Evaluation of Susceptibility of the Human Pathogen Helicobacter pylori to the Antibiotic Capreomycin.

Helicobacter pylori infection causes gastritis, peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma, and gastric cancer and can also promote thrombosis. It is estimated that approximately 4.5 billion individuals are infected, thus rendering H. pylori the most prevalent microbial pathogen. Currently established regimes for antibiotic treatment are massively challenged by increasing drug resistance and the development of novel antimicrobial therapies is urgently required. The antibiotic capreomycin is clinically used against multiple drug-resistant strains of Mycobacterium tuberculosis. It targets the complex between TlyA, a hemolysin- and RNA-binding protein, and the bacterial rRNA. In this study we have explored the possible antibacterial effects of capreomycin against several strains of H. pylori and found only moderate activity which was comparable to metronidazole-resistant strains. Molecular docking of capreomycin to TlyA proteins from H. pylori and M. tuberculosis identified several residues within TlyA which interact with the drug; however, binding affinities of H. pylori- TlyA for capreomycin appear to be higher than those of Mycobacterium- TlyA. The data suggest that capreomycin may warrant further investigations into its potential use as antibiotic against H. pylori.

Optimized high-purity protein preparation of biologically active recombinant VacA cytotoxin variants from Helicobacter pylori.

Vacuolating cytotoxin A (VacA) is a highly polymorphic virulence protein produced by the human gastric pathogen Helicobacter pylori which can cause gastritis, peptic ulcer and gastric cancer. Here, we present an optimized protein preparation of the mature full-length VacA variants (m1-and m2-types) and their 33-kDa N-terminal and 55/59-kDa C-terminal domains as biologically active recombinant proteins fused with an N-terminal His(6) tag. All recombinant VacA constructs were over-expressed in Escherichia coli as insoluble inclusions which were soluble when phosphate buffer (pH 7.4) was supplemented with 5-6 M urea. Upon immobilized-Ni2+ affinity purification under 5-M urea denaturing conditions, homogenous products (>95% purity) of 55/59-kDa domains were consistently obtained while only ~80% purity of both mature VacA variants and the 33-kDa truncate was achieved, thus requiring additional purification by size-exclusion chromatography. After successive refolding via optimized stepwise dialysis, all refolded VacA proteins were proven to possess both cytotoxic and vacuolating activity against cultured human gastric epithelial cells albeit the activity observed for VacA-m2 was lower than the m1-type variant. Such an optimized protocol described herein was effective for production of high-purity recombinant VacA proteins in large amounts (~30-40 mg per liter culture) that would pave the way for further studies on sequence-structure and function relationships of different VacA variants.

Importance of the Cys124-Cys128 intermolecular disulfide bonding for oligomeric assembly and hemolytic activity of the Helicobacter pylori TlyA hemolysin.

Although the TlyA hemolysin from Helicobacter pylori has been implicated as a potential virulence factor involved in mediating host cell colonization and hence disease progression, its structural determinants underlying the biological activity are still largely uncertain. In this study, an important role of the formation of a particular disulfide bond for functional oligomeric assembly of the H. pylori TlyA toxin was evidently elucidated. The 27-kDa TlyA recombinant protein was overexpressed in Escherichia coli, subsequently purified to near homogeneity by cation exchange chromatography, and proven to be hemolytically active against sheep erythrocytes. Additionally, TlyA-induced hemolytic activity was significantly diminished under conditions of disulfide bond reduction with a thiol-reducing agent, dithiothreitol. When the purified TlyA protein was subjected to modified SDS-PAGE under non-reducing conditions, the presence of an oligomeric state of this protein was clearly revealed by its apparent molecular mass of ∼48 kDa. Recombinant E. coli cells expressing TlyA also displayed contact-dependent hemolysis of erythrocytes, suggesting TlyA localization at the bacterial outer membrane and thus supporting the formation of disulfide-bonded TlyA. Homology-based modeling and in silico structural assembly analysis of TlyA signified potential intermolecular, rather than intramolecular, disulfide bonding through Cys124 and Cys128. Subsequently, single substitution of either of these Cys residues with Ser severely affected the oligomeric assembly of both TlyA mutants and hence abolished their hemolytic activity. Altogether, our present data provide pivotal evidence that the formation of intermolecular disulfide bonding between Cys124 and Cys128 plays a critical role in structural assembly of a biologically active-TlyA oligomer.

