Structural analysis of CACHE domain of the McpA chemoreceptor from Leptospira interrogans.

Leptospira is a genus of spirochete bacteria highly motile that includes pathogenic species responsible to cause leptospirosis disease. Chemotaxis and motility are required for Leptospira infectivity, pathogenesis, and invasion of bacteria into the host. In prokaryotes, the most common chemoreceptors are methyl-accepting chemotaxis proteins that have a role play to detect the chemical signals and move to a favorable environment for its survival. Here, we report the first crystal structure of CACHE domain of the methyl-accepting chemotaxis protein (McpA) of L. interrogans. The structural analysis showed that McpA adopts similar α/ß architecture of several other bacteria chemoreceptors. We also found a typical dimerization interface that appears to be functionally crucial for signal transmission and chemotaxis. In addition to McpA structural analyses, we have identified homologous proteins and conservative functional regions using bioinformatics techniques. These results improve our understanding the relationship between chemoreceptor structures and functions of Leptospira species.

Identification of a novel protein in the genome sequences of Leptospira interrogans with the ability to interact with host's components.

BACKGROUND: Leptospirosis is an infectious disease that affects humans and animals worldwide. The etiological agents of this disease are the pathogenic species of the genus Leptospira. The mechanisms involved in the leptospiral pathogenesis are not full understood. The elucidation of novel mediators of host-pathogen interaction is important in the detection of virulence factors involved in the pathogenesis of leptospirosis. OBJECTIVE: This work focused on identification and characterization of a hypothetical protein of Leptospira encoded by the gene LIC10920. METHODS: The protein of unknown function was predicted to be surface exposed. Therefore, the LIC10920 gene was cloned and the protein expressed in Escherichia coli BL21 (DE3) Star pLysS strain. The recombinant protein was purified by metal affinity chromatography and evaluated with leptospirosis human serum samples. The interaction with host components was also performed. RESULTS: The recombinant protein was recognized by antibodies present in leptopsirosis human serum, suggesting its expression during infection. Immunofluorescence and intact bacteria assays indicated that the bacterial protein is surface-exposed. The recombinant protein interacted with human laminin, in a dose-dependent and saturable manner and was named Lsa24.9, for Leptospiral surface adhesin, followed by its molecular mass. Lsa24.9 also binds plasminogen (PLG) in a dose-dependent and saturable fashion, fulfilling receptor ligand interaction. Moreover, Lsa24.9 has the ability to acquire PLG from normal human serum, exhibiting similar profile as observed with the human purified component. PLG bound Lsa24.9 was able of generating plasmin, which could increase the proteolytic power of the bacteria. CONCLUSIONS: This novel leptospiral protein may function as an adhesin at the colonization steps and may help the invasion process by plasmin generation at the bacterial cell surface.

Crystal structure and biochemical characterization of O-acetylhomoserine acetyltransferase from Mycobacterium smegmatis ATCC 19420.

Mycobacterium smegmatis is a good model for studying the physiology and pathogenesis of Mycobacterium tuberculosis due to its genetic similarity. As methionine biosynthesis exists only in microorganisms, the enzymes involved in methionine biosynthesis can be a potential target for novel antibiotics. Homoserine O-acetyltransferase from M. smegmatis (MsHAT) catalyzes the transfer of acetyl-group from acetyl-CoA to homoserine. To investigate the molecular mechanism of MsHAT, we determined its crystal structure in apo-form and in complex with either CoA or homoserine and revealed the substrate binding mode of MsHAT. A structural comparison of MsHAT with other HATs suggests that the conformation of the α5 to α6 region might influence the shape of the dimer. In addition, the active site entrance shows an open or closed conformation and might determine the substrate binding affinity of HATs.

Crystallization of FcpA from Leptospira, a novel flagellar protein that is essential for pathogenesis.

