Blocking pathogenic Leptospira invasion with aptamer molecules targeting outer membrane LipL32 protein.

This study aimed to develop aptamers targeting LipL32, a most abundant lipoprotein in pathogenic Leptospira, to hinder bacterial invasion. The objectives were to identify high-affinity aptamers through SELEX and evaluate their specificity and inhibitory effects. SELEX was employed to generate LipL32 aptamers (L32APs) over 15 rounds of selection. L32APs' binding affinity and specificity for pathogenic Leptospira were assessed. Their ability to inhibit LipL32-ECM interaction and Leptospira invasion was investigated. Animal studies were conducted to evaluate the impact of L32AP treatment on survival rates, Leptospira colonization, and kidney damage. Three L32APs with strong binding affinity were identified. They selectively detected pathogenic Leptospira, sparing non-pathogenic strains. L32APs inhibited LipL32-ECM interaction and Leptospira invasion. In animal studies, L32AP administration significantly improved survival rates, reduced Leptospira colonies, and mitigated kidney damage compared to infection alone. This pioneering research developed functional aptamers targeting pathogenic Leptospira. The identified L32APs exhibited high affinity, pathogen selectivity, and inhibition of invasion and ECM interaction. L32AP treatment showed promising results, enhancing survival rates and reducing Leptospira colonization and kidney damage. These findings demonstrate the potential of aptamers to impede pathogenic Leptospira invasion and aid in recovery from Leptospira-induced kidney injury (190 words).

Crystal structure of Leptospira LSS_01692 reveals a dimeric structure and induces inflammatory responses through Toll-like receptor 2-dependent NF-κB and MAPK signal transduction pathways.

Leptospirosis is a commonly overlooked zoonotic disease that occurs in tropical and subtropical regions. Recent studies have divided the Leptospira spp. into three groups based on virulence, including pathogenic, intermediate, and saprophytic species. Pathogenic species express a protein family with leucine-rich repeat (LRR) domains, which are less expressed or absent in nonpathogenic species, highlighting the importance of this protein family in leptospirosis. However, the role of LRR domain proteins in the pathogenesis of leptospirosis is still unknown and requires further investigation. In this study, the 3D structure of LSS_01692 (rLRR38) was obtained using X-ray crystallography at a resolution of 3.2 Å. The results showed that rLRR38 forms a typical horseshoe structure with 11 α-helices and 11 ß-sheets and an antiparallel dimeric structure. The interactions of rLRR38 with extracellular matrix and cell surface receptors were evaluated using ELISA and single-molecule atomic force microscopy. The results showed that rLRR38 interacted with fibronectin, collagen IV, and Toll-like receptor 2 (TLR2). Incubating HK2 cells with rLRR38 induced two downstream inflammation responses (IL-6 and MCP-1) in the TLR2 signal transduction pathway. The TLR2-TLR1 complex showed the most significant upregulation effects under rLRR38 treatment. Inhibitors also significantly inhibited nuclear factor κB and mitogen-activated protein kinases signals transduction under rLRR38 stimulation. In conclusion, rLRR38 was determined to be a novel LRR domain protein in 3D structure and demonstrated as a TLR2-binding protein that induces inflammatory responses. These structural and functional studies provide a deeper understanding of the pathogenesis of leptospirosis.

Transcriptome landscape reveals the chronic inflammatory response in kidneys affected by the combinatory effect of leptospirosis and nephrotoxic injury.

Leptospirosis can cause chronic kidney damage, putting patients at risk of additional kidney injury due to other factors that can lead to renal failure. To understand the combined effect, the transcriptome profiles of kidneys of mice with adenine-induced and chronically Leptospira-infected kidneys were analysed. Chronic inflammation and T-helper 17 immune responses were activated and a high-level expression of Indoleamine 2,3-dioxygenase 1 protein was found. The results indicate that the combination may predispose patients to chronic inflammation, kidney function disruption, and symptoms seen in progressive chronic kidney disease (CKD). Furthermore, immunometabolic regulation may contribute to renal injury caused by chronic leptospirosis with secondary nephrotoxic injury. This study identified several significantly disrupted genes that could serve as potential targets for the diagnosis or treatment of CKD. Our work provides insight into the combined effect of leptospirosis and secondary kidney damage and the molecular basis for rapid progression of CKD.

