subunit complex is targeted to a pore located in the outer membrane, called the usher, that serves as assembly platform. There, pilus subunits dissociate from the chaperone and polymerize, resulting in a surface organelle, the pilus, that protrudes out of the usher. Here, we have determined the structure of the major subunit of the P pilus, PapA. The P pilus, produced in uropathogenic Escherichia coli, displays the adhesin PapG responsible for targeting the bacterium to the kidney epithelium. We have determined the structure of PapA either bound to its cognate chaperone, PapD, or bound to another PapA subunit. These structures provide a view of PapA before and after its assembly in the pilus and shed light on the mechanism of PapA assembly.National Institutes of Health (DE 09761, GM040388, DE 09161); Committee of Scientific Research (3 PO4A 003 24, 2 P05A 137 24); Foundation for Polish Science (SUBSYDIUM PROFESORSKIE award); Swedish Rheumatism Association; Nanna Svartz Foundation; King Gustaf V Foundatio

The Pseudomonas syringae HrpJ protein controls the secretion of type III translocator proteins and has a virulence role inside plant cells

The bacterial plant pathogen Pseudomonas syringae injects effector proteins into plant cells via a type III secretion system (T3SS), which is required for pathogenesis. The protein HrpJ is secreted by P. syringae and is required for a fully functional T3SS. A hrpJ mutant is non-pathogenic and cannot inject effectors into plant cells or secrete the harpin HrpZ1. Here we show that the hrpJ mutant also cannot secrete the harpins HrpW1 and HopAK1 or the translocator HrpK1, suggesting that these proteins are required in the translocation (injection) of effectors into plant cells. Complementation of the hrpJ mutant with secretion incompetent HrpJ derivatives restores the secretion of HrpZ1 and HrpW1 and the ability to elicit a hypersensitive response, a measure of translocation. However, growth in planta and disease symptom production is only partially restored, suggesting that secreted HrpJ may have a direct role in virulence. Transgenic Arabidopsis plants expressing HrpJ-HA complemented the virulence phenotype of the hrpJ mutant expressing a secretion incompetent HrpJ derivative and were reduced in their immune responses. Collectively, these data indicate that HrpJ has a dual role in P. syringae: inside bacterial cells HrpJ controls the secretion of translocator proteins and inside plant cells it suppresses plant immunity

Type III secretion: The bacteria-eukaryotic cell express

Type III secretion (T3S) is an export pathway used by Gram-negative pathogenic bacteria to inject bacterial proteins into the cytosol of eukaryotic host cells. This pathway is characterized by (i) a secretion nanomachine related to the bacterial flagellum, but usually topped by a stiff needle-like structure; (ii) the assembly in the eukaryotic cell membrane of a translocation pore formed by T3S substrates; (iii) a non-cleavable N-terminal secretion signal; (iv) T3S chaperones, assisting the secretion of some substrates; (v) a control mechanism ensuring protein delivery at the right place and time. Here, we review these different aspects focusing in open questions that promise exciting findings in the near futur

Feedback Inhibition in the PhoQ/PhoP Signaling System by a Membrane Peptide

The PhoQ/PhoP signaling system responds to low magnesium and the presence of certain cationic antimicrobial peptides. It regulates genes important for growth under these conditions, as well as additional genes important for virulence in many gram-negative pathogens. PhoQ is a sensor kinase that phosphorylates and activates the transcription factor PhoP. Since feedback inhibition is a common theme in stress-response circuits, we hypothesized that some members of the PhoP regulon may play such a role in the PhoQ/PhoP pathway. We therefore screened for PhoP-regulated genes that mediate feedback in this system. We found that deletion of mgrB (yobG), which encodes a 47 amino acid peptide, results in a potent increase in PhoP-regulated transcription. In addition, over-expression of mgrB decreased transcription at both high and low concentrations of magnesium. Localization and bacterial two-hybrid studies suggest that MgrB resides in the inner-membrane and interacts directly with PhoQ. We further show that MgrB homologs from Salmonella typhimurium and Yersinia pestis also repress PhoP-regulated transcription in these organisms. In cell regulatory circuits, feedback has been associated with modulating the induction kinetics and/or the cell-to-cell variability in response to stimulus. Interestingly, we found that elimination of MgrB-mediated feedback did not have a significant effect on the kinetics of reporter protein production and did not decrease the variability in expression among cells. Our results indicate MgrB is a broadly conserved membrane peptide that is a critical mediator of negative feedback in the PhoQ/PhoP circuit. This new regulator may function as a point of control that integrates additional input signals to modulate the activity of this important signaling system

