Hematúria macroscópica em equinos associada à infecção por Leptospira interrogans / Macroscopic haematuria in horses associated with Leptospira interrogans infection

Hematúria é uma grave manifestação clínica de doença do sistema urinário, ocorrendo sob as formas micro ou macroscópica. Neste artigo relatam-se dois casos de hematúria macroscópica associada à infecção por Leptospira interrogans sorogrupo Canicola. O exame clínico inicial revelou hematúria macroscópica, taquicardia, taquipneia, febre, elevação do tempo de perfusão capilar, hipomotilidade intestinal, além de icterícia da mucosa oral. Leucocitose, proteinúria, glicosúria, piúria e azotemia foram achados comuns aos dois casos. Teste de Soroaglutinação Microscópica foi realizado para titulação de anticorpos contra Leptospira interrogans. Tratamento incluiu medidas terapêuticas de suporte (fluidoterapia), controle da hematúria e antibioticoterapia. Sete dias após manifestação dos sinais clínicos iniciais, ambos animais receberam alta hospitalar após remissão dos sinais clínicos.

Haematuria is a serious clinical manifestation of urinary system disease, occurring in micro or macroscopic forms. In this article two cases of macroscopic haematuria associated with Leptospira interrogans serogroup Canicolainfection are related. The initial clinical examination revealed macroscopic haematuria, tachycardia, tachypnea, fever, increased capillary perfusion time, intestinal hypomotility, in addition to jaundice of the oral mucosa. Leukocytosis, proteinuria, glycosuria, pyuria and azotemia were common findings in both cases. Microscopic serum agglutination test was performed for titration of antibodies against Leptospira interrogans. Treatment included supportive therapeutic measures (fluid therapy), hematuria control and antibiotic therapy. Seven days after the manifestation of the initial clinical signs, both animals were discharged from the hospital without complications.

The Role of Properdin in Killing of Non-Pathogenic Leptospira biflexa.

Properdin (P) is a positive regulatory protein that stabilizes the C3 convertase and C5 convertase of the complement alternative pathway (AP). Several studies have suggested that properdin can bind directly to the surface of certain pathogens regardless of the presence of C3bBb. Saprophytic Leptospira are susceptible to complement-mediated killing, but the interaction of properdin with Leptospira spp. has not been evaluated so far. In this work, we demonstrate that properdin present in normal human serum, purified properdin, as well as properdin oligomers P2, P3, and P4, interact with Leptospira. Properdin can bind directly to the bacterial surface even in the absence of C3b. In line with our previous findings, AP activation was shown to be important for killing non-pathogenic L. biflexa, and properdin plays a key role in this process since this microorganism survives in P-depleted human serum and the addition of purified properdin to P-depleted human serum decreases the number of viable leptospires. A panel of pathogenic L.interrogans recombinant proteins was used to identify putative properdin targets. Lsa30, an outer membrane protein from L. interrogans, binds to unfractionated properdin and to a lesser extent to P2-P4 properdin oligomers. In conclusion, properdin plays an important role in limiting bacterial proliferation of non-pathogenic Leptospira species. Once bound to the leptospiral surface, this positive complement regulatory protein of the AP contributes to the formation of the C3 convertase on the leptospire surface even in the absence of prior addition of C3b.

M16-Type Metallopeptidases Are Involved in Virulence for Invasiveness and Diffusion of Leptospira interrogans and Transmission of Leptospirosis.