Leptospirosis: A Potential Culprit for Chronic Kidney Disease of Uncertain Etiology.

PURPOSE: Chronic kidney disease of uncertain etiology (CKDu) is an environmental nephropathy in which the etiological factors are yet uncertain. Leptospirosis, a spirochetal infection that is common among agricultural communities, has been identified as a potential etiology for CKDu beyond environmental nephropathy. Although CKDu is a chronic kidney disease, in endemic regions, an increasing number of cases are reported with features suggestive of acute interstitial nephritis without any known reason (AINu), with or without background CKD. The study hypothesizes that exposure to pathogenic leptospires is one of the causative factors for the occurrence of AINu. METHOD: This study was carried out using 59 clinically diagnosed AINu patients, 72 healthy controls from CKDu endemic region (endemic controls [ECs]), and 71 healthy controls from CKDu non-endemic region (non-endemic controls [NECs]). RESULTS: The seroprevalence of 18.6, 6.9, and 7.0% was observed in the AIN (or AINu), EC, and NEC groups, respectively, from the rapid IgM test. Among 19 serovars tested, the highest seroprevalence was observed at 72.9, 38.9, and 21.1% in the AIN (AINu), EC, and NEC groups, respectively, by microscopic agglutination test (MAT), particularly for serovar Leptospira santarosai serovar Shermani. This emphasizes the presence of infection in AINu patients, and this also suggests that Leptospira exposure might play an important role in AINu. CONCLUSION: These data suggest that exposure to Leptospira infection could be one of the possible causative factors for the occurrence of AINu, which may lead to CKDu in Sri Lanka.

Implication of the IL-10-Expression Signature in the Pathogenicity of Leptospira-Infected Macrophages.

Leptospirosis, an emerging infectious disease caused by pathogenic Leptospira spp., occurs in ecoregions with heavy rainfall and has public health implications. Macrophages are the major anti-Leptospira phagocytes that infiltrate the kidneys during renal leptospirosis, which is caused by leptospires residing in the renal tubules. The pathogenicity of Leptospira spp. in immune effector cells such as macrophages is not well understood. To evaluate this pathogenesis, we characterized and compared the transcriptome-wide alterations in macrophages infected with pathogenic and nonpathogenic Leptospira spp. Using transcriptome data and quantitative reverse transcription PCR analysis, at 2 h postinfection, the hypoxia-inducible factor-1α-dependent glycolysis pathway was implicated in pathogenic Leptospira-infected macrophages but not in nonpathogenic leptospiral infections. Immune-related biological processes were mostly activated in pathogenic Leptospira-infected macrophages, and flow cytometry investigations revealed that classically activated macrophages represent the predominant polarization status. At 24 h after infection, biological pathways associated with interleukin-10, IL-10, signaling the induction of macrophage tolerance, as well as higher levels of IL-10 mRNA and protein expression, were observed in nonpathogenic Leptospira-infected macrophages compared to in pathogenic leptospiral infection. Following leptospiral infection of macrophages, strong IL-10-expressing transcriptome signatures were observed following nonpathogenic leptospiral infection. The transcriptional programs generated in Leptospira-infected macrophages revealed an inflammatory milieu following the production of a critical anti-inflammatory cytokine, IL-10, which is implicated in controlling the pathogenicity of activated macrophages. These findings imply that IL-10-mediated anti-inflammatory responses and tolerance in activated macrophages induced by nonpathogenic Leptospira spp. infection reduce inflammation and tissue damage, thus providing a potential therapeutic target for leptospirosis. IMPORTANCE Activation of macrophages by Leptospira spp. infection is thought to be involved in the pathogenesis of leptospirosis. To evaluate the innate macrophage responses to Leptospira spp., specifically pathogenic versus nonpathogenic Leptospira spp., we characterized the entire transcriptome-wide alterations in infected macrophages. We showed that hypoxia-inducible factor-1α and immune-related pathways are activated in pathogenic leptospiral-infected macrophages. We confirmed the significantly high levels of IL-10-expressing signatures and tolerance in activated macrophages caused by nonpathogenic Leptospira infection. Furthermore, nonpathogenic leptospiral infections attenuated macrophage activation responses. These findings suggest a potential therapeutic strategy for the immune microenvironment caused by macrophage activation driven by IL-10 overexpression, which may contribute to regulating inflammation in leptospirosis.