A New Means To Identify Type 3 Secreted Effectors: Functionally Interchangeable Class IB Chaperones Recognize a Conserved Sequence

Many Gram-negative bacteria utilize specialized secretion systems to inject proteins (effectors) directly into host cells. Little is known regarding how bacteria ensure that only small subsets of the thousands of proteins they encode are recognized as substrates of the secretion systems, limiting their identification through bioinformatic analyses. Many of these proteins require chaperones to direct their secretion. Here, using the newly described protein interaction platform assay, we demonstrate that type 3 secretion system class IB chaperones from one bacterium directly bind their own effectors as well as those from other species. In addition, we observe that expression of class IB homologs from seven species, including pathogens and endosymbionts, mediate the translocation of effectors from Shigella directly into host cells, demonstrating that class IB chaperones are often functionally interchangeable. Notably, class IB chaperones bind numerous effectors. However, as previously proposed, they are not promiscuous; rather they recognize a defined sequence that we designate the conserved chaperone-binding domain (CCBD) sequence [(LMIF)1XXX(IV)5XX(IV)8X(N)10]. This sequence is the first defined amino acid sequence to be identified for any interspecies bacterial secretion system, i.e., a system that delivers proteins directly into eukaryotic cells. This sequence provides a new means to identify substrates of type III secretion systems. Indeed, using a pattern search algorithm for the CCBD sequence, we have identified the first two probable effectors from an endosymbiont, Sodalis glossinidius

Mechanistic and Structural Studies of Salicylate Biosynthesis in Pseudomonas aeruginosa

Iron is an essential element for most pathogenic bacteria. To survive and establish infections in host tissues, these pathogens must compete with the host organism for iron. One strategy is to excrete iron-chelator siderophores with very high affinity to ferric iron in the low iron environment of the host. The phenolate type siderophore, such as pyochelin in Pseudomonas aeruginosa, uses salicylate derived from chorismate as a precursor. Studies have shown that the salicylate activation by adenylation for incorporation to siderophores is associated with the growth and virulence of some pathogens. The inhibition of salicylate biosynthesis, and hence, siderophore production is considered an attractive target for the development of novel antimicrobial agents. In P. aeruginosa, salicylate is derived from chorismate via isochorismate by two enzymes: isochorismate synthase (PchA) and isochorismate-pyruvate lyase (IPL, PchB). PchB eliminates the enolpyruvyl side chain from isochorismate through a biologically unusual pericyclic reaction mechanism. PchB can also perform an adventitious pericyclic reaction that rearranges chorismate to prephenate possibly due to the homology to the E. coli chorismate mutase (CM). The primary contribution to lower the activation energy for enzymatic pericyclic reactions is controversial and may be arise from electrostatic stabilization of the transition state, or conformational stabilization of the reactive substrate. Structural and mutational studies on a key residue, lysine 42, of the active site loop suggest that rate enhancement of the two pericyclic reactions (IPL and CM) performed by PchB results from both the transition state stabilization and the reactive substrate conformation, but the relative contributions are different for each reaction. A mutation with less active site loop mobility, A43P indicates that the loop dynamics is related to catalysis. The I87T structure reveals a larger disordered region compared to the wild type structure, suggesting that conformational mobility may play a role in catalysis. PchA is an isochorismate synthase (ICS) in P. aeruginosa that removes the C4 hydroxyl group and adds a hydroxyl group to C2-chorismate. PchA is homologous to salicylate synthases from Yersinia spp. and Mycobacterium tuberculosis that convert chorismate to salicylate without requirement of an additional lyase such as PchB in P. aeruginosa. A sequence comparison between PchA with salicylate synthases of known structure suggests that two conserved residues are directly involved in the general acid and base chemistry in PchA: K221 as the general base and E269 as the general acid. Replacement of K221 and E269 with alanine respectively led to catalytically inactive enzymes, suggesting that K221 and E269 are critical for ICS catalysis. Preliminary pH dependence data for PchA supports the general acid and base mechanism of PchA catalysis. Two nonconserved residues A375 and D310 were also examined. Replacement of A375 by threonine, the corresponding residue in salicylate synthase, resulted in only residual ICS activity, indicating that A375 is not associated with the IPL-deficiency in PchA. The D310E mutant leads to two additional activities, IPL and CM. The additional activities in three reactions may due to the preferential orientation of substrates in the active site