Purification and characterization of the antibacterial peptidase lysostaphin from Staphylococcus simulans: Adverse influence of Zn2+ on bacteriolytic activity.

Lysostaphin, a bacteriolytic toxin from Staphylococcus simulans, is a Zn2+-dependent endopeptidase that cleaves pentaglycine cross-bridges found in peptidoglycan of certain Staphylococci. Here, we have investigated a critical influence of Zn2+ ions on lysostaphin-induced bioactivity. Initially, we succeeded in producing a large amount with high purity of the 28-kDa His-tagged mature lysostaphin via soluble expression in Escherichia coli and subsequent purification via immobilized-Ni2+ affinity chromatography (IMAC). The purified monomeric bacteriocin exhibited concentration-dependent bioactivity against S. aureus and its methicillin-resistant strain through cell-wall hydrolysis rather than membrane perturbation. Following pre-incubation of the purified lysostaphin with exogenous Zn2+, a marked inhibition in staphylolytic activity was observed. When the pre-mixture was exposed to 1,10-phenanthroline (PNT, a Zn2+-chelator), the adverse effect of the exogenous Zn2+ on bioactivity was greatly decreased. Conversely, lysostaphin pre-treated with excess PNT retained relatively high bioactivity, indicating ineffective chelation of PNT to detach the catalytic Zn2+ from the active-site pocket. Structural analysis of the lysostaphin-catalytic domain together with amino acid sequence alignments of lysostaphin-like endopeptidases revealed a potential extraneous Zn2+-binding site found in close proximity to the Zn2+-coordinating active site. Overall our results provide more insights into an adverse influence of exogenous Zn2+ ions on staphylolytic activity of the purified Zn2+-dependent endopeptidase lysostaphin, implicating the presence of an extraneous inhibitory metal-binding site.

A Helicobacter pylori Vacuolating Cytotoxin A: Mouse DHFR Fusion Protein Triggers Dye Release from Liposomes.

The membrane perturbing action of the VacA toxin from Helicobacter pylori is responsible for vacuole formation in intracellular compartments and the induction of apoptosis. The VacA toxin contains 2 major domains, p33 and p55, which are involved in receptor binding and membrane pore formation, respectively. Improved methodologies for VacA purification and assays are urgently needed for further detailed investigations on the mechanism of action of this significant virulence factor. We found that by fusing mouse DHFR with the N-terminus of the full-length (p88) VacA toxin, expression levels in recombinant E. coli were substantially increased when compared to the conventional (His)6-tagged protein. The DHFR-VacA fusion protein was active in sulforhodamine dye-release assays using liposomes at acidic pH in a concentration-dependent manner. Enzymatic activity of DHFR in the fusion protein was comparable to a commercial reference sample of purified DHFR; however, activity was insensitive to inhibition by methotrexate. Our findings suggest that the VacA p88 toxin with a modified N-terminus still maintains its capability for membrane insertion and that pH-dependent conformational changes occur during interaction of VacA with membranes.

Acylation of the Bordetella pertussis CyaA-hemolysin: Functional implications for efficient membrane insertion and pore formation.