The protein FcpA is a unique component of the flagellar filament of spirochete bacteria belonging to the genus Leptospira. Although it plays an essential role in translational motility and pathogenicity, no structures of FcpA homologues are currently available in the PDB. Its three-dimensional structure will unveil the novel motility mechanisms that render pathogenic Leptospira particularly efficient at invading and disseminating within their hosts, causing leptospirosis in humans and animals. FcpA from L. interrogans was purified and crystallized, but despite laborious attempts no useful X ray diffraction data could be obtained. This challenge was solved by expressing a close orthologue from the related saprophytic species L. biflexa. Three different crystal forms were obtained: a primitive and a centred monoclinic form, as well as a hexagonal variant. All forms diffracted X-rays to suitable resolutions for crystallographic analyses, with the hexagonal type typically reaching the highest limits of 2.0 Šand better. A variation of the quick-soaking procedure resulted in an iodide derivative that was instrumental for single-wavelength anomalous diffraction methods.

Immune response in hamsters immunised with a recombinant fragment of LigA from Leptospira interrogans, associated with carrier molecules.

Immunisation with the C-terminal region of leptospiral immunoglobulin-like A protein (LigANI) has shown promising results against leptospirosis. We evaluated the humoral immune response and protection induced by LigANI associated with carboxyl multi-walled carbon nanotubes (COOH-MWCNTs), CpG oligodeoxynucleotides (CpG ODNs), or Alhydrogel. Animals immunised with CpG ODNs were unable to develop a humoral immune response, whereas immunisation with LigANI and COOH-MWCNTs produced a high level of IgG antibodies, similar to that with LigANI and Alhydrogel, but it was not protective. The use of carbon nanotubes as an adjuvant in subunit vaccines against leptospirosis is a novel approach for improving specific IgG production.

Evaluation of combined B cell specific N-terminal immunogenic domains of LipL21 for diagnosis of leptospirosis.

Leptospiral outer membrane protein LipL21 and its truncated N-terminal immunogenic region (I-LipL21) were evaluated for diagnosis of leptospirosis. The complete coding sequence of LipL21 nucleotide sequence was subjected to BCPred and VaxiJen analysis for determination of B cell specific immunogenic epitopes. Epitope1 ACS STD TGQ KDA TTV GDG (1.8837), Epitope2 WGG PPE QRN DGK TPR DTN (0.9483), Epitope3 VKG VGV YEC KAT GSG SDP (1.4077) and Epitope4 NEW ECQ CVI YAK FPG GKD (0.4462) were predicted. LipL21 and N-terminal fragment having B-cell specific epitopes with higher VaxiJen score >0.9 as truncated I-LipL21 were cloned independently in pET15b and expressed in Escherichia coli. IgM ELISA and dot blot assay was performed for sera samples collected from Delhi-NCR for leptospiral whole cell lysate (WCL), recombinant LipL21 and I-LipL21. The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were found to be 92.5%, 92.8%, 83.3%, and 97% respectively for recombinant I-LipL21 by IgM-ELISA. 11-14.8% increased sensitivity was observed over LipL21 and WCL. The I-LipL21 dot blot assay showed a further increased sensitivity of 3.8% over the IgM-ELISA. Therefore I-LipL21 may be the ideal candidate protein for diagnosis of leptospirosis.

Dual nuclease activity of a Cas2 protein in CRISPR-Cas subtype I-B of Leptospira interrogans.

Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 carries a set of cas genes associated with CRISPR-Cas subtype I-B. Herein, we report for the first time active transcription of a set of cas genes (cas1 to cas8) of L. interrogans where cas4, cas1, cas2 and cas6, cas3, cas8, cas7, cas5 are clustered together in two independent operons. As an initial step toward comprehensive understanding of CRISPR-Cas system in spirochete, the biochemical study of one of the core Leptospira Cas2 proteins (Lep_Cas2) showed nuclease activity on both DNA and RNA in a nonspecific manner. Additionally, unlike other known Cas2 proteins, Lep_Cas2 showed metal-independent RNase activity and preferential activity on RNA over DNA. These results provide insight for understanding Cas2 diversity existing in the prokaryotic adaptive immune system.

Solution structure of leptospiral LigA4 Big domain.