BACKGROUND: Leptospirosis is a global zoonotic infectious disease caused by Leptospira interrogans. The pathogen rapidly invades into hosts and diffuses from bloodstream into internal organs and excretes from urine to cause transmission of leptospirosis. However, the mechanism of leptospiral invasiveness remains poorly understood. METHODS: Proteolytic activity of M16-type metallopeptidases (Lep-MP1/2/3) of L. interrogans was determined by spectrophotometry. Expression and secretion of Lep-MP1/2/3 during infection of cells were detected by quantitative reverse-transcription polymerase chain reaction, Western blot assay, and confocal microscopy. Deletion and complementation mutants of the genes encoding Lep-MP1/2/3 were generated to determine the roles of Lep-MP1/2/3 in invasiveness using transwell assay and virulence in hamsters. RESULTS: Leptospira interrogans but not saprophytic Leptospira biflexa strains were detectable for Lep-MP-1/2/3-encoding genes. rLep-MP1/2/3 hydrolyzed extracellular matrix proteins, but rLep-MP1/3 displayed stronger proteolysis than rLep-MP2, with 123.179/340.136 µmol/L Km and 0.154/0.159 s-1 Kcat values. Expression, secretion and translocation of Lep-MP1/2/3 during infection of cells were increased. ΔMP1/3 but not ΔMP2 mutant presented attenuated transmigration through cell monolayers, decreased leptospiral loading in the blood, lungs, liver, kidneys, and urine, and 10/13-fold decreased 50% lethal dose and milder histopathologic injury in hamsters. CONCLUSIONS: Lep-MP1 and 3 are involved in virulence of L. interrogans in invasion into hosts and diffusion in vivo, and transmission of leptospirosis.

Haemolysin Sph2 of Leptospira interrogans induces cell apoptosis via intracellular reactive oxygen species elevation and mitochondrial membrane injury.

Leptospira interrogans causes widespread leptospirosis in humans and animals, with major symptoms of jaundice and haemorrhage. Sph2, a member of the sphingomyelinase haemolysins, is an important virulence factor for leptospire. In this study, the function and mechanism of Sph2 in the pathogenesis of leptospirosis were investigated to further understand the pathogenesis of leptospire. Real-time PCR analysis of expression levels during cell invasion showed that sph2 gene expression was transiently induced in human umbilical vein endothelial cells (HUVECs), human embryo liver cells (L02), and human epithelial lung cells (L132), with expression levels reaching a peak after 45 min of infection. Further functional analysis of recombinant Sph2 (rSph2) by LDH assays and confocal microscopy showed that rSph2 can be internalised by cells both by causing cell membrane damage and by a damage-independent clathrin-mediated endocytosis pathway. Subsequently, rSph2 is able to translocate to mitochondria, which led to an increase in the levels of reactive oxygen species (ROS) and a decrease of the mitochondrial membrane potential (ΔΨm ). Further flowcytometry analyses after rSph2 exposure showed that 28.7%, 31%, and 27.3% of the HUVEC, L02, and L132 cells, respectively, became apoptotic. Because apoptosis could be decreased with the ROS inhibitor N-acetyl cysteine, these experiments suggested that rSph2 triggers apoptosis through mitochondrial membrane damage and ROS elevation. The ability of leptospiral haemolysin rSph2 to cause apoptosis likely contributes to the pathogenesis of leptospirosis.

Macrophages and Galectin 3 Control Bacterial Burden in Acute and Subacute Murine Leptospirosis That Determines Chronic Kidney Fibrosis.

Previous studies have suggested that macrophages may contribute to acute Leptospira dissemination, as well as having a major role in kidney fibrosis. Our aim was to characterize the role of macrophages and galectin 3 (Gal-3) on the survival, clinical course, bacterial burden, interstitial nephritis, and chronic kidney fibrosis in Leptospira interrogans serovar Copenhageni (LIC)-induced experimental murine leptospirosis. C57BL/6J mice depleted of macrophages by liposome-encapsulated clodronate treatment and infected with LIC presented a higher bacterial burden, had reduced subacute nephritis and enhanced chronic kidney fibrosis relative to untreated, infected mice. Moreover, LIC infection in mice whose Gal-3 was disrupted (Lgals3-/-) had a higher bacterial burden and enhanced subacute nephritis and chronic kidney fibrosis when compared to C57BL/6J wild-type mice. Chronic fibrosis did not correlate with higher transcription levels of TGF-ß1 or IL-13 in the kidneys. Kidney fibrosis was found in chronically infected rats as well as in wild infected rats. On the other hand, human fibroblast cultures exhibited enhanced differentiation to myofibroblasts after treatment with LIC. Our results demonstrate that macrophages and Gal-3 play a critical role in controlling the LIC burden but has a minor role in subsequent fibrosis. Instead, kidney fibrosis was better correlated with bacterial burden. Taken together, our results do not support a role for macrophages to disseminate leptospires during acute infection, nor in chronic kidney fibrosis.