Previously, the ~130-kDa CyaA-hemolysin domain (CyaA-Hly) from Bordetella pertussis co-expressed with CyaC-acyltransferase in Escherichia coli was demonstrated to be palmitoylated at Lys983 and thus activated its hemolytic activity against target erythrocytes. Here, we report the functional importance of Lys983-palmitoylation for membrane insertion and pore formation of CyaA-Hly. Intrinsic fluorescence emissions of both non-acylated CyaA-Hly (NA/CyaA-Hly) and CyaA-Hly were indistinguishable, suggesting no severe conformational change upon acylation at Lys983. Following pre-incubation of sheep erythrocytes with NA/CyaA-Hly, there was a drastic decrease in CyaA-Hly-induced hemolysis. Direct interactions between NA/CyaA-Hly and target erythrocyte membranes were validated via membrane-binding assays along with Western blotting, suggestive of acylation-independent capability of NA/CyaA-Hly to interact with erythrocyte membranes. As compared with CyaA-Hly, NA/CyaA-Hly displayed a slower rate of incorporation into DOPC:DOPE:Ch or DiPhyPC bilayers under symmetrical conditions (1M KCl, 10mM HEPES, pH7.4) and formed channels exhibiting different conductance. Further analysis revealed that channel-open lifetime in DOPC:DOPE:Ch bilayers of NA/CyaA-Hly was much shorter than that of the acylated form, albeit slightly shorter lifetime found in DiPhyPC bilayers. Sequence alignments of the Lys983-containing CyaA-segment with those of related RTX-cytolysins revealed a number of highly conserved hydrophobic residues and a Lys/Arg cluster that is predicted be important for toxin-membrane interactions. Altogether, our data disclosed that the Lys983-linked palmitoyl group is not directly involved in either binding to target erythrocyte membranes or toxin-induced channel conductivity, but rather required for efficient membrane insertion and pore formation of the acylated CyaA-Hly domain.

The Forgotten Virulence Factor: The 'non-conventional' Hemolysin TlyA And Its Role in Helicobacter pylori Infection.

Helicobacter pylori is a human-specific Gram-negative pathogenic bacterium which colonizes the gastric mucosal layer in the stomach causing diseases such as peptic ulcer, adenocarcinoma, and gastric lymphoma. It is estimated that approximately half of the world's population is infected with H. pylori making it the most intensively characterized microbial pathogen up to now. Hemolysis has been suggested to significantly contribute to colonization of the stomach and disease progression by H. pylori. In a number of earlier studies, TlyA was characterized as a putative pore-forming cytolysin. Although a few observations in the literature suggest a role for TlyA as significant virulence factor of H. pylori, the molecular and structural characterization of this protein is much curtailed at present. Given the intensive characterization of numerous H. pylori virulence factors over the past decade, surprisingly little information exists for the TlyA toxin and its significance for pathogenesis. This review provides a brief overview on microbial hemolysis and its role for pathogenesis and discusses recent research efforts aimed at an improved understanding of the role of the 'non-conventional' hemolysin and its associated RNA methyltransferase TlyA from H. pylori.

Importance of polarity of the α4-α5 loop residue-Asn(166) in the pore-forming domain of the Bacillus thuringiensis Cry4Ba toxin: implications for ion permeation and pore opening.

Bacillus thuringiensis Cry4Ba toxin is lethal to mosquito-larvae by forming ion-permeable pores in the target midgut cell membrane. Previously, the polarity of Asn(166) located within the α4-α5 loop composing the Cry4Ba pore-forming domain was shown to be crucial for larvicidal activity. Here, structurally stable-mutant toxins of both larvicidal-active (N166D) and inactive (N166A and N166I) mutants were FPLC-purified and characterized for their relative activities in liposomal-membrane permeation and single-channel formation. Similar to the 65-kDa trypsin-activated wild-type toxin, the N166D bio-active mutant toxin was still capable of releasing entrapped calcein from lipid vesicles. Conversely, the two other bio-inactive mutants showed a dramatic decrease in causing membrane permeation. When the N166D mutant was incorporated into planar lipid bilayers (under symmetrical conditions at 150mM KCl, pH8.5), it produced single-channel currents with a maximum conductance of about 425pS comparable to the wild-type toxin. However, maximum conductances for single K(+)-channels formed by both bio-inactive mutants (N166I and N166A) were reduced to approximately 165-205pS. Structural dynamics of 60-ns simulations of a trimeric α4-α5 pore model in a fully hydrated-DMPC system revealed that an open-pore structure could be observed only for the simulated pores of the wild type and N166D. Additionally, the number of lipid molecules interacting with both wild-type and N166D pores is relatively higher than those of N166A and N166I pores. Altogether, our results further signify that the polarity at the α4-α5 loop residue-Asn(166) is directly involved in ion permeation through the Cry4Ba toxin-induced ionic pore and pore opening at the membrane-water interface.

