Outer Membrane Protein LipL32 Research Articles

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.

Molecular evidence for the presence of Leptospira borgpetersenii in synanthropic rodents in the Nautla region, Veracruz, México

The genus Leptospira encompasses ten species of spirochetes capable of infecting mammals, particularly rodents. In Mexico, studies focused on the detection of Leptospira sp. in rodents are scarce, all of them restricted to three states of the Gulf of Mexico. For this reason, this work aimed to identify the diversity of Leptospira species associated with synanthropic rodents in Veracruz, a state where leptospirosis is endemic. Rodents were sampled with Sherman traps placed in 10 Production Units across the Nautla region. Animals were euthanized and their kidneys removed. Subsequently, a 474-bp segment of the outer membrane protein LipL32, present in all pathogenic species, was amplified and sequenced. Sequences were compared vs. reference using the BLAST algorithm: a phylogenetic reconstruction was carried out using the Maximum Likelihood method. In addition, the prevalence of infection in each Production Unit was estimated. Twenty eight rodents of a single species (Mus musculus) were caught. Leptospira DNA was detected in 17 samples (62.9 %, CI95 % 42.3 to 80.6) from seven localities in the Nautla region. The sequences recovered exhibited 99-100% identity to each other and 99 % identity with Leptospira borgspetersenii sequences deposited in GenBank. This study confirms the presence of L. borgspetersenii in rodents, particularly in M. musculus, in Mexico. This study increases the inventory of pathogenic leptospires for the state of Veracruz to three species.

Discovery of 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-ones as a new class of pathogen specific anti-leptospiral agents

A simple and efficient method for the synthesis of a series of 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-one derivatives starting from 5-carboalkoxy-2,3-dihydropyranone (5-CDHPs) has been developed. Pyranoisoxazolones 10a-j, dihydronaphthopyran-4-one (DHNPs) class of natural product 12b and 12c and its analogues 12a and 13a-c were preliminarily screened against pathogenic leptospiral serovar Autumnalis strain N2 at various concentrations. Six pyranoisoxazolones, 10b, 10d, 10f, 10g, 10i and 10j which displayed very good anti-leptospiral activity was taken for secondary screening against twelve strains of pathogenic and one non-pathogenic leptospiral serovars. While all the compounds displayed significant anti-leptospiral activity against the pathogenic serovars at MIC of 62.5–500 μg/mL. Compounds 10d, 10g and 10j did not show any significant effect on non-pathogenic serovar. Inhibition of leptospires at a significant level by pyranoisoxazolone 10g was confirmed using RT-qPCR assay. In vivo treatment of BALB/c mice with compound 10g revealed that, it has 95% survivability against the pathogenic strain Canicola and also showed inhibition of renal colonization of leptospires. Compound 10g was found to show cytotoxicity against THP-1 cells only at higher concentration (≥75 μg/mL). Effective binding of compound 10g with leptospiral outer membrane protein LipL32 observed via in silico molecular docking provided a suitable explanation for pathogen specificity of compound 10g. Antibiotics acting against leptospirosis in human are very few. The results obtained from in vitro, in vivo and in silico study reveals that 6,7-dihydro-3H-pyrano[4,3-c]isoxazol-3-ones class of compounds are lead molecules for further development as pathogen specific anti-leptospiral agents.

Leptospiral outer membrane protein LipL32 induces inflammation and kidney injury in zebrafish larvae

Leptospirosis is an often overlooked cause of acute kidney injury that can lead to multiple organ failure and even death. The principle protein that conserved in many pathogenic leptospires is the outer membrane protein LipL32. However, the role of LipL32 in the pathogenesis of renal injury in leptospirosis is not entirely clear. Here we studied the effects of LipL32 on the developing kidney in zebrafish larvae. Incubation of zebrafish larvae with Leptospira santarosai serovar Shermani induced acute tubular injury predominantly in the proximal pronephric ducts. Furthermore, microinjection of lipl32 mRNA or recombinant LipL32 protein into zebrafish larvae increased macrophage accumulation and disrupted the basolateral location of NA-K-ATPase in pronephric ducts. These changes led to substantial impairment of the pronephric kidney structure. We further demonstrated that morpholino knockdown of tlr2, but not tlr4, reduced the LipL32-induced leukocyte infiltration and kidney injury. These data demonstrate that LipL32 contributes to the renal pathology in leptospirosis and gives some clues to the potential virulence of LipL32. Our results support the use of zebrafish as a model organism for studying the disease mechanism of leptospiral infection. This model might permit the future exploration of the virulence and molecular pathways of different leptospiral outer membrane proteins.