Proteomic Analysis of Urine from California Sea Lions ( Zalophus californianus): A Resource for Urinary Biomarker Discovery.

Urinary markers for the assessment of kidney diseases in wild animals are limited, in part, due to the lack of urinary proteome data, especially for marine mammals. One of the most prevalent kidney diseases in marine mammals is caused by Leptospira interrogans, which is the second most common etiology linked to stranding of California sea lions ( Zalophus californianus). Urine proteins from 11 sea lions with leptospirosis kidney disease and eight sea lions without leptospirosis or kidney disease were analyzed using shotgun proteomics. In total, 2694 protein groups were identified, and 316 were differentially abundant between groups. Major urine proteins in sea lions were similar to major urine proteins in dogs and humans except for the preponderance of resistin, lysozyme C, and PDZ domain containing 1, which appear to be over-represented. Previously reported urine protein markers of kidney injury in humans and animals were also identified. Notably, neutrophil gelatinase-associated lipocalin, osteopontin, and epidermal fatty acid binding protein were elevated over 20-fold in the leptospirosis-infected sea lions. Consistent with leptospirosis infection in rodents, urinary proteins associated with the renin-angiotensin system were depressed, including neprilysin. This study represents a foundation from which to explore the clinical use of urinary protein markers in California sea lions.

Characterizing interactions of Leptospira interrogans with proximal renal tubule epithelial cells.

BACKGROUND: Leptospira interrogans is a pathogenic, spirochetal bacterium that is responsible for leptospirosis, an emerging worldwide zoonosis. Leptospires colonize the renal proximal tubules and chronically infect the kidney. Live bacteria are excreted into urine, contaminating the environment. While it is well known that leptospires can persist in the kidneys without signs of disease for several months, the interactions of leptospires with the proximal renal epithelial tubule cells that allow the chronic renal colonization have not been elucidated yet. In the present study, we compared the interactions between a virulent, low passage (LP) strain and a cultured-attenuated, high passage (HP) strain with renal proximal tubule epithelial cells (RPTECs) to elucidate the strategies used by Leptospira to colonize the kidney. RESULTS: Kinetics analysis of kidney colonization in a mouse model of chronic infection performed by quantitative real-time PCR and immunofluorescence, showed that the LP strain reached the kidney by 3 days post infection (pi) and attached to the basal membrane side of the renal epithelial cells. At 10 days pi, some leptospires were attached to the luminal side of the tubular epithelia and the number of colonizing leptospires gradually increased. On the other hand, the HP strain was cleared during hematogenous dissemination and did not colonize the kidney. Transmission electron microscopy analysis of LP-infected kidneys at 25 days pi showed aggregated leptospires and membrane vesicles attached to the epithelial brush border. Leptospiral kidney colonization altered the organization of the RPTEC brush border. An in vitro model of infection using TCMK-1 cells, showed that leptospiral infection induced a host stress response, which is delayed in LP-infected cells. CONCLUSIONS: After hematogenous dissemination, leptospires create protective and replicative niches in the base membrane and luminal sides of the RPTECs. During the long-term colonization, leptospires attached to the RPTEC brush borders and membrane vesicles might be involved in the formation of a biofilm-like structure in vivo. Our results also suggested that the virulent strain is able to manipulate host cell stress responses to promote renal colonization.

Bid-Induced Release of AIF/EndoG from Mitochondria Causes Apoptosis of Macrophages during Infection with Leptospira interrogans.