Pathogenic Leptospiraspecies express immunoglobulin-like proteins which serve as adhesins to bind to the extracellular matrices of host cells. Leptospiral immunoglobulin-like protein A (LigA), a surface exposed protein containing tandem repeats of bacterial immunoglobulin-like (Big) domains, has been proved to be involved in the interaction of pathogenic Leptospira with mammalian host. In this study, the solution structure of the fourth Big domain of LigA (LigA4 Big domain) from Leptospira interrogans was solved by nuclear magnetic resonance (NMR). The structure of LigA4 Big domain displays a similar bacterial immunoglobulin-like fold compared with other Big domains, implying some common structural aspects of Big domain family. On the other hand, it displays some structural characteristics significantly different from classic Ig-like domain. Furthermore, Stains-all assay and NMR chemical shift perturbation revealed the Ca(2+) binding property of LigA4 Big domain.

Structural characterization of a novel subfamily of leucine-rich repeat proteins from the human pathogen Leptospira interrogans.

Pathogenic Leptospira spp. are the agents of leptospirosis, an emerging zoonotic disease. Analyses of Leptospira genomes have shown that the pathogenic leptospires (but not the saprophytes) possess a large number of genes encoding proteins containing leucine-rich repeat (LRR) domains. In other pathogenic bacteria, proteins with LRR domains have been shown to be involved in mediating host-cell attachment and invasion, but their functions remain unknown in Leptospira. To gain insight into the potential function of leptospiral LRR proteins, the crystal structures of four LRR proteins that represent a novel subfamily with consecutive stretches of a 23-amino-acid LRR repeat motif have been solved. The four proteins analyzed adopt the characteristic α/ß-solenoid horseshoe fold. The exposed residues of the inner concave surfaces of the solenoid, which constitute a putative functional binding site, are not conserved. The various leptospiral LRR proteins could therefore recognize distinct structural motifs of different host proteins and thus serve separate and complementary functions in the physiology of these bacteria.

Phenotypic and Molecular Characterization of Leptospira interrogans Isolated from Canis familiaris in Southern Brazil.

Leptospirosis is a zoonotic disease caused by pathogenic spirochetes from the genus Leptospira, which includes 20 species and more than 300 serovars. Canines are important hosts of pathogenic leptospires and can transmit the pathogen to humans via infected urine. Here, we report the phenotypic and molecular characterization of Leptospira interrogans isolated from Canis familiaris in Southern Brazil. The isolated strain was characterized by variable-number tandem-repeats analysis as L. interrogans, serogroup Icterohaemorrhagiae. In addition, the isolate was recognized by antibodies from human and canine serum samples previously tested by microscopic agglutination test. Ultimately, the expression of membrane-associated antigens (LipL32 and leptospiral immunoglobulin-like proteins) from pathogenic leptospires using monoclonal antibodies was detected by indirect immunofluorescence assay. In conclusion, identification of new strains of Leptospira can help in the diagnosis and control of leptospirosis.

Pathogenic Leptospira species acquire factor H and vitronectin via the surface protein LcpA.

Upon infection, pathogenic Leptospira species bind several complement regulators in order to overcome host innate immunity. We previously characterized a 20-kDa leptospiral surface protein which interacts with C4b binding protein (C4BP): leptospiral complement regulator-acquiring protein A (LcpA). Here we show that LcpA also interacts with human factor H (FH), which remains functionally active once bound to the protein. Antibodies directed against short consensus repeat 20 (SCR20) inhibited binding of FH to LcpA by approximately 90%, thus confirming that this particular domain is involved in the interaction. We have also shown for the first time that leptospires bind human vitronectin and that the interaction is mediated by LcpA. Coincubation with heparin blocked LcpA-vitronectin interaction in a dose-dependent manner, strongly suggesting that binding may occur through the heparin binding domains of vitronectin. LcpA also bound to the terminal pathway component C9 and inhibited Zn(2+)-induced polymerization and membrane attack complex (MAC) formation. Competitive binding assays indicated that LcpA interacts with C4BP, FH, and vitronectin through distinct sites. Taken together, our findings indicate that LcpA may play a role in leptospiral immune evasion.