Immunogenicity of a DNA and Recombinant Protein Vaccine Combining LipL32 and Loa22 for Leptospirosis Using Chitosan as a Delivery System.

Leptospirosis is a worldwide zoonotic disease caused by pathogenic Leptospira, a genus of which more than 250 serovars have been identified. Commercial bacterin vaccines are limited in that they lack both cross-protection against heterologous serovars and long-term protection. This study investigated in mice the immunogenicity of an anti-leptospirosis vaccine, using the outer membrane proteins LipL32 and Loa22 as antigens. The immunogenicity of this vaccine formulation was compared with those induced by vaccines based on LipL32 or Loa22 alone. A DNA-encapsulated chitosan nanoparticle was used for in vivo DNA delivery. Using a unique DNA plasmid expressing both lipL32 and loa22 for vaccination, higher antibody responses were induced than when combining plasmids harboring each gene separately. Therefore, this formulation was used to test the immunogenicity when administered by a heterologous prime (DNA)-boost (protein) immunization regimen. The specific antibody responses against LipL32 (total IgG and IgG1) and Loa22 (IgG1) were higher in mice receiving two antigens in combination than in those vaccinated with a single antigen alone. Although no significant difference in splenic CD4+ T cell proliferation was observed among all groups of vaccinated mice, splenocytes from mice vaccinated with two antigens exhibited higher interferon-γ and IL-2 production than when using single antigens alone upon in vitro restimulation. Taken together, the immunogenicity induced by LipL32 and Loa22 antigens in a heterologous primeboost immunization regimen using chitosan as a DNA delivery system induces higher immune response, and may be useful for developing a better vaccine for leptospirosis.

Vaccination with Leptospiral Outer Membrane Lipoprotein LipL32 Reduces Kidney Invasion of Leptospira interrogans Serovar Canicola in Hamsters

The Leptospira interrogans vaccines currently available are serovar specific and require regular booster immunizations to maintain protection of the host. In addition, a hamster challenge batch potency test is necessary to evaluate these vaccines prior to market release, requiring the use of a large number of animals, which is ethically and financially undesirable. Our previous work showed that the N terminus of the outer membrane protein LipL32 was altered in Leptospira interrogans serovar Canicola vaccines that fail the hamster challenge test, suggesting that it may be involved in the protective immune response. The aim of this study was to determine if vaccination with LipL32 protein alone could provide a protective response against challenge with L. interrogans serovar Canicola to hamsters. Recombinant LipL32, purified from an Escherichia coli expression system, was assessed for protective immunity in five groups of hamsters (n = 5) following a challenge with the virulent L. interrogans serovar Canicola strain Kito as a challenge strain. However, no significant survival against the L. interrogans serovar Canicola challenge was observed compared to that of unvaccinated negative controls. Subsequent histological analysis revealed reduced amounts of L. interrogans in the kidneys from the hamsters vaccinated with recombinant LipL32 protein prior to challenge; however, no significant survival against the L. interrogans serovar Canicola challenge was observed compared to that of unvaccinated negative controls. This finding corresponded to a noticeably reduced severity of renal lesions. This study provides evidence that LipL32 is involved in the protective response against L. interrogans serovar Canicola in hamsters and is the first reported link to LipL32-induced protection against kidney invasion.

Analysis of Multiple Leptospira interrogans Serovar Canicola Vaccine Proteomes and Identification of LipL32 as a Biomarker for Potency

The current batch potency test for Leptospira interrogans serovar Canicola vaccines requires the use of a large number of hamsters and has severe effects (i.e., hepatic and renal failure resulting in death); while this vaccine is effective, a safer, cheaper, more ethical replacement is desired. The aim of this study was to analyze vaccine proteomes and identify target molecules common to all L. interrogans serovar Canicola vaccines which could be used to design an in vitro potency test. Initial analysis of L. interrogans serovar Canicola vaccines (A to E) from different manufacturers, using the Limulus amebocyte lysate assay and silver-stained sodium dodecyl sulfate polyacrylamide gels, indicated that lipopolysaccharide was not present in all vaccines, preventing it from being a suitable target molecule. The protein contents of vaccines A to E were therefore determined by two-dimensional liquid chromatography mass spectrometry ([2D-LC/MS] 221 ± 31, 9 ± 8, 34 ± 4, 21 ± 5, and 34 ± 17 proteins [mean ± 1 standard deviation] found, respectively). The outer membrane protein LipL32 was established to be common to all and to be present at a significantly higher (P ≤ 0.05) relative spectral abundance in a batch of vaccine which passed the in vivo potency test than in one which had failed. Further analysis using multiple reaction monitoring revealed that the concentration of the N terminus of LipL32 was significantly lower (P ≤ 0.01) in failed batches (n = 2) of vaccine than in passed batches (n = 2); the concentration of the C terminus between the two batches was approximately the same. An in vitro Leptospira vaccine potency test, based on N-terminal amino acid quantification of LipL32, was subsequently developed.

