Characterization of the putative type III secretion ATPase CdsN (Cpn0707) of Chlamydophila pneumoniae

Type III secretion (T3S) is utilized by a wide range of gram-negative bacterial pathogens to allow the efficient delivery of effector proteins into the host cell cytoplasm through the use of a syringe-like injectisome. Chlamydophila pneumoniae is a gram-negative, obligate intracellular pathogen that has the structural genes coding for a T3S system, but the functionality of the system has not yet been demonstrated. T3S is dependent on ATPase activity, which catalyzes the unfolding of proteins and the secretion of effector proteins through the injectisome. CdsN (Cpn0707) is predicted to be the T3S ATPase of C. pneumoniae based on sequence similarity to other T3S ATPases. Full-length CdsN and a C-terminal truncation of CdsN were cloned as glutathione S-transferase (GST)-tagged constructs and expressed in Escherichia coli. The GST-tagged C-terminal truncation of CdsN possessed ATPase activity, catalyzing the release of ADP and P(i) from ATP at a rate of 0.55 +/- 0.07 micromol min(-1) mg(-1) in a time- and dose-dependent manner. CdsN formed oligomers and high-molecular-weight multimers, as assessed by formaldehyde fixation and nondenaturing polyacrylamide gel electrophoresis. Using bacterial two-hybrid and GST pull-down assays, CdsN was shown to interact with CdsD, CdsL, CdsQ, and CopN, four putative structural components of the C. pneumoniae T3S system. CdsN also interacted with an unannotated protein, Cpn0706, a putative CdsN chaperone. Interactions between CdsN, CdsD, and CopN represent novel interactions not previously reported for other bacterial T3S systems and may be important in the localization and/or function of the ATPase at the inner membrane of C. pneumoniae.     

Chlamydophila pneumoniae PknD exhibits dual amino acid specificity and phosphorylates Cpn0712, a putative type III secretion YscD homolog

Chlamydophila pneumoniae is an obligate intracellular bacterium that causes bronchitis, pharyngitis, and pneumonia and may be involved in atherogenesis and Alzheimer's disease. Genome sequencing has identified three eukaryote-type serine/threonine protein kinases, Pkn1, Pkn5, and PknD, that may be important signaling molecules in Chlamydia. Full-length PknD was cloned and expressed as a histidine-tagged protein in Escherichia coli. Differential centrifugation followed by sodium carbonate treatment of E. coli membranes demonstrated that His-PknD is an integral membrane protein. Fusions of overlapping PknD fragments to alkaline phosphatase revealed that PknD contains a single transmembrane domain and that the kinase domain is in the cytoplasm. To facilitate solubility, the kinase domain was cloned and expressed as a glutathione S-transferase (GST) fusion protein in E. coli. Purified GST-PknD kinase domain autophosphorylated, and catalytic mutants (K33G, D156G, and K33G-D156G mutants) and activation loop mutants (T185A and T193A) were inactive. PknD phosphorylated recombinant Cpn0712, a type III secretion YscD homolog that has two forkhead-associated domains. Thin-layer chromatography revealed that the PknD kinase domain autophosphorylated on threonine and tyrosine and phosphorylated the FHA-2 domain of Cpn0712 on serine and tyrosine. To our knowledge, this is the first demonstration of a bacterial protein kinase with amino acid specificity for both serine/threonine and tyrosine residues and this is the first study to show phosphorylation of a predicted type III secretion structural protein.     

Interactions between partner switcher orthologs BtrW and BtrV regulate type III secretion in Bordetella

We have recently described a multicomponent cascade that regulates type III secretion in Bordetella. This cascade includes a group of proteins, BtrU, BtrW, and BtrV, that contain an array of domains that define partner-switching complexes previously characterized in gram-positive bacteria. BtrU contains a PP2C-like serine phosphatase domain, BtrW contains a serine kinase/anti-sigma factor motif, and BtrV includes an anti-sigma factor antagonist domain. On the basis of genetic studies and sequence similarity with the RsbU-RsbW-RsbV and SpoIIE-SpoIIAB-SpoIIAA partner switchers of Bacillus subtilis, a series of interactions between Bordetella orthologs have been proposed. Bacterial two-hybrid analysis, tagged protein pull-downs, and in vitro phosphorylation assays were used to characterize interactions between BtrW and BtrV. In addition, BtrV mutants predicted to mimic a constitutively phosphorylated form of BtrV or to be nonphosphorylatable and BtrW mutants defective in serine kinase activity or the ability to bind BtrV were constructed and analyzed. Our results demonstrate that (i) BtrW and BtrV interact with each other, (ii) BtrW phosphorylates BtrV at serine S55, (iii) the conserved serine residue S55 of BtrV plays a key role in BtrV-BtrW interactions, and (iv) the ability of BtrW to phosphorylate BtrV and disrupt BtrV-BtrW binding is essential for the type III secretion process.     