Injection of hybrid 3D spheroids composed of podocytes, mesenchymal stem cells, and vascular endothelial cells into the renal cortex improves kidney function and replenishes glomerular podocytes.

Podocytes are highly differentiated epithelial cells that are crucial for maintaining the glomerular filtration barrier in the kidney. Podocyte injury followed by depletion is the major cause of pathological progression of kidney diseases. Although cell therapy has been considered a promising alternative approach to kidney transplantation for the treatment of kidney injury, the resultant therapeutic efficacy in terms of improved renal function is limited, possibly owing to significant loss of engrafted cells. Herein, hybrid three-dimensional (3D) cell spheroids composed of podocytes, mesenchymal stem cells, and vascular endothelial cells were designed to mimic the glomerular microenvironment and as a cell delivery vehicle to replenish the podocyte population by cell transplantation. After creating a native glomerulus-like condition, the expression of multiple genes encoding growth factors and basement membrane factors that are strongly associated with podocyte maturation and functionality was significantly enhanced. Our in vivo results demonstrated that intrarenal transplantation of podocytes in the form of hybrid 3D cell spheroids improved engraftment efficiency and replenished glomerular podocytes. Moreover, the proteinuria of the experimental mice with hypertensive nephropathy was effectively reduced. These data clearly demonstrated the potential of hybrid 3D cell spheroids for repairing injured kidneys.

Transcriptomic signatures of exacerbated progression in leptospirosis subclinical chronic kidney disease with secondary nephrotoxic injury.

High-incidence regions of leptospirosis caused by Leptospira spp. coincide with chronic kidney disease. This study investigated whether asymptomatic leptospirosis is an emerging culprit that predisposes to progressive chronic kidney disease when superimposed on secondary nephrotoxic injury. Kidney histology/function and whole transcriptomic profiles were evaluated for Leptospira-infected C57/BL6 mice with adenine-induced kidney injury. The extent of tubulointerstitial kidney lesions and expression of inflammation/fibrosis genes in infected mice with low-dose (0.1%) adenine, particularly in high-dose (0.2%) adenine-fed superimposed on Leptospira-infected mice, were significantly increased compared with mice following infection or adenine diet alone, and the findings are consistent with renal transcriptome analysis. Pathway enrichment findings showed that integrin-ß- and fibronectin-encoding genes had distinct expression within the integrin-linked kinase-signaling pathway, which were upregulated in 0.2% adenine-fed Leptospira-infected mice but not in 0.2% adenine-fed mice, indicating that background subclinical Leptospiral infection indeed enhanced subsequent secondary nephrotoxic kidney injury and potential pathogenic molecules associated with secondary nephrotoxic leptospirosis. Comparative analysis of gene expression patterns with unilateral ureteric obstruction-induced mouse renal fibrosis and patients with chronic kidney disease showed that differentially expressed orthologous genes such as hemoglobin-α2, PDZ-binding kinase, and DNA topoisomerase II-α were identified in infected mice fed with low-dose and high-dose adenine, respectively, revealing differentially expressed signatures identical to those found in the datasets and may serve as markers of aggravated kidney progression. This study indicates that background subclinical leptospirosis, when subjected to various degrees of subsequent secondary nephrotoxic injury, may predispose to exacerbated fibrosis, mimicking the pathophysiological process of progressive chronic kidney disease.NEW & NOTEWORTHYLeptospira-infected mice followed by secondary nephrotoxic injury exacerbated immune/inflammatory responses and renal fibrosis. Comparison with the murine model revealed candidates involved in the progression of renal fibrosis in chronic kidney disease (CKD). Comparative transcriptome study suggests that secondary nephrotoxic injury in Leptospira-infected mice recapitulates the gene expression signatures found in CKD patients. This study indicates that secondary nephrotoxic injury may exacerbate CKD in chronic Leptospira infection implicating in the progression of CKD of unknown etiology.