Leptospirosis is a global zoonotic infectious disease caused by pathogenic Leptospira species. Leptospire-induced macrophage apoptosis through the Fas/FasL-caspase-8/3 pathway plays an important role in the survival and proliferation of the pathogen in hosts. Although, the release of mitochondrial apoptosis-inducing factor (AIF) and endonuclease G (EndoG) in leptospire-infected macrophages has been described, the mechanisms linking caspase and mitochondrion-related host-cell apoptosis has not been determined. Here, we demonstrated that leptospire-infection induced apoptosis through mitochondrial damages in macrophages. Apoptosis was caused by the mitochondrial release and nuclear translocation of AIF and/or EndoG, leading to nuclear DNA fragmentation. However, the mitochondrion-related CytC-caspase-9/3 pathway was not activated. Next, we found that the release and translocation of AIF and/or EndoG was preceded by the activation of the BH3-interacting domain death agonist (Bid). Furthermore, our data demonstrated that caspase-8 was activated during the infection and caused the activation of Bid. Meanwhile, high reactive oxygen species (ROS) trigged by the infection caused the dephosphorylation of Akt, which also activated Bid. In conclusion, Bid-mediated mitochondrial release of AIF and/or EndoG followed by nuclear translocation is a major mechanism of leptospire- induced apoptosis in macrophages, and this process is modulated by both caspase-8 and ROS-Akt signal pathways.

Inhibitory Effects of Emodin, Thymol, and Astragalin on Leptospira interrogans-Induced Inflammatory Response in the Uterine and Endometrium Epithelial Cells of Mice.

Leptospirosis is a systemic infection that causes, among others, acute kidney injury, acute liver disease, muscle pain, vasculitis, bleeding disorders, and reproductive loss. In an effort to reduce uterine inflammatory responses induced by Leptospira, we evaluated the anti-inflammation effects of emodin, thymol, and astragalin in a mouse model. Our results showed that treatment with emodin, thymol, and astragalin alleviated uterine inflammation induced by leptospira infection via suppression of pro-inflammatory cytokine expression and prevented tissue damage. Furthermore, we used primary endometrium epithelial cells to show that treatment with these chemicals inhibited the expression of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 using enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Western blot results showed that these chemicals suppressed the phosphorylation of p38, p65, extracellular signal-regulated kinase, and c-Jun N-terminal kinase. These results indicate that treatment with emodin, thymol, and astragalin suppressed inflammatory response by regulating NF-κB and mitogen-activated protein kinase signaling pathways in leptospira-infected uterine and endometrium epithelial cells of mice.

Functional thioredoxin reductase from pathogenic and free-living Leptospira spp.

Low molecular mass thiols and antioxidant enzymes have essential functions to detoxify reactive oxygen and nitrogen species maintaining cellular redox balance. The metabolic pathways for redox homeostasis in pathogenic (Leptospira interrogans) and free-living (Leptospira biflexa) leptospires species were not functionally characterized. We performed biochemical studies on recombinantly produced proteins to in depth analyze kinetic and structural properties of thioredoxin reductase (LinTrxR) and thioredoxin (LinTrx) from L. interrogans, and two TrxRs (LbiTrxR1 and LbiTrxR2) from L. biflexa. All the TrxRs were characterized as homodimeric flavoproteins, with LinTrxR and LbiTrxR1 catalyzing the NADPH dependent reduction of LinTrx and DTNB. The thioredoxin system from L. interrogans was able to use glutathione disulfide, lipoamide disulfide, cystine and bis-γ-glutamyl cysteine and homologous peroxiredoxin as substrates. Classic TrxR activity of LinTrxR2 had not been evidenced in vitro, but recombinant Escherichia coli cells overexpressing LbiTrxR2 showed high tolerance to oxidative stress. The enzymatic systems herein characterized could play a key role for the maintenance of redox homeostasis and the function of defense mechanisms against reactive oxidant species in Leptospira spp. Our results contribute to the general knowledge about redox biochemistry in these bacteria, positioning TrxR as a critical molecular target for the development of new anti-leptospiral drugs.