The extended structure of the periplasmic region of CdsD,a structural protein of the type III secretion system of Chlamydia trachomatis

The type III secretion system (T3SS) is required for the virulence of many gram‐negative bacterial human pathogens. It is composed of several structural proteins, forming the secretion needle and its basis, the basal body. In Chlamydia spp., the T3SS inner membrane ring (IM‐ring) of the basal body is formed by the periplasmic part of CdsD (outer ring) and CdsJ (inner ring). Here we describe the crystal structure of the C‐terminal, periplasmic part of CdsD, not including the last 60 residues. Two crystal forms were obtained, grown in three different crystallization conditions. In both crystal forms there is one molecule per asymmetric unit adopting a similar extended structure. The structures consist of three periplasmic domains (PDs) of similar αββαβ topology as seen also in the structures of the homologous PrgH (Salmonella typhimurium) and YscD (Yersinia enterocolitica). Only in the C2 crystal form, there is a C‐terminal additional helix after the PD3 domain. The relative orientation of the three subsequent CdsD PD domains with respect to each other is more extended than in PrgH but less extended than in YscD. Small‐angle X‐ray scattering data show that also in solution this CdsD construct adopts the same elongated shape. In both crystal forms the CdsD molecules are packed in a parallel fashion, using translational crystallographic symmetry. The most extensive crystal contacts are preserved in both crystal forms, suggesting a possible mode of assembly of the CdsD periplasmic part into a 24‐mer complex forming the outer ring of the IM‐ring of the T3SS.     

pH sensitivity of type III secretion system tip proteins

Many pathogenic gram-negative bacteria employ type III secretion systems to transport proteins into the host cell membrane and cytoplasm to subvert normal cellular functions. The type III secretion apparatus consists of a basal body spanning the inner and outer bacterial membranes and a needle which extends away from the bacterium. Recent work has found that a special class of proteins localizes to the tip of the needle to control secretion of effector proteins. Five of these tip proteins are IpaD (Shigella flexneri), BipD (Burkholderia pseudomallei), SipD (Salmonella spp.), LcrV (Yersinia spp.), and PcrV (Pseudomonas aeruginosa). In this study, the conformational stability of these proteins was characterized as a function of pH and temperature. Understanding the stability of the proteins in different pH environments is particularly important since they are expected to encounter different pH environments in their passage through the gastrointestinal tract and are exposed to low pH microenvironments near the surface of target cell membranes. Secondary and tertiary structural changes were monitored using the spectroscopic techniques of far-UV circular dichroism, Trp fluorescence, ANS fluorescence, and ultraviolet absorption spectroscopy. Optical density and right angle scattering measurements were also used to evaluate protein association/dissociation. Empirical phase diagrams were then applied to mathematically combine data from the various spectroscopic techniques to provide a global picture of the proteins' structural behavior in solution. The responses of the proteins to changes in temperature and pH conditions reveal two distinct subfamilies in terms of stability. The first is that of IpaD, BipD, and SipD whose corresponding phase diagrams show conformational differences at pH 5-6. The conserved pH dependence in this subfamily suggests possible common mechanistic function. In the second subfamily (LcrV and PcrV), conformational stability is directly related to pH, also indicating mechanistic similarities.     