Peptidoglycan mediates Leptospira outer membrane protein Loa22 to toll-like receptor 2 for inflammatory interaction: a novel innate immune recognition.

Leptospirosis is an overlooked zoonotic disease caused by pathogenic Leptospira depended on virulence of Leptospira and the host-pathogen interaction. Kidney is the major organ infected by Leptospira which causes tubulointerstitial nephritis. Leptospira outer membrane contains several virulence factors and an outer membrane protein A (OmpA) like protein (Loa22) is essential for virulence. Pull-down assays suggested that Loa22 was a potential Toll-Like Receptor 2 (TLR2) binding candidates from pathogenic Leptospira. Confocal microscopy was employed to observe the co-localization of TLR2 and Loa22-LPGN (Leptospira peptidoglycan) complexes. Atomic force microscopy (AFM), side-directed mutagenesis, and enzyme-linked immunosorbent assay (ELISA) were performed to investigate the affinity between rLoa22, LPGN, and TLR2. Real time PCR was applied to measure the cytokines expression. Downstream signal transduction components were verified by western blot to evaluate the gene regulations. Mutation of two Loa22 key residues (Asp122 and Arg143) attenuated the affinities for LPGN. rLoa22-LPGN complexes were observed to co-localize with TLR2 and provoked inflammatory responses including CXCL8/IL8, hCCL2/MCP-1, and hTNF-α. Affinity studies suggested that Loa22-LPGN complexes elevated the affinity to TLR2 as compared to Loa22 protein. Downstream signals from TLR2 including p38, ERK, and JNK were regulated under rLoa22-LPGN complexes treatments. This study identified LPGN mediates interactions between Loa22 and TLR2 and induces downstream signals to trigger inflammatory responses. rLoa22-LPGN-TLR2 complexes reveal a novel binding mechanism for the innate immune system.

Enhanced early immune response of leptospiral outer membrane protein LipL32 stimulated by narrow band mid-infrared exposure.

Previous studies revealed significant impact on cancer cell by mid-infrared (MIR) radiation. However, the effects of narrow band MIR on immune reaction and infectious disease are still unknown. In this study, an enhanced innate immune response was observed through the interaction between Leptospiral outer membrane protein (LipL32) and toll-like receptor 2 (TLR2). Thereafter, human kidney proximal tubular cells (HK-2 cells) initiated a serial reaction of enhanced MCP-1 production. The 6 µm narrow bandwidth light source emitted by waveguide thermal emitter (WTE) was applied to induce carbonyl group (CO bond) stretching vibration during the stage of antigen-receptor complex formation. The amount of MCP-1 gene expression had 2.5 folds increase after narrow band MIR illumination comparing to non-MIR illumination at low dose LipL32 condition. Besides, both ELISA and confocal microscopy results also revealed that the chemokine concentration increased significantly after narrow band MIR illumination either at low or high concentration of LipL32. Furthermore, a specific phenomenon that narrow band MIR can amplify the signal of weak immune response by enhancing sensitivity of the interaction between antigen and receptor was observed. This study exhibits clear evidence that the narrow band MIR exposure can modulate the early immune response of infectious disease and play a potential role to develop host-directed therapy in the future.

A Microfluidic Platform for Investigating Transmembrane Pressure-Induced Glomerular Leakage.

Transmembrane pressure across the glomerular filter barrier may underlie renal failure. However, studies of renal failure have been difficult owing to a lack of in vitro models to capture the transmembrane pressure in a controlled approach. Here we report a microfluidic platform of podocyte culture to investigate transmembrane pressure induced glomerular leakage. Podocytes, the glomerular epithelial cells essential for filtration function, were cultivated on a porous membrane supplied with transmembrane pressure ΔP. An anodic aluminum oxide membrane with collagen coating was used as the porous membrane, and the filtration function was evaluated using dextrans of different sizes. The results show that dextran in 20 kDa and 70 kDa can penetrate the podocyte membrane, whereas dextran in 500 kDa was blocked until ΔP ≥ 60 mmHg, which resembles the filtration function when ΔP was in the range of a healthy kidney (ΔP < 60 mmHg) as well as the hypertension-induced glomerular leakage (ΔP ≥ 60 mmHg). Additionally, analysis showed that synaptopodin and actin were also downregulated when ΔP > 30 mmHg, indicating that the dysfunction of renal filtration is correlated with the reduction of synaptopodin expression and disorganized actin cytoskeleton. Taking together, our microfluidic platform enables the investigation of transmembrane pressure in glomerular filter membrane, with potential implications for drug development in the future.