Bacillus thuringiensis Cry4Aa insecticidal protein: functional importance of the intrinsic stability of the unique α4-α5 loop comprising the Pro-rich sequence.

The long loop connecting transmembrane α4 and α5 of the Bacillus thuringiensis Cry4Aa toxin possesses a unique feature with Pro-rich sequence (Pro(193)Pro(194)_Pro(196)) which was shown to be crucial for toxicity. Here, the structural role in the intrinsic stability of the Pro-rich sequence toward toxin activity was investigated. Three Val-substituted mutants (P193V, P194V and P196V) and one Phe-substituted mutant (P193F) were generated and over-expressed in Escherichia coli as inclusions at levels equal to the wild-type. Bioassays demonstrated that all mutants, particularly P193V and P193F whose inclusions were hardly soluble in carbonate buffer (pH9.0), exhibited reduced toxicity, suggesting an essential role in toxin function by the specific cyclic structure of individual Pro residues. Analysis of the 65-kDa Cry4Aa structure from 10-ns molecular dynamics (MD) simulations revealed that the α4-α5 loop is substantially stable as it showed low structural fluctuation with a 1.2-Å RMSF value. When the flexibility of the α4-α5 loop was increased through P193G, P194G and P196G substitutions, decreased toxicity was also observed for all mutants, mostly for the P193G mutant with low alkali-solubility, suggesting a functional importance of loop-rigidity attributed by individual Pro-cyclic side-chains, particularly Pro(193). Further MD simulations revealed that the most critical residue-Pro(193) for which mutations vastly affect toxin solubility and larval toxicity is in close contact with several surrounding residues, thus playing an additional role in the structural arrangement of the Cry4Aa toxin molecule. Altogether, our data signify that the intrinsic stability of the unique Cry4Aa α4-α5 loop structure comprising the Pro-rich sequence plays an important role in toxin activity.

Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity.

The interaction between Bacillus thuringiensis Cry toxins and their receptors on midgut cells of susceptible insect larvae is the critical determinant in toxin specificity. Besides GPI-linked alkaline phosphatase in Aedes aegypti mosquito-larval midguts, membrane-bound aminopeptidase N (AaeAPN) is widely thought to serve as a Cry4Ba receptor. Here, two full-length AaeAPN isoforms, AaeAPN2778 and AaeAPN2783, predicted to be GPI-linked were cloned and successfully expressed in Spodoptera frugiperda (Sf9) cells as 112- and 107-kDa membrane-bound proteins, respectively. In the cytotoxicity assay, Sf9 cells expressing each of the two AaeAPN isoforms showed increased sensitivity to the Cry4Ba mosquito-active toxin. Double immunolocalization revealed specific binding of Cry4Ba to each individual AaeAPN expressed on the cell membrane surface. Sequence analysis and homology-based modeling placed these two AaeAPNs to the M1 aminopeptidase family as they showed similar four-domain structures, with the most conserved domain II being the catalytic component. Additionally, the most variable domain IV containing negatively charged surface patches observed only in dipteran APNs could be involved in insect specificity. Overall results demonstrated that these two membrane-bound APN isoforms were responsible for mediating Cry4Ba toxicity against AaeAPN-expressed Sf9 cells, suggesting their important role as functional receptors for the toxin counterpart in A. aegypti mosquito larvae.

Isolated CyaA-RTX subdomain from Bordetella pertussis: Structural and functional implications for its interaction with target erythrocyte membranes.