Simultaneous detection of LipL32 and LipL21 genes of pathogenic leptospira from serum samples of bovines by multiplex PCR

Leptospirosis is a worldwide zoonotic disease of cattle associated with pathogenic leptospiral infection. This study focuses in the use of a molecular tool to detect pathogenic leptospiral infection in bovines by targeting the outer membrane proteins LipL32 and LipL21 simultaneously in a multiplex PCR. Sixteen pathogenic reference strains and 10 bovine serum samples were analyzed for simultaneous detection of both genes at appropriate annealing conditions. These findings are suggestive of the fact that multiplex PCR can be used to detect major outer membrane proteins of pathogenic leptospira from serum samples. Further it aided in the differentiation of pathogenic and non-pathogenic species of leptospires too. This study will definitely serve as a valuable tool, as it suggests the importance of LipL32 genes as potential candidates for vaccine development to control animal Leptospirosis.

Simultaneous detection of LipL32 and LipL21 genes of pathogenic leptospira from serum samples of bovines by multiplex PCR

<p>Leptospirosis is a worldwide zoonotic disease of cattle associated with pathogenic leptospiral infection. This study focuses in the use of a molecular tool to detect pathogenic leptospiral infection in bovines by targeting the outer membrane proteins LipL32 and LipL21 simultaneously in a multiplex PCR. Sixteen pathogenic reference strains and 10 bovine serum samples were analyzed for simultaneous detection of both genes at appropriate annealing conditions. These findings are suggestive of the fact that multiplex PCR can be used to detect major outer membrane proteins of pathogenic leptospira from serum samples. Further it aided in the differentiation of pathogenic and non-pathogenic species of leptospires too. This study will definitely serve as a valuable tool, as it suggests the importance of <em>LipL32</em> genes as potential candidates for vaccine development to control animal Leptospirosis.</p>

In Vitro Identification of Novel Plasminogen-Binding Receptors of the Pathogen Leptospira interrogans

BackgroundLeptospirosis is a multisystem disease caused by pathogenic strains of the genus Leptospira. We have reported that Leptospira are able to bind plasminogen (PLG), to generate active plasmin in the presence of activator, and to degrade purified extracellular matrix fibronectin.Methodology/Principal FindingsWe have now cloned, expressed and purified 14 leptospiral recombinant proteins. The proteins were confirmed to be surface exposed by immunofluorescence microscopy and were evaluated for their ability to bind plasminogen (PLG). We identified eight as PLG-binding proteins, including the major outer membrane protein LipL32, the previously published rLIC12730, rLIC10494, Lp29, Lp49, LipL40 and MPL36, and one novel leptospiral protein, rLIC12238. Bound PLG could be converted to plasmin by the addition of urokinase-type PLG activator (uPA), showing specific proteolytic activity, as assessed by its reaction with the chromogenic plasmin substrate, D-Val-Leu-Lys 4-nitroanilide dihydrochloride. The addition of the lysine analog 6-aminocaproic acid (ACA) inhibited the protein-PLG interaction, thus strongly suggesting the involvement of lysine residues in plasminogen binding. The binding of leptospiral surface proteins to PLG was specific, dose-dependent and saturable. PLG and collagen type IV competed with LipL32 protein for the same binding site, whereas separate binding sites were observed for plasma fibronectin.Conclusions/SignificancePLG-binding/activation through the proteins/receptors on the surface of Leptospira could help the bacteria to specifically overcome tissue barriers, facilitating its spread throughout the host.