Murine Renal Transcriptome Profiles Upon Leptospiral Infection: Implications for Chronic Kidney Diseases.

Background: Leptospirosis caused by pathogenic Leptospira spp leads to kidney damage that may progress to chronic kidney disease. However, how leptospiral infections induced renal damage is unclear. Methods: We apply microarray and next-generation sequencing technologies to investigate the first murine transcriptome-wide, leptospires-mediated changes in renal gene expression to identify biological pathways associated with kidney damage. Results: Leptospiral genes were detected in renal transcriptomes of mice infected with Leptospira interrogans at day 28 postinfection, suggesting colonization of leptospires within the kidney with propensity of chronicity. Comparative differential gene expression and pathway analysis were investigated in renal transcriptomes of mice infected with pathogens and nonpathogens. Pathways analysis showed that Toll-like receptor signaling, complements activation, T-helper 1 type immune response, and T cell-mediated immunity/chemotaxis/proliferation were strongly associated with progressive tubulointerstitial damage caused by pathogenic leptospiral infection. In addition, 26 genes related with complement system, immune function, and cell-cell interactions were found to be significantly up-regulated in the L interrogans-infected renal transcriptome. Conclusions: Our results provided comprehensive knowledge regarding the host transcriptional response to leptospiral infection in murine kidneys, particularly the involvement of cell-to-cell interaction in the immune response. It would provide valuable resources to explore functional studies of chronic renal damage caused by leptospiral infection.

Active Components of Leptospira Outer Membrane Protein LipL32 to Toll-Like Receptor 2.

Proteins belonging to the toll-like receptor (TLR) family, particularly TLR2, are the major components of innate immunity against Leptospira infection. The ligands for TLR2 harbor several conserved patterns such as lipidation molecules, leucine-rich repeat (LRR) domains, TLR2 binding motifs, and TLR2 binding structure. In Leptospira, LipL32 interacts with TLR2 on human kidney cells concomitantly stimulating inflammatory responses. However, the binding mechanism of LipL32 to TLR2 is unknown. The computational prediction suggests that ß1ß2, loop-α3-loop, and α4 domains of LipL32 play vital roles in LipL32-TLR2 complex formation. To test these predictions, protein truncation experiments revealed that LipL32ΔNß1ß2 significantly decreased the affinity to TLR2 while LipL32ΔCα4 slightly reduced it. Interestingly, LipL32ΔCenα3 retained affinity to TLR2 in the absence of Ca2+ ions, indicating that Cenα3 play a role preventing the interaction between LipL32 and TLR2. Furthermore, the critical residues of LipL32 involved in interacting with TLR2 suggested that V35S, L36S and L263S variants significantly decreased the affinity to TLR2. The results further confirm that LipL32 interacts with TLR2 through Nß1ß2 and Cα4 domains of LipL32 as well as LipL32-TLR2 complex formation results from hydrophobic interactions. This study provides a detailed mechanism of the interaction between LipL32 and TLR2 and the residues involved in complex formation.

Inhibition of spleen tyrosine kinase (syk) suppresses renal fibrosis through anti-inflammatory effects and down regulation of the MAPK-p38 pathway.