The 126-kDa Bordetella pertussis CyaA-hemolysin (CyaA-Hly) was previously expressed in Escherichia coli as a soluble precursor that can be acylated to retain hemolytic activity. Here, we investigated structural and functional characteristics of a ∼100-kDa isolated RTX (Repeat-in-ToXin) subdomain (CyaA-RTX) of CyaA-Hly. Initially, we succeeded in producing a large amount with high purity of the His-tagged CyaA-RTX fragment and in establishing the interaction of acylated CyaA-Hly with sheep red blood cell (sRBC) membranes by immuno-localization. Following pre-incubation of sRBCs with non-acylated CyaA-Hly or with the CyaA-RTX fragment that itself produces no hemolytic activity, there was a dramatic decrease in CyaA-Hly-induced hemolysis. When CyaA-RTX was pre-incubated with anti-CyaA-RTX antisera, the capability of CyaA-RTX to neutralize the hemolytic activity of CyaA-Hly was greatly decreased. A homology-based model of the 100-kDa CyaA-RTX subdomain revealed a loop structure in Linker II sharing sequence similarity to human WW domains. Sequence alignment of Linker II with the human WW-domain family revealed highly conserved aromatic residues important for protein-protein interactions. Altogether, our present study demonstrates that the recombinant CyaA-RTX subdomain retains its functionality with respect to binding to target erythrocyte membranes and the WW-homologous region in Linker II conceivably serves as a functional segment required for receptor-binding activity.

Design and evaluation of substrate-based octapeptide and non substrate-based tetrapeptide inhibitors of dengue virus NS2B-NS3 proteases.

A series of 45 peptide inhibitors was designed, synthesized, and evaluated against the NS2B-NS3 proteases of the four subtypes of dengue virus, DEN-1-4. The design was based on proteochemometric models for Michaelis (Km) and cleavage rate constants (kcat) of protease substrates. This led first to octapeptides showing submicromolar or low micromolar inhibitory activities on the four proteases. Stepwise removal of cationic substrate non-prime side residues and variations in the prime side sequence resulted finally in an uncharged tetrapeptide, WYCW-NH2, with inhibitory Ki values of 4.2, 4.8, 24.4, and 11.2 µM for the DEN-1-4 proteases, respectively. Analysis of the inhibition data by proteochemometric modeling suggested the possibility for different binding poses of the shortened peptides compared to the octapeptides, which was supported by results of docking of WYCW-NH2 into the X-ray structure of DEN-3 protease.

Molecular Mechanisms of Antibiotic Resistance and Novel Treatment Strategies for Helicobacter pylori Infections.

Helicobacter pylori infects approximately 50% of the world's population and is considered the major etiological agent of severe gastric diseases, such as peptic ulcers and gastric carcinoma. Increasing resistance to standard antibiotics has now led to an ever-decreasing efficacy of eradication therapies and the development of novel and improved regimens for treatment is urgently required. Substantial progress has been made over the past few years in the identification of molecular mechanisms which are conducive to resistant phenotypes as well as for efficient strategies to counteract strain resistance and to avoid the use of ineffective antibiotics. These involve molecular testing methods, improved salvage therapies, and the discovery of novel and potent antimicrobial compounds. High rates of prevalence and gastric cancer are currently observed in Asian countries, including Japan, China, Korea, and Taiwan, where concomitantly intensive research efforts were initiated to explore advanced eradication regimens aimed at reducing the risk of gastric cancer. In this review, we present an overview of the known molecular mechanisms of antibiotic resistance and discuss recent intervention strategies for H. pylori diseases, with a view of the research progress in Asian countries.

Correction: Srisuphanunt et al. Molecular Mechanisms of Antibiotic Resistance and Novel Treatment Strategies for Helicobacter pylori Infections. Trop. Med. Infect. Dis. 2023, 8, 163.

In the original publication [...].

Structural platform for the autolytic activity of an intact NS2B-NS3 protease complex from dengue virus.