Evaluation of the Leptospira interrogans Outer Membrane Protein OmpL37 as a Vaccine Candidate.

The identification of potential vaccine candidates against leptospirosis remains a challenge. However, one such candidate is OmpL37, a potentially surface-exposed antigen that has the highest elastin-binding ability described to date, suggesting that it plays an important role in host colonization. In order to evaluate OmpL37's ability to induce a protective immune response, prime-boost, DNA and subunit vaccine strategies were tested in the hamster model of lethal leptospirosis. The humoral immune response was evaluated using an indirect ELISA test, and the cytokine profile in whole blood was determined by quantitative real-time PCR. Unlike the DNA vaccine, the administration of recombinant OmpL37 induced a strong IgG antibody response. When individually administrated, both formulations stimulated a TNF-α mediated inflammatory response. However, none of the OmpL37 formulations or vaccination strategies induced protective immunity. Further studies are required towards the identification of new vaccine targets against leptospirosis.

WC1 is a hybrid γδ TCR coreceptor and pattern recognition receptor for pathogenic bacteria.

WC1 proteins are uniquely expressed on γδ T cells and belong to the scavenger receptor cysteine-rich (SRCR) superfamily. While present in variable, and sometimes high, numbers in the genomes of mammals and birds, in cattle there are 13 distinct genes (WC1-1 to WC1-13). All bovine WC1 proteins can serve as coreceptors for the TCR in a tyrosine phosphorylation dependent manner, and some are required for the γδ T cell response to Leptospira. We hypothesized that individual WC1 receptors encode Ag specificity via coligation of bacteria with the γδ TCR. SRCR domain binding was directly correlated with γδ T cell response, as WC1-3 SRCR domains from Leptospira-responsive cells, but not WC1-4 SRCR domains from Leptospira-nonresponsive cells, bound to multiple serovars of two Leptospira species, L. borgpetersenii, and L. interrogans. Three to five of eleven WC1-3 SRCR domains, but none of the eleven WC1-4 SRCR domains, interacted with Leptospira spp. and Borrelia burgdorferi, but not with Escherichia coli or Staphylococcus aureus. Mutational analysis indicated that the active site for bacterial binding in one of the SRCR domains is composed of amino acids in three discontinuous regions. Recombinant WC1 SRCR domains with the ability to bind leptospires inhibited Leptospira growth. Our data suggest that WC1 gene arrays play a multifaceted role in the γδ T cell response to bacteria, including acting as hybrid pattern recognition receptors and TCR coreceptors, and they may function as antimicrobials.

[PRODUCTION OF CERTAIN PRO- AND ANTI-INFLAMMATORY CYTOKINES DURING STIMULATION BY LIVE AND INACTIVATED LEPTOSPIRA PATHOGENS IN THE MODEL OF HUMAN WHOLE BLOOD].

AIM: Study of the ability of clinical isolates of leptospira to cause production of certain pro- and antiinflammatory cytokines in the model of human whole blood. MATERIALS AND METHODS: Leptospira interrogans strain was taken for the experiment. Cytokine content was determined by a method based on xMAP technology using a standard panel, composed of 9 analytes: TNF-α, MCP-1, IL-8, IL-4, IL-6, IL-10, IL-IRa, IL- 12 (p70), IFN-γ. RESULTS: An optimal concentration of L. interrogans was selected for stimulation of human whole blood--1 x 10(6) leptospirae/ml. For the first time in the model of human whole blood it was determined, that at early stages of incubation IFN-γ, IL-12(p70), IL-4 and IL-1Ra are more actively produced; at later stages (6 hour incubation)--IL-8 and TNF-α. CONCLUSION: A differential pattern of cytokine production stimulation was shown in the model of human whole blood by live and inactivated leptospirae.

RETRACTED: ChpK and MazF of the toxin-antitoxin modules are involved in the virulence of Leptospira interrogans during infection.