Renal fibrosis results from an excessive accumulation of extracellular matrix that occurs in most types of chronic kidney disease. Among the many fibrogenic factors that regulate renal fibrotic processes, transforming growth factor-ß1 (TGF-ß1) and inflammation after injury play critical roles. Spleen tyrosine kinase (Syk) is important for signaling processes implicated in autoimmune, inflammatory, and allergic diseases. We examined the effects of Syk inhibition on renal fibrosis in vivo and on TGF-ß1-induced renal fibroblast activation in vitro. A unilateral ureteral obstruction (UUO) model was induced in male B6 mice. Mice with UUO were administered a Syk inhibitor or saline intraperitoneally 1 day before UUO surgery and daily thereafter. Both kidneys were harvested 7 days after surgery for further analysis. For the in vitro experiments, NRK-49F rat fibroblasts were pre-incubated with a Syk inhibitor before TGF-ß1 stimulation. The inhibitory effects of Syk inhibition on signaling pathways down-stream of TGF-ß1 were analyzed. In the UUO mouse model, administration of a Syk inhibitor attenuated extracellular matrix protein deposition and expression of α-smooth muscle actin, type I collagen, and fibronectin in a dose-dependent manner. In addition, macrophage infiltration in UUO kidney was reduced by Syk inhibition. Pre-incubation of NRK-49F cells with a Syk inhibitor suppressed TGF-ß1-induced myofibroblast activation. Furthermore, inhibitory effects of Syk inhibition on TGF-ß1-mediated myofibroblast activation were associated with down-regulation of MAPK-p38. These results suggest that Syk inhibition reduces tubulointerstitial fibrosis in UUO mice and inhibits TGF-ß1-induced kidney myofibroblast activation. Syk inhibition could have therapeutic potential for the treatment of renal tubulointerstitial fibrosis.

Overlooked Risk for Chronic Kidney Disease after Leptospiral Infection: A Population-Based Survey and Epidemiological Cohort Evidence.

BACKGROUND: Leptospirosis is the most widespread zoonosis. Chronic human infection and asymptomatic colonization have been reported. However, renal involvement in those with leptospira chronic exposure remains undetermined. METHODS AND FINDINGS: In 2007, a multistage sampling survey for chronic kidney disease (CKD) was conducted in a southern county of Taiwan, an area with a high prevalence of dialysis. Additionally, an independent cohort of 88 participants from a leptospira-endemic town was followed for two years after a flooding in 2009. Risks of CKD, stages of CKD, associated risk factors as well as kidney injury markers were compared among adults with anti-leptospira antibody as defined by titers of microscopic agglutination test (MAT). Of 3045 survey participants, the individuals with previous leptospira exposure disclosed a lower level of eGFR (98.3 ± 0.4 vs 100.8 ± 0.6 ml/min per 1.73 m2, P < 0.001) and a higher percentage of CKD, particularly at stage 3a-5 (14.4% vs 8.5%), than those without leptospira exposure. Multivariable linear regression analyses indicated the association of leptospiral infection and lower eGFR (95% CI -4.15 to -1.93, P < 0.001). In a leptospiral endemic town, subjects with a MAT titer ≥ 400 showed a decreased eGFR and higher urinary kidney injury molecule-1 creatinine ratio (KIM1/Cr) level as compared with those having lower titers of MAT (P < 0.05). Furthermore, two participants with persistently high MAT titers had positive urine leptospira DNA and deteriorating renal function. CONCLUSIONS AND SIGNIFICANCE: Our data are the first to show that chronic human exposure of leptospirosis is associated significantly with prevalence and severity of CKD and may lead to deterioration of renal function. This study also shed light on the search of underlying factors in areas experiencing CKD of unknown aetiology (CKDu) such as Mesoamerican Nephropathy.

The AMPK agonist AICAR inhibits TGF-ß1 induced activation of kidney myofibroblasts.