Dengue virus completes its protein synthesis inside human cells on the endoplasmic reticulum membrane by processing the single-chain polyprotein precursor into 10 functional proteins. This vital process relies on the two-component virus-encoded protease complex; nonstructural protein 3 (NS3) possesses the proteolytic activity in its N-terminus, and NS2B acts as a fundamental activator and membrane-anchoring subunit. The membrane-associated NS2B-NS3 complex has essentially not yet been isolated or studied. We describe here a useful protocol for the preparation of the full-length NS2B-NS3 complex from dengue serotype 2 virus by utilizing a Mistic-fusion expression cassette in Escherichia coli. The protease complex was successfully solubilized and stabilized from the bacterial membrane and purified with the use of fos-choline-14 detergent. The detergent-solubilized protease complex retained autolytic activity and, intriguingly, exists as a robust trimer, implying a molecular assembly in the membrane. We further conducted a random mutagenesis study to efficiently scan for entire residues and motifs contributing to autocleavage and provide evidence of the importance of the two distal ß-hairpins in the activity of the viral protease. Our results provide the first comprehensive view of an active dengue protease in the membrane-bound form.

Correction: Srisuphanunt et al. Prognostic Indicators for the Early Prediction of Severe Dengue Infection: A Retrospective Study in a University Hospital in Thailand. Trop. Med. Infect. Dis. 2022, 7, 162.

In the original publication [...].

Prognostic Indicators for the Early Prediction of Severe Dengue Infection: A Retrospective Study in a University Hospital in Thailand.

This study aimed to develop simple diagnostic guidelines which would be useful for the early detection of severe dengue infections. Retrospective data of patients with dengue infection were reviewed. Patients with diagnosed dengue infection were categorized in line with the International Statistical Classification of Diseases (ICD-10): A90, dengue fever; A91, dengue hemorrhagic fever; and A910, dengue hemorrhagic fever with shock. A total of 302 dengue-infected patients were enrolled, of which 136 (45%) were male and 166 (55%) were female. Multivariate analysis was conducted to determine independent diagnostic predictors of severe dengue infection and to convert simple diagnostic guidelines into a scoring system for disease severity. Coefficients for significant predictors of disease severity generated by ordinal multivariable logistic regression analysis were transformed into item scores. The derived total scores ranged from 0 to 38.6. The cut-off score for predicting dengue severity was higher than 14, with an area under the receiver operating curve (AUROC) of 0.902. The predicted positive value (PPV) was 68.7% and the negative predictive value (NPV) was 94.1%. Our study demonstrates that several diagnostic parameters can be effectively combined into a simple score sheet with predictive value for the severity evaluation of dengue infection.

Leptospiral Leucine-Rich Repeat Protein-Based Lateral Flow for Assessment of Canine Leptospiral Immunoglobulin G.

The recombinant, modified leucine-rich repeat protein rhKU_Sej_LRR_2271 has been suggested as a candidate for leptospiral vaccine development since it was predicted to be a transmembrane protein containing leucine-rich repeat motifs and immunogenic epitopes. The immunogenic epitopes showed binding affinities with lower IC50 values than peptides of known antigenic proteins, e.g., LipL32. Moreover, this protein was immunoreactive with hyperimmune sera against several serovars. In this study, we aimed to develop a lateral flow strip test using the rhKU_Sej_LRR_2271 protein for the detection of anti-leptospiral IgG in dogs. The lateral flow assay was performed with 184 dog plasma samples and evaluated with a culture method, 16S ribosomal RNA gene (rss) analysis real-time PCR, and LipL32 ELISA. The culture method failed to detect leptospires in the dog blood samples. Six of nine symptomatic dogs gave positive results with the real-time PCR assay. The lateral flow assay and LipL32 ELISA gave positive results with 59 and 50 dogs, respectively. The sensitivity, specificity, and accuracy of the rhKU_Sej_LRR_2271 lateral flow strip test were 70.00, 82.09, and 78.80%, respectively, when compared with LipL32 ELISA. There was a significant association between the LipL32 ELISA and the rhKU_Sej_LRR_2271 lateral flow assay. The rhKU_Sej_LRR_2271 lateral flow strip test has therefore demonstrated a good potential to detect anti-leptospiral IgG in dogs.