Interactions of the type III secretion pathway proteins LcrV and LcrG from Yersinia pestis are mediated by coiled-coil domains

The type III secretion system is used by pathogenic Yersinia to translocate virulence factors into the host cell. A key component is the multifunctional LcrV protein, which is present on the bacterial surface prior to host cell contact and up-regulates translocation by blocking the repressive action of the LcrG protein on the cytosolic side of the secretion apparatus. The functions of LcrV are proposed to involve self-interactions (multimerization) and interactions with other proteins including LcrG. Coiled-coil motifs predicted to be present are thought to play a role in mediating these protein-protein interactions. We have purified recombinant LcrV, LcrG, and site-directed mutants of LcrV and demonstrated the structural integrity of these proteins using circular dichroism spectroscopy. We show that LcrV interacts both with itself and with LcrG and have obtained micromolar and nanomolar affinities for these interactions, respectively. The effects of LcrV mutations upon LcrG binding suggest that coiled-coil interactions indeed play a significant role in complex formation. In addition, comparisons of secretion patterns of effector proteins in Yersinia, arising from wild type and mutants of LcrV, support the proposed role of LcrG in titration of LcrV in vivo but also suggest that other factors may be involved.     

Interactions between type III secretion apparatus components from Yersinia pestis detected using the yeast two-hybrid system

Interactions among the Yersinia secretion (Ysc) proteins of Yersinia pestis were explored using the yeast two-hybrid system. Various pairwise combinations of the yscEFGHIKLN and Q genes fused to the DNA-binding or activation domain of the yeast GAL4 gene were introduced into yeast, and expression of a reporter gene encoding beta-galactosidase was detected. Combinations of yscN and yscL, yscL and yscQ, and yscQ and yscK resulted in high levels of reporter gene activation. These results suggest that YscL interacts with both YscN and YscQ, and that YscQ interacts with both YscL and YscK. Three-hybrid analyses using plasmid pDELA to target a third hybrid protein to the yeast nucleus was used to detect the formation of ternary protein complexes. Using the three-hybrid system, YscQ expressed from plasmid pDELA was able to bring together the YscK and YscL fusion proteins. In a similar manner, YscL expressed from plasmid pDELA was able to bring together the YscN and YscQ fusion proteins. Together, these results suggest that a complex composed of YscN, YscQ, YscK and YscL is involved in the assembly and/or function of the Y. pestis type III secretion apparatus.     

Interactions between flagellar and type III secretion proteins in <Emphasis Type="Italic">Chlamydia pneumoniae</Emphasis>

Background  

Flagellar secretion systems are utilized by a wide variety of bacteria to construct the flagellum, a conserved apparatus that allows for migration towards non-hostile, nutrient rich environments. Chlamydia pneumoniae is an obligate, intracellular pathogen whose genome contains at least three orthologs of flagellar proteins, namely FliI, FlhA and FliF, but the role of these proteins remains unknown.     

A putative lytic transglycosylase tightly regulated and critical for the EHEC type three secretion

Open reading frame l0045 in the pathogenic island of enterohemorrhagic Escherichia coli O157:H7 has been predicted to encode a lytic transglycosylase that is homologous to two different gene products encoded by the same bacteria at loci away from the island. To deduce the necessity of the presence in the island, we created an l0045-deleted strain of EHEC and observed that both the level of cytosolic EspA and that of the other type III secreted proteins in the media were affected. In a complementation assay, a low level-expressing L0045 appeared to recover efficiently the type III secretion (TTS). On the other hand, when l0045 was driven to express robustly, the intracellular levels of representative TTS proteins were severely suppressed. This suppression is apparently caused by the protein of L0045 per se since introducing an early translational termination codon abolished the suppression. Intriguingly, the authentic L0045 was hardly detected in all lysates of EHEC differently prepared while the same construct was expectedly expressed in the K-12 strain. A unique network must exist in EHEC to tightly regulate the presence of L0045, and we found that a LEE regulator (GrlA) is critically involved in this regulation.     