Leptospiral lipopolysaccharide stimulates the expression of toll-like receptor 2 and cytokines in pig fibroblasts.

Pigs throughout the world are afflicted with leptospirosis, causing serious economic losses and potential hazards to human health. Although it has been known that leptospiral lipopolysaccharide (L-LPS) is involved in an immunological reaction between an antigen and a host cell, little is known about how the immune system of pigs can respond to L-LPS. Here, we stimulated pig fibroblasts by L-LPS and then quantitatively measured gene and protein expression levels of two toll-like receptors (TLRs), TLR2 and TLR4, by real-time PCR and Western blotting. As a result, expression of TLR2 was found to be significantly up-regulated within 24 h after L-LPS stimulation whereas induction of TLR4 expression was relatively weak. We also revealed that of myeloid differentiation primary response gene 88 (MyD88), interleukin 6 (IL-6) and IL-8 gene expressions were markedly up-regulated by L-LPS stimulation. These results may suggest that the pig cell can activate TLR2 rather than TLR4 by L-LPS stimulation, thereby inducing expression of cytokines.

VapC from the leptospiral VapBC toxin-antitoxin module displays ribonuclease activity on the initiator tRNA.

The prokaryotic ubiquitous Toxin-Antitoxin (TA) operons encode a stable toxin and an unstable antitoxin. The most accepted hypothesis of the physiological function of the TA system is the reversible cessation of cellular growth under stress conditions. The major TA family, VapBC is present in the spirochaete Leptospira interrogans. VapBC modules are classified based on the presence of a predicted ribonucleasic PIN domain in the VapC toxin. The expression of the leptospiral VapC in E. coli promotes a strong bacterial growth arrestment, making it difficult to express the recombinant protein. Nevertheless, we showed that long term induction of expression in E. coli enabled the recovery of VapC in inclusion bodies. The recombinant protein was successfully refolded by high hydrostatic pressure, providing a new method to obtain the toxin in a soluble and active form. The structural integrity of the recombinant VapB and VapC proteins was assessed by circular dichroism spectroscopy. Physical interaction between the VapC toxin and the VapB antitoxin was demonstrated in vivo and in vitro by pull down and ligand affinity blotting assays, respectively, thereby indicating the ultimate mechanism by which the activity of the toxin is regulated in bacteria. The predicted model of the leptospiral VapC structure closely matches the Shigella's VapC X-ray structure. In agreement, the ribonuclease activity of the leptospiral VapC was similar to the activity described for Shigella's VapC, as demonstrated by the cleavage of tRNAfMet and by the absence of unspecific activity towards E. coli rRNA. This finding suggests that the cleavage of the initiator transfer RNA may represent a common mechanism to a larger group of bacteria and potentially configures a mechanism of post-transcriptional regulation leading to the inhibition of global translation.

Understanding the highly efficient catalysis of prokaryotic peptide deformylases by shedding light on the determinants specifying the low activity of the human counterpart.

Peptide deformylases (PDFs), which are essential and ubiquitous enzymes involved in the removal of the N-formyl group from nascent chains, are classified into four subtypes based on the structural and sequence similarity of specific conserved domains. All PDFs share a similar three-dimensional structure, are functionally interchangeable in vivo and display similar properties in vitro, indicating that their molecular mechanism has been conserved during evolution. The human mitochondrial PDF is the only exception as despite its conserved fold it reveals a unique substrate-binding pocket together with an unusual kinetic behaviour. Unlike human PDF, the closely related mitochondrial PDF1As from plants have catalytic efficiencies and enzymatic parameters that are similar to those of other classes of PDFs. Here, the aim was to identify the structural basis underlying the properties of human PDF compared with all other PDFs by focusing on plant mitochondrial PDF1A. The construction of a chimaera composed of plant PDF1A with the nonrandom substitutions found in a conserved motif of its human homologue converted it into an enzyme with properties similar to the human enzyme, indicating the crucial role of these positions. The crystal structure of this human-like plant PDF revealed that substitution of two residues leads to a reduction in the volume of the ligand-binding site together with the introduction of negative charges, unravelling the origin of the weak affinity of human PDF for its substrate. In addition, the substitution of the two residues of human PDF modifies the transition state of the reaction through alteration of the network of interactions between the catalytic residues and the substrate, leading to an overall reduced reaction rate.