Leptospira borgpetersenii Leucine-Rich Repeat Proteins and Derived Peptides in an Indirect ELISA Development for the Diagnosis of Canine Leptospiral Infections.

Domestic and stray dogs can be frequently infected by Leptospira, and thus may represent a source for transmission of this zoonotic disease in Thailand. Here, we have used peptides derived from a recombinant leucine-rich repeat (LRR) protein of Leptospira, rKU_Sej_LRR_2012M, for the development of an indirect enzyme-linked immunosorbent assay (ELISA) aimed at detecting antibodies against Leptospira interrogans, L. borgpetersenii, and L. biflexa, the three major seroprevalences in Thai dogs. The rKU_Sej_LRR_2012M protein is recognized by hyperimmune sera against several leptospiral serovars. The epitope peptides of the rKU_Sej_LRR_2012M showed binding affinities with lower IC50 values than peptides of known antigenic protein LipL32. Four peptides, 2012-3T, 2012-4B, 2012-5B and pool 2012-B, were specifically recognized by rabbit hyperimmune sera against nine serovars from three Leptospira spp. The indirect peptide-based ELISAs with these four peptides were evaluated with the LipL32 ELISA by using a receiver-operator curve (ROC) analysis. All peptides had an area under the curve of ROC (AUC) greater than 0.8, and the sum of sensitivity and specificity for each peptide was greater than 1.5. The degree of agreement of 2012-3T and pool 2012-B and 2012-4B and 2012-5B peptides were in moderate-to-good levels with kappa values of 0.41-0.60 and 0.61-0.80, when compared with LipL32, respectively. This finding would suggest an excellent capability of the 2012-4B and 2012-5B peptide-based ELISAs assay for the diagnosis of canine leptospiral infections.

Leptospira borgpetersenii Leucine-Rich Repeat Proteins Provide Strong Protective Efficacy as Novel Leptospiral Vaccine Candidates.

Leucine-rich repeat (LRR) proteins are advocated for being assessed in vaccine development. Leptospiral LRR proteins were identified recently in silico from the genome of Leptospira borgpetersenii serogroup Sejroe, the seroprevalence of leptospiral infections of cattle in Thailand. Two LRR recombinant proteins, rKU_Sej_LRR_2012M (2012) and rhKU_Sej_LRR_2271 (2271), containing predicted immunogenic epitopes, were investigated for their cross-protective efficacies in an acute leptospirosis model with heterologous Leptospira serovar Pomona, though, strains from serogroup Sejroe are host-adapted to bovine, leading to chronic disease. Since serovar Pomona is frequently reported as seropositive in cattle, buffaloes, pigs, and dogs in Thailand and causes acute and severe leptospirosis in cattle by incidental infection, the serogroup Sejroe LRR proteins were evaluated for their cross-protective immunity. The protective efficacies were 37.5%, 50.0%, and 75.0% based on the survival rate for the control, 2012, and 2271 groups, respectively. Sera from 2012-immunized hamsters showed weak bactericidal action compared to sera from 2271-immunized hamsters (p < 0.05). Therefore, bacterial tissue clearances, inflammatory responses, and humoral and cell-mediated immune (HMI and CMI) responses were evaluated only in 2271-immunized hamsters challenged with virulent L. interrogans serovar Pomona. The 2271 protein induced prompt humoral immune responses (p < 0.05) and leptospiral tissue clearance, reducing tissue inflammation in immunized hamsters. In addition, protein 2271 and its immunogenic peptides stimulated splenocyte lymphoproliferation and stimulated both HMI and CMI responses by activating Th1 and Th2 cytokine gene expression in vaccinated hamsters. Our data suggest that the immunogenic potential renders rhKU_Sej_LRR_2271 protein a promising candidate for the development of a novel cross-protective vaccine against animal leptospirosis.