Outer and inner membrane proteins compose an arginine-agmatine exchange system in Chlamydophila pneumoniae

Most chlamydial strains have a pyruvoyl-dependent decarboxylase protein that converts L-arginine to agmatine. However, chlamydiae do not produce arginine, so they must import it from their host. Chlamydophila pneumoniae has a gene cluster encoding a putative outer membrane porin (CPn1033 or aaxA), an arginine decarboxylase (CPn1032 or aaxB), and a putative cytoplasmic membrane transporter (CPn1031 or aaxC). The aaxC gene was expressed in Escherichia coli producing an integral cytoplasmic membrane protein that catalyzed the exchange of L-arginine for agmatine. Expression of the aaxA gene produced an outer membrane protein that enhanced the arginine uptake and decarboxylation activity of cells coexpressing aaxB and aaxC. This chlamydial arginine/agmatine exchange system complemented an E. coli mutant missing the native arginine-dependent acid resistance system. These cells survived extreme acid shock in the presence of L-arginine. Biochemical and evolutionary analysis showed the aaxABC genes evolved convergently with the enteric arginine degradation system, and they could have a different physiological role in chlamydial cells. The chlamydial system uniquely includes an outer membrane porin, and it is most active at a higher pH from 3 to 5. The chlamydial AaxC transporter was resistant to cadaverine, L-lysine and L-ornithine, which inhibit the E. coli AdiC antiporter.     

A new family of highly variable proteins in the Chlamydophila pneumoniae genome

Chlamydiaceae are obligate intracellular bacterial pathogens characterized by a wide range of vertebrate host, tissue tropism and spectrum of diseases. To get insights into the biological mechanisms involved in these differences, we have put forward a computational and experimental procedure to identify the genome recombination hotspots, as frequent sequence variation allows rapid adaptation to environmental changes. We find a larger potential for recombination in Chlamydophila pneumoniae genomes as compared with Chlamydia trachomatis or Chlamydia muridarum. Such potential is mostly concentrated in a family of seven previously uncharacterized species-specific elements that we named ppp for C.pneumoniae polymorphic protein genes, which have the potential to vary by homologous recombination and slipped-mispair. Experimentally, we show that these sequences are indeed highly polymorphic among a collection of nine C.pneumoniae strains of very diverse geographical and pathological origins, mainly by slippage of a poly(C) tract. We also show that most elements are transcribed during infection. In silico analyses suggest that Ppps correspond to outer membrane proteins. Given their species specificity, their putative location in the outer membrane and their extreme polymorphism, Ppps are most likely to be important in the pathogenesis of C.pneumoniae and could represent targets for future vaccine development.     

SpiC is required for secretion of Salmonella Pathogenicity Island 2 type III secretion system proteins

Replication of Salmonella typhimurium in host cells depends in part on the action of the Salmonella Pathogenicity Island 2 (SPI-2) type III secretion system (TTSS), which translocates bacterial effector proteins across the membrane of the Salmonella-containing vacuole (SCV). We have shown previously that one activity of the SPI-2 TTSS is the assembly of a coat of F-actin in the vicinity of bacterial microcolonies. To identify proteins involved in SPI-2 dependent actin polymerization, we tested strains carrying mutations in each of several genes whose products are proposed to be secreted through the SPI-2 TTSS, for their ability to assemble F-actin around intracellular bacteria. We found that strains carrying mutations in either sseB, sseC, sseD or spiC were deficient in actin assembly. The phenotypes of the sseB-, sseC- and sseD- mutants can be attributed to their requirement for translocation of SPI-2 effectors. SpiC was investigated further in view of its proposed role as an effector. Transient expression of a myc::SpiC fusion protein in Hela cells did not induce any significant alterations to the host cell cytoskeleton, and failed to restore actin polymerization around intracellular spiC- mutant bacteria. However, the same protein did complement the mutant phenotype when expressed from a plasmid within bacteria. Furthermore, spiC was found to be required for SPI-2 mediated secretion of SseB, SseC and SseD in vitro. An antibody against SpiC detected the protein on immunoblots from total cell lysates of S. typhimurium expressing SpiC from a plasmid, but it was not detected in secreted fractions after exposure of cells to conditions that result in secretion of other SPI-2 effector proteins. Investigation of the trafficking of SCVs containing a spiC- mutant in macrophages revealed only a low level of association with the lysosomal marker cathepsin D, similar to that of wild-type bacteria. Together, these results show that SpiC is involved in the process of SPI-2 secretion and indicate that phenotypes associated with a spiC- mutant are caused by the inability of this strain to translocate effector proteins, thus calling for further investigation into the function(s) of this protein.     