Coiled to diffuse: Brownian motion of a helical bacterium.

We employ real-time three-dimensional confocal microscopy to follow the Brownian motion of a fixed helically shaped Leptospira interrogans (LI) bacterium. We extract from our measurements the translational and the rotational diffusion coefficients of this bacterium. A simple theoretical model is suggested, perfectly reproducing the experimental diffusion coefficients, with no tunable parameters. An older theoretical model, where edge effects are neglected, dramatically underestimates the observed rates of translation. Interestingly, the coiling of LI increases its rotational diffusion coefficient by a factor of 5, compared to a (hypothetical) rectified bacterium of the same contour length. Moreover, the translational diffusion coefficients would have decreased by a factor of ~1.5, if LI were rectified. This suggests that the spiral shape of the spirochaete bacteria, in addition to being employed for their active twisting motion, may also increase the ability of these bacteria to explore the surrounding fluid by passive Brownian diffusion.

Lsa30, a novel adhesin of Leptospira interrogans binds human plasminogen and the complement regulator C4bp.

Pathogenic Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. Surface proteins have the potential to promote several activities, including adhesion. This work aimed to study the leptospiral coding sequence (CDS) LIC11087, genome annotated as hypothetical outer membrane protein. The LIC11087 gene was cloned and expressed in Escherichia coli BL21 (DE3) strain by using the expression vector pAE. The recombinant protein tagged with N-terminal 6XHis was purified by metal-charged chromatography and characterized by circular dichroism (CD) spectroscopy. The recombinant protein has the ability to mediate attachment to the extracellular matrix (ECM) components, laminin and plasma fibronectin, and was named Lsa30 (Leptospiral surface adhesin of 30 kDa). Lsa30 binds to laminin and to plasma fibronectin in a dose-dependent and saturable manner, with dissociation equilibrium constants (K(D)) of 292 ± 24 nm and 157 ± 35 nm, respectively. Moreover, the Lsa30 is a plasminogen (PLG) receptor, capable of generating plasmin, in the presence of activator. This protein may interfere with the complement cascade by interacting with C4bp regulator. The Lsa30 is probably a new surface protein of Leptospira as revealed by immunofluorescence assays with living organisms and the reactivity with antibodies present in serum samples of experimentally infected hamsters. Thus, Lsa30 is a novel versatile protein that may play a role in mediating adhesion and may help pathogenic Leptospira to overcome tissue barriers and to escape the immune system.

Using iRT, a normalized retention time for more targeted measurement of peptides.

Multiple reaction monitoring (MRM) has recently become the method of choice for targeted quantitative measurement of proteins using mass spectrometry. The method, however, is limited in the number of peptides that can be measured in one run. This number can be markedly increased by scheduling the acquisition if the accurate retention time (RT) of each peptide is known. Here we present iRT, an empirically derived dimensionless peptide-specific value that allows for highly accurate RT prediction. The iRT of a peptide is a fixed number relative to a standard set of reference iRT-peptides that can be transferred across laboratories and chromatographic systems. We show that iRT facilitates the setup of multiplexed experiments with acquisition windows more than four times smaller compared to in silico RT predictions resulting in improved quantification accuracy. iRTs can be determined by any laboratory and shared transparently. The iRT concept has been implemented in Skyline, the most widely used software for MRM experiments.

A LigA three-domain region protects hamsters from lethal infection by Leptospira interrogans.