Effect of Leptospira interrogans outer membrane proteins LipL32 on HUVEC

Leptospira cause disease through a toxin-mediated process by inducing vascular injury, particularly a small-vessel vasculitis. Breakdown of vessel endothelial cell integrity may increase vessel permeability which is correlated with the changes of tight junction and/or apoptosis in vessel endothelial cells. The specific toxin responsible remains unidentified. In this study, we amplified outer membrane protein LipL32 from the genome of Leptospira interrogans serovar Lai, and it was subcloned in pET32a(+) vector to express thioredoxin(Trx)–LipL32 fusion protein in Escherichia coli BL21(DE3). The protein was expressed and purified, and Trx–LipL32 was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the role of leptospiral outer membrane proteins in vessel endothelial cell. The purified recombinant protein was capable to increase the permeability of HUVECs. And the protein was able to decrease the expression of ZO-1 and induce F-actin in HUVECs display thickening and clustering. Moreover, apoptosis of HUVEC was significantly accelerated. But the fusion partner had no effect in these regards. It is possible that LipL32 is involved in the vessel lesions.

Monoclonal Antibodies to LipL32 Protect Against HeterologousLeptospiraspp. Challenge

A non-culture-based leptospirosis vaccine that cross-protects against infection caused by heterologous Leptospira spp. should replace the currently available products, which are qualitatively and quantitatively inadequate. With that in mind, two murine hybridomas secreting monoclonal antibodies (MAb) binding only to homogenates of pathogenic Leptospira spp., and not of the saprophytic L. biflexa, serogroup Patoc, serovar Patoc, were produced. The MAbs of both clones neutralized Leptospira-mediated human red blood cell lysis in vitro and rescued hamsters from lethal infection with heterologous Leptospira spp. The orthologous Leptospira spp. protein carrying the MAb epitope(s) was identified by two-dimensional gel electrophoresis (2DE)-based proteomics and database search. The epitopes of the MAbs were located on the major outer membrane protein LipL32 of the pathogenic Leptospira spp. The MAbs in their humanized version are potential leptospirosis immunotherapeutics. They are also suitable as detection reagents in antigen-based assays for the rapid diagnosis of leptospirosis. Recombinant LipL32 is a good candidate for a broad spectrum, non-culture-based leptospirosis vaccine.

Immunohistochemical Identification and Pathologic Findings in Natural Cases of Equine Abortion Caused by Leptospiral Infection

The aim of this study was to examine the utility of immunohistochemistry (IHC) in the diagnosis of leptospiral equine abortion and to compare IHC to silver staining and serology of the aborted mares. Ninety-six fetuses from 57 farms were examined using all 3 diagnostic techniques, revealing evidence of leptospiral infection in 3 fetuses (3.1%) from 3 (5.3%) different farms. A new finding in 1 of these confirmed cases of leptospiral abortion was the presence of macroscopic pinpoint grayish-white nodules that had a histologic correlate of hepatic necrosis; other histologic findings were consistent with those previously reported. IHC performed using 2 different leptospiral antisera (multivalent whole-cell rabbit antiserum and rabbit antiserum against the major outer membrane protein LipL32) yielded similar results. IHC was more sensitive (19/21 [90.5%] tissue samples) than silver staining (8/21 [38.1%] tissue samples), and more specific than serology performed using the microscopic agglutination test. The primary advantage of IHC over silver staining was the ability of IHC to identify leptospiral antigen not only as morphologically intact spiral forms.

LipL21 is a novel surface-exposed lipoprotein of pathogenic Leptospira species.

Leptospira is the etiologic agent of leptospirosis, a bacterial zoonosis distributed worldwide. Leptospiral lipopolysaccharide is a protective immunogen, but the extensive serological diversity of leptospires has inspired a search for conserved outer membrane proteins (OMPs) that may stimulate heterologous immunity. Previously, a global analysis of leptospiral OMPs (P. A. Cullen, S. J. Cordwell, D. M. Bulach, D. A. Haake, and B. Adler, Infect. Immun. 70:2311-2318, 2002) identified pL21, a novel 21-kDa protein that is the second most abundant constituent of the Leptospira interrogans serovar Lai outer membrane proteome. In this study, we identified the gene encoding pL21 and found it to encode a putative lipoprotein; accordingly, the protein was renamed LipL21. Southern hybridization analysis revealed the presence of lipL21 in all of the pathogenic species but in none of the saprophytic species examined. Alignment of the LipL21 sequence from six strains of Leptospira revealed 96 to 100% identity. When specific polyclonal antisera to recombinant LipL21 were used, LipL21 was isolated together with other known leptospiral OMPs by both Triton X-114 extraction and sucrose density gradient membrane fractionation. All nine strains of pathogenic leptospires investigated by Western blotting, whether culture attenuated or virulent, were found to express LipL21. In contrast, the expression of LipL21 or an antigenically related protein could not be detected in nonpathogenic L. biflexa. Infected hamster sera and two of eight human leptospirosis sera tested were found to react with recombinant LipL21. Native LipL21 was found to incorporate tritiated palmitic acid, consistent with the prediction of a lipoprotein signal peptidase cleavage site. Biotinylation of the leptospiral surface resulted in selective labeling of LipL21 and the previously known OMPs LipL32 and LipL41. These findings show that LipL21 is a surface-exposed, abundant outer membrane lipoprotein that is expressed during infection and conserved among pathogenic Leptospira species.