Activation of interstitial myofibroblasts and excessive production of extracellular matrix proteins are common pathways that contribute to chronic kidney disease. In a number of tissues, AMP-activated kinase (AMPK) activation has been shown to inhibit fibrosis. Here, we examined the inhibitory effect of the AMPK activator, 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR), on renal fibrosis in vivo and TGF-ß1-induced renal fibroblasts activation in vitro. A unilateral ureteral obstruction (UUO) model was induced in male BALB/c mice. Mice with UUO were administered AICAR (500 mg/Kg/day) or saline intraperitoneally 1 day before UUO surgery and daily thereafter. Both kidneys were harvested 7 days after surgery for further analysis. For the in vitro studies, NRK-49F rat fibroblasts were pre-incubated with AICAR before TGF-ß1 stimulation. The inhibitory effects of AICAR on signaling pathways down-stream of TGF-ß1 were analyzed. In UUO model mice, administration of AICAR attenuated extracellular matrix protein deposition and the expression of α-smooth muscle actin (α-SMA), type I collagen and fibronectin. Pre-incubation of NRK-49F cells with AICAR inhibited TGF-ß1-induced myofibroblast activation. Silencing of AMPKα1 by siRNA or by blocking AMPK activation with Compound C diminished the inhibitory effect of AICAR. Moreover, the inhibitory effects of AICAR on TGF-ß1-mediated myofibroblast activation were associated with down-regulation of ERK 1/2 and STAT3. Our results suggest that AICAR reduces tubulointerstitial fibrosis in UUO mice and inhibits TGF-ß1-induced kidney myofibroblast activation. AMPK activation by AICAR may have therapeutic potential for the treatment of renal tubulointerstitial fibrosis.

Potential impact on kidney infection: a whole-genome analysis of Leptospira santarosai serovar Shermani.

Leptospira santarosai serovar Shermani is the most frequently encountered serovar, and it causes leptospirosis and tubulointerstitial nephritis in Taiwan. This study aims to complete the genome sequence of L. santarosai serovar Shermani and analyze the transcriptional responses of L. santarosai serovar Shermani to renal tubular cells. To assemble this highly repetitive genome, we combined reads that were generated from four next-generation sequencing platforms by using hybrid assembly approaches to finish two-chromosome contiguous sequences without gaps by validating the data with optical restriction maps and Sanger sequencing. Whole-genome comparison studies revealed a 28-kb region containing genes that encode transposases and hypothetical proteins in L. santarosai serovar Shermani, but this region is absent in other pathogenic Leptospira spp. We found that lipoprotein gene expression in both L. santarosai serovar Shermani and L. interrogans serovar Copenhageni were upregulated upon interaction with renal tubular cells, and LSS19962, a L. santarosai serovar Shermani-specific gene within a 28-kb region that encodes hypothetical proteins, was upregulated in L. santarosai serovar Shermani-infected renal tubular cells. Lipoprotein expression during leptospiral infection might facilitate the interactions of leptospires within kidneys. The availability of the whole-genome sequence of L. santarosai serovar Shermani would make it the first completed sequence of this species, and its comparison with that of other Leptospira spp. may provide invaluable information for further studies in leptospiral pathogenesis.

LipL41, a hemin binding protein from Leptospira santarosai serovar Shermani.

Leptospirosis is one of the most widespread zoonotic diseases in the world. It is caused by the pathogen Leptospira that results in multiple-organ failure, in particular of the kidney. Outer membrane lipoprotein is the suspected virulence factor of Leptospira. In Leptospira spp LipL41 is one major lipoprotein and is highly conserved. Previous study suggests that LipL41 bears hemin-binding ability and might play a possible role in iron regulation and storage. However, the characterization of hemin-binding ability of LipL41 is still unclear. Here the hemin-binding ability of LipL41 was examined, yielding a K d = 0.59 ± 0.14 µM. Two possible heme regulatory motifs (HRMs), C[P/S], were found in LipL41 at (140)Cys-Ser and (220)Cys-Pro. The mutation study indicates that Cys140 and Cys220 might be cooperatively involved in hemin binding. A supramolecular assembly of LipL41 was determined by transmission electron microscopy. The LipL41 oligomer consists of 36 molecules and folds as a double-layered particle. At the C-terminus of LipL41, there are two tetratricopeptide repeats (TPRs), which might be involved in the protein-protein interaction of the supramolecular assembly.

Essential calcium-binding cluster of Leptospira LipL32 protein for inflammatory responses through the Toll-like receptor 2 pathway.