The leptospiral LigA protein consists of 13 bacterial immunoglobulin-like (Big) domains and is the only purified recombinant subunit vaccine that has been demonstrated to protect against lethal challenge by a clinical isolate of Leptospira interrogans in the hamster model of leptospirosis. We determined the minimum number and location of LigA domains required for immunoprotection. Immunization with domains 11 and 12 was found to be required but insufficient for protection. Inclusion of a third domain, either 10 or 13, was required for 100% survival after intraperitoneal challenge with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130. As in previous studies, survivors had renal colonization; here, we quantitated the leptospiral burden by qPCR to be 1.2×10(3) to 8×10(5) copies of leptospiral DNA per microgram of kidney DNA. Although renal histopathology in survivors revealed tubulointerstitial changes indicating an inflammatory response to the infection, blood chemistry analysis indicated that renal function was normal. These studies define the Big domains of LigA that account for its vaccine efficacy and highlight the need for additional strategies to achieve sterilizing immunity to protect the mammalian host from leptospiral infection and its consequences.

Immuno-fluorescence assay of leptospiral surface-exposed proteins.

Bacterial surface proteins are involved in direct contact with host cells and in uptake of nutrients from the environment (1). For this reason, cellular localization can provide insights into the functional role of bacterial proteins. Surface localization of bacterial proteins is a key step towards identification of virulence factors involved in mechanisms of pathogenicity. Methods for fractionating leptospiral membranes (2-5) may be selective for a certain class of outer-membrane proteins (OMPs), such as lipoproteins vs. transmembrane OMPs, and therefore lead to misclassification. This likely is due to structural differences and how they are associated to the outer membrane. Lipoproteins are associated with membranes via a hydrophobic interaction between the N-terminal lipid moiety (three fatty acids) and the lipid bilayer phospholipids (6, 7). In contrast, transmembrane OMPs are typically integrated into the lipid bilayer by amphipathic ß-sheets arranged in a barrel-like structure (8, 9). In addition, presence of a protein in the outer-membrane does not necessarily guarantee that the protein or its domains are exposed on the surface. Spirochetal outer membranes are known to be fragile and therefore necessitate methods involving gentle manipulation of cells and inclusion of sub-surface protein controls to assess the integrity of the outer membrane. Here, we present an immunofluorescence assay (IFA) method to directly assess surface exposure of proteins on intact leptospires. This method is based on recognition of leptospiral surface proteins by antigen-specific antibodies. Herein, antibodies specific for OmpL54(10) are detetcted aftero binding to native, surface exposed epitopes. Comparison of antibody reactivity to intact versus permeabilized cells enables evaluation of cellular distribution and whether or not a protein is selectively present on leptospiral surface. The integrity of outer membrane should be assessed using antibody to one or more subsurface proteins, preferably located in the periplasm. The surface IFA method can be used to analyze surface exposure of any leptospiral protein to which specific antibodies are available. Both the usefulness and limitation of the method depends on whether the antibodies employed are able to bind to native epitopes. Since antibodies often are raised against recombinant proteins, epitopes of native, surface-exposed proteins may not be recognized. Nevertheless, the surface IFA method is a valuable tool for studying components of intact bacterial surfaces. This method can be applied not only for leptospires but also other spirochetes and gram-negative bacteria. For stronger conclusions regarding surface-exposure of OMPs, a comprehensive approach involving several cell localization methods is recommended (10).

Characterization of the immunogenic and antigenic potential of putative lipoproteins from Leptospira interrogans.

The search for a vaccine capable of conferring heterologous protection, through the identification of conserved and cross-protective antigens, remains an ongoing priority in leptospirosis research. In the present study, an in silico analysis was used to identify potentially protective lipoproteins from Leptospira interrogans serovar Copenhageni. Eight putative lipoproteins were selected (LIC10009, LIC10054, LIC10091, LIC11058, LIC11567, LIC13059, LIC13305, and LIC20172), cloned and expressed in Escherichia coli and purified by affinity chromatography. The recombinant proteins were used to inoculate mice and the subsequent humoral immune response was evaluated by ELISA. Seven of the potential lipoproteins induced a significant IgG response. Furthermore, all of the recombinant proteins were recognized by antibodies present in the sera of severe leptospirosis patients. These putative lipoproteins exhibited potential for further evaluation as prospective vaccine candidates.