Hunger for iron: the alternative siderophore iron scavenging systems in highly virulent Yersinia

Low molecular weight siderophores are used by many living organisms to scavenge scarcely available ferric iron. Presence of at least a single siderophore-based iron acquisition system is usually acknowledged as a virulence associated trait and a pre-requisite to become an efficient and successful pathogen. Currently, it is assumed that yersiniabactin (Ybt) is the solely functional endogenous siderophore iron uptake system in highly virulent Yersinia (Yersinia pestis, Y pseudotuberculosis, and Y enterocolitica biotype 1B). Genes responsible for biosynthesis, transport, and regulation of the yersiniabactin (ybt) production are clustered on a mobile genetic element, the High Pathogenicity Island (HPI) that is responsible for broad dissemination of the ybt genes in Enterobacteriaceae. However, the ybt gene cluster is absent from nearly half of Y pseudotuberculosis 03 isolates and epidemic Y pseudotuberculosis 01 isolates responsible for the Far East Scarlet like Fever. Several potential siderophore-mediated iron uptake gene clusters are documented in Yersinia genomes, however, neither of them have been proven to be functional. It has been suggested that at least two siderophores alternative to Ybt may operate in the highly virulent Yersinia pestis/Y. pseudotuberculosis group, and are referred to as pseudochelin (Pch) and yersiniachelin (Ych). Furthermore, most sporadic Y pseudotuberculosis 01 strains possess gene clusters encoding all three iron scavenging systems. Thus, the Ybt system appears not to be the sole endogenous siderophore iron uptake system in the highly virulent yersiniae and may be efficiently substituted and/or supplemented by alternative iron siderophore scavenging system

THE \u3ci\u3ePSEUDOMONAS SYRINGAE\u3c/i\u3e TYPE III SECRETION SYSTEM: THE TRANSLOCATOR PROTEINS, THEIR SECRETION, AND THE RESTRICTION OF TRANSLOCATION BY THE PLANT IMMUNE SYSTEM

Pseudomonas syringae is a Gram-negative plant pathogen whose virulence is dependent upon its type III secretion system (T3SS), a nanosyringe that facilitates translocation, or injection, of type III effector (T3E) proteins into eukaryotic cells. The primary function of P. syringae T3E proteins is suppression of plant immunity. Bacterial proteins called translocators form a translocon that forms a pore in the host plasma membrane which is traversed by T3Es. HrpK1, a putative P. syringae translocator, is a type III-secreted protein important for virulence and T3E injection, but not secretion of T3Es. Harpins are a group of proteins specific to plant pathogens that are also important for T3E translocation. P. syringae pv. tomato DC3000 has 4 harpins – HrpZ1, HrpW1, HopAK1, and HopP1. Here, HrpK1 is confirmed to be a translocator. HrpK1 had a greater impact on T3E translocation than the harpins. HrpK1 and HrpZ1 disrupted liposomes. Both proteins interacted with phosphatidic acid which interfered with T3E translocation. HrpJ, a type III-secreted protein required for HrpZ1 secretion, was also required for secretion of HrpK1, HrpW1, and HopAK1. A hrpJ mutant secreted elevated levels of the Hrp pilus protein HrpA1. HrpJ appears to control transition from Hrp pilus secretion to translocator secretion. Secretion was complemented by secretion incompetent HrpJ derivatives indicating that HrpJ controls secretion from inside the bacteria. The hrpJ mutant expressing secretion incompetent HrpJ was reduced in virulence but was complemented by HrpJ expressed inside plant cells. Additionally, transgenic Arabidopsis plants expressing HrpJ were reduced in their immune responses indicating that HrpJ can suppress plant immunity. Plants pretreated with an inducer of pathogen-associated molecular pattern-triggered immunity are unable to produce an HR. Plants, as an immune response, have evolved the ability to block T3E translocation when plant immunity has been induced prior to bacterial inoculation. This is especially true in non-host interactions whereas virulent bacteria appear to be able to attenuate injection restriction in host plants via T3E activity. Adviser: James R. Alfan