Leptospirosis is the most widespread zoonosis caused by the pathogenic Leptospira worldwide. LipL32, a 32-kDa lipoprotein, is the most abundant protein on the outer membrane of Leptospira and has an atypical poly(Asp) motif ((161)DDDDDGDD(168)). The x-ray crystallographic structure of LipL32 revealed that the calcium-binding cluster of LipL32 includes several essential residues Asp(132), Thr(133), Asp(164), Asp(165), and Tyr(178). The goals of this study were to determine possible roles of the Ca(2+)-binding cluster for the interaction of LipL32 and Toll-like receptor 2 (TLR2) in induced inflammatory responses of human kidney cells. Site-directed mutagenesis was employed to individually mutate Ca(2+)-binding residues of LipL32 to Ala, and their effects subsequently were observed. These mutations abolished primarily the structural integrity of the calcium-binding cluster in LipL32. The binding assay and atomic force microscopy analysis further demonstrated the decreased binding capability of LipL32 mutants to TLR2. Inflammatory responses induced by LipL32 variants, as determined by TLR2 pathway intermediates hCXCL8/IL-8, hCCL2/MCP-1, hMMP7, and hTNF-α, were also lessened. In conclusion, the calcium-binding cluster of LipL32 plays essential roles in presumably sustaining LipL32 conformation for its proper association with TLR2 to elicit inflammatory responses in human renal cells.

Sequence of Leptospira santarosai serovar Shermani genome and prediction of virulence-associated genes.

Leptospirosis, a widespread zoonosis, is a re-emerging infectious disease caused by pathogenic Leptospira species. In Taiwan, Leptospira santarosai serovar Shermani is the most frequently isolated serovar, causing both renal and systemic infections. This study aimed to generate a L. santarosai serovar Shermani genome sequence and categorize its hypothetical genes, particularly those associated with virulence. The genome sequence consists of 3,936,333 nucleotides and 4033 predicted genes. Additionally, 2244 coding sequences could be placed into clusters of orthologous groups and the number of genes involving cell wall/membrane/envelope biogenesis and defense mechanisms was higher than that of other Leptospira spp. Comparative genetic analysis based on BLASTX data revealed that about 73% and 68.8% of all coding sequences have matches to pathogenic L. interrogans and L. borgpetersenii, respectively, and about 57.6% to saprophyte L. biflexa. Among the hypothetical proteins, 421 have a transmembrane region, 172 have a signal peptide and 17 possess a lipoprotein signature. According to PFAM prediction, 32 hypothetical proteins have properties of toxins and surface proteins mediated bacterial attachment, suggesting they may have roles associated with virulence. The availability of the genome sequence of L. santarosai serovar Shermani and the bioinformatics re-annotation of leptospiral hypothetical proteins will facilitate further functional genomic studies to elucidate the pathogenesis of leptospirosis and develop leptospiral vaccines.

Leptospiral outer membrane lipoprotein LipL32 binding on toll-like receptor 2 of renal cells as determined with an atomic force microscope.

Leptopirosis is a renal disease caused by pathogenic Leptospira that primarily infects the renal proximal tubules, consequently resulting in severe tubular injuries and malfunctions. The protein extracted from the outer membrane of this pathogenic strain contains a major component of a 32 kDa lipoprotein (LipL32), which is absent in the counter membrane of nonpathogenic strains and has been identified as a crucial factor for host cell infection. Previous studies showed that LipL32 induced inflammatory responses and interacted with the extracellular matrix (ECM) of the host cell. However, the exact relationship between LipL32-mediated inflammatory responses and ECM binding is still unknown. In this study, an atomic force microscope with its tip modified by purified LipL32 was used to assess the interaction between LipL32 and cell surface receptors. Furthermore, an antibody neutralization technique was employed to identify Toll-like receptor 2 (TLR2) but not TLR4 as the major target of LipL32 attack. The interaction force between LipL32 and TLR2 was measured as approximately 59.5 +/- 8.7 pN, concurring with the theoretical value for a single-pair molecular interaction. Moreover, transformation of a TLR deficient cell line with human TLR2 brought the interaction force from the basal level to approximately 60.4 +/- 11.5 pN, confirming unambiguously TLR2 as counter receptor for LipL32. The stimulation of CXCL8/IL-8 expression by full-length LipL32 as compared to that without the N-terminal signal peptide domain suggests a significant role of the signal peptide of the protein in the inflammatory responses. This study provides direct evidence that LipL32 binds to TLR2, but not TLR4, on the cell surface, and a possible mechanism for the virulence of leptospirosis is accordingly proposed.