The Leptospira outer membrane protein LipL32 induces tubulointerstitial nephritis-mediated gene expression in mouse proximal tubule cells.

Tubulointerstitial nephritis is a main renal manifestation caused by pathogenic leptospira that accumulate mostly in the proximal tubules, thereby inducing tubular injury and tubulointerstitial nephritis. To elucidate the role of leptospira outer membrane proteins in tubulointerstitial nephritis, outer membrane proteins from pathogenic Leptospira shermani and nonpathogenic Leptospira patoc extracted by Triton X-114 were administered to cultured mouse proximal tubule cells. A dose-dependent increase of monocyte chemoattractant protein-1 (MCP-1), RANTES, nitrite, and tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant was observed 48 h after incubating Leptospira shermani outer membrane proteins with mouse proximal tubule cells. RT competitive-PCR experiments showed that Leptospira shermani outer membrane proteins (0.2 microg/ml) increased the expression of MCP-1, nitric oxide synthase (iNOS), RANTES, and TNF-alpha mRNA by 3.0-, 9.4-, 2.5-, and 2.5-fold, respectively, when compared with untreated cells. Outer membrane proteins extract from avirulent Leptospira patoc did not induce significant effects. The pathogenic outer membrane proteins extract contain a major component of a 32-kD lipoprotein (LipL32), which is absent in the nonpathogenic leptospira outer membrane. An antibody raised against LipL32 prevented the stimulatory effect of Leptospira shermani outer membrane proteins extract on MCP-1 and iNOS mRNA expression in cultured proximal tubule cells, whereas recombinant LipL32 significantly stimulated the expression of MCP-1 and iNOS mRNAs and augmented nuclear binding of nuclear factor-kappaB (NF-kappaB) and AP-1 transcription factors in proximal tubule cells. An antibody raised against LipL32 also blunted the effects induced by the recombinant LipL32. This study demonstrates that LipL32 is a major component of pathogenic leptospira outer membrane proteins involved in the pathogenesis of tubulointerstitial nephritis.

The leptospiral major outer membrane protein LipL32 is a lipoprotein expressed during mammalian infection.

We report the cloning of the gene encoding the 32-kDa lipoprotein, designated LipL32, the most prominent protein in the leptospiral protein profile. We obtained the N-terminal amino acid sequence of a staphylococcal V8 proteolytic-digest fragment to design an oligonucleotide probe. A Lambda-Zap II library containing EcoRI fragments of Leptospira kirschneri DNA was screened, and a 5.0-kb DNA fragment which contained the entire structural lipL32 gene was identified. Several lines of evidence indicate that LipL32 is lipid modified in a manner similar to that of other procaryotic lipoproteins. The deduced amino acid sequence of LipL32 would encode a 272-amino-acid polypeptide with a 19-amino-acid signal peptide, followed by a lipoprotein signal peptidase cleavage site. LipL32 is intrinsically labeled during incubation of L. kirschneri in media containing [(3)H]palmitate. The linkage of palmitate and the amino-terminal cysteine of LipL32 is acid labile. LipL32 is completely solubilized by Triton X-114 extraction of L. kirschneri; phase separation results in partitioning of LipL32 exclusively into the hydrophobic, detergent phase, indicating that it is a component of the leptospiral outer membrane. CaCl(2) (20 mM) must be present during phase separation for recovery of LipL32. LipL32 is expressed not only during cultivation but also during mammalian infection. Immunohistochemistry demonstrated intense LipL32 reactivity with L. kirschneri infecting proximal tubules of hamster kidneys. LipL32 is also a prominent immunogen during human leptospirosis. The sequence and expression of LipL32 is highly conserved among pathogenic Leptospira species. These findings indicate that LipL32 may be important in the pathogenesis, diagnosis, and prevention of leptospirosis.