Interactions between C ring proteins and export apparatus components: a possible mechanism for facilitating type III protein export

The flagellar switch proteins of Salmonella, FliG, FliM and FliN, participate in the switching of motor rotation, torque generation and flagellar assembly/export. FliN has been implicated in the flagellar export process. To address this possibility, we constructed 10-amino-acid scanning deletions and larger truncations over the C-terminal domain of FliN. Except for the last deletion variant, all other variants were unable to complement a fliN null strain or to restore the export of flagellar proteins. Most of the deletions showed strong negative dominance effects on wild-type cells. FliN was found to associate with FliH, a flagellar export component that regulates the ATPase activity of FliI. The binding of FliM to FliN does not interfere with this FliN-FliH interaction. Furthermore, a five-protein complex consisting of FliG, His-tagged FliM, FliN, FliH and FliI was purified by nickel-affinity chromatography. FliJ, a putative general chaperone, is bound to FliM even in the absence of FliH. The importance of the C ring as a possible docking site for export substrates, chaperones and FliI through FliH for their efficient delivery to membrane components of the export apparatus is discussed.     

Self-chaperoning of the type III secretion system needle tip proteins IpaD and BipD

Bacteria expressing type III secretion systems (T3SS) have been responsible for the deaths of millions worldwide, acting as key virulence elements in diseases ranging from plague to typhoid fever. The T3SS is composed of a basal body, which traverses both bacterial membranes, and an external needle through which effector proteins are secreted. We report multiple crystal structures of two proteins that sit at the tip of the needle and are essential for virulence: IpaD from Shigella flexneri and BipD from Burkholderia pseudomallei. The structures reveal that the N-terminal domains of the molecules are intramolecular chaperones that prevent premature oligomerization, as well as sharing structural homology with proteins involved in eukaryotic actin rearrangement. Crystal packing has allowed us to construct a model for the tip complex that is supported by mutations designed using the structure.     

A monoclonal antibody against a carbohydrate epitope in lipopolysaccharide differentiates Chlamydophila psittaci from Chlamydophila pecorum, Chlamydophila pneumoniae, and Chlamydia trachomatis

Lipopolysaccharide (LPS) of Chlamydophila psittaci but not of Chlamydophila pneumoniae or Chlamydia trachomatis contains a tetrasaccharide of 3-deoxy-alpha-d-manno-oct-2-ulopyranosonic acid (Kdo) of the sequence Kdo(2-->8)[Kdo(2-->4)] Kdo(2-->4)Kdo. After immunization with the synthetic neoglycoconjugate antigen Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4) Kdo-BSA, we obtained the mouse monoclonal antibody (mAb) S69-4 which was able to differentiate C. psittaci from Chlamydophila pecorum, C. pneumoniae, and C. trachomatis in double labeling experiments of infected cell monolayers and by enzyme-linked immunosorbent assay (ELISA). The epitope specificity of mAb S69-4 was determined by binding and inhibition assays using bacteria, LPS, and natural or synthetic Kdo oligosaccharides as free ligands or conjugated to BSA. The mAb bound preferentially Kdo(2-->8)[Kdo(2-->4)]Kdo(2-->4)Kdo(2-->4) with a K(d) of 10 microM, as determined by surface plasmon resonance (SPR) for the monovalent interaction using mAb or single chain Fv. Cross-reactivity was observed with Kdo(2-->4)Kdo(2-->4) Kdo but not with Kdo(2-->8)Kdo(2-->4)Kdo, Kdo disaccharides in 2-->4- or 2-->8-linkage, or Kdo monosaccharide. MAb S69-4 was able to detect LPS on thin-layer chromatography (TLC) plates in amounts of <10 ng by immunostaining. Due to the high sensitivity achieved in this assay, the antibody also detected in vitro products of cloned Kdo transferases of Chlamydia. The antibody can therefore be used in medical and veterinarian diagnostics, general microbiology, analytical biochemistry, and studies of chlamydial LPS biosynthesis. Further contribution to the general understanding of carbohydrate-binding antibodies was obtained by a comparison of the primary structure of mAb S69-4 to that of mAb S45-18 of which the crystal structure in complex with its ligand has been elucidated recently (Nguyen et al., 2003, Nat. Struct. Biol., 10, 1019-1025).     

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.