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the corresponding author and the editorial office of Microbes and Infection. An independent reviewer of the retraction request was also appointed given that one of the authors is the Editor-in- Chief. For figure 1C, Lanes 1 and 2 appear to share some unexpected similarities, except for the bottom band, which also appear to be the band of interest. Sections of Figure 2C appear similar to sections of Figure 5D of a paper that had already appeared in Molecular Microbiology, volume 83, issue 5 (2012) 1006-1023. https://doi.org/10.1111/j.1365-2958.2012.07985.x. In figure 3A, Flow cytograms share identical/similar patterns highlighted in various colours. Peculiarly, some of these patterns can be seen as horizontal rotations of others along the axis that separates different quadrants. (ie red green & purple). Moreover, some quadrants appear to have very high densities of events that are suprisingly limited by quadrant gates (most noticeably quadrants B2 from the second column of panels. Figure 5A-B it was found that there were duplicated bands were produced. Figures 5C and 5D, it was found that bands across each individual gel appear identical. One of the conditions of submission of a paper for publication is that authors declare explicitly that the paper has not been previously published and is not under consideration for publication elsewhere. Re-use of any data should be appropriately cited. As such this article represents a misuse of the scientific publishing system. The scientific community takes a very strong view on this matter and apologies are offered to readers of the journal that this was not detected during the submission process".

Genotipificación y evaluación de la dinámica de infección de un aislamiento colombiano de Leptospira santarosai en el modelo experimental en hámster / Genotyping and evaluation of infection dynamics in a Colombian isolate of Leptospira santarosai in hamster as an experimental model

Introducción. Es necesario desarrollar modelos de estudio de la leptospirosis. Objetivo. Genotipificar un aislamiento de Leptospira proveniente de una persona con síndrome de Weil y evaluar, con el modelo experimental en Mesocricetus auratus , su dinámica de infección. Materiales y métodos. Se hizo la genotipificación por análisis de las secuencias génicas rrs 16S y lipL32 . Se determinó la dosis letal media en hámster inoculada por vía intraperitoneal. Se identificaron los patrones de química clínica, la duración de la leptospiremia, la leptospiruria y la histopatología, comparados con el mismo modelo inoculado con la cepa de Leptospira interrogans (Fiocruz L1-130). Resultados. Mediante análisis molecular se determinó que el aislamiento correspondía a la especie patógena Leptospira santarosai . La bacteria se recuperó a partir de tejido de riñón y de pulmón, y se detectó por medio de PCR lipL32 en el tercer día después de la infección. La proteína C reactiva aumentó en el quinto día después de la infección (3,25 mg/dl; valor normal: 0,3 mg/dl) con una disminución en el día 18 (2,60 mg/dl; valor normal: 0,8 mg/dl). Los biomarcadores de urea mostraron alteraciones indicativas de falla renal aguda (día 5 después de la infección: 49,01 mg/dl y día 18: 53,71 mg/dl). La histopatología mostró neumonía intersticial con diferentes grados de hemorragia, así como nefritis intersticial. Conclusión. Se identificó la presencia de la especie L. santarosai con capacidad patógena comparable con la cepa Fiocruz L1-130 de L. interrogans , de reconocida virulencia y tropismo pulmonar, en cuanto a los aspectos histopatológicos de tropismo a pulmón y riñón. Nunca antes se había evaluado en un modelo experimental un aislamiento de origen local bajo estos criterios biológicos.

Introduction: Is necessary to develop models for the study of leptospirosis. Objective: To genotype a Colombian strain of Leptospira isolated from a human with Weil´s syndrome and to evaluate its infection dynamics in the hamster experimental model. Materials and methods: Genotyping was performed by amplification and sequence analysis of the rrs 16S and lipL32 genes. The median lethal dose was determined in intraperitoneally inoculated hamsters. The patterns of clinical chemistry, the duration of leptospiremia, leptospiruria and pathological findings were studied and compared in the same animal model infected with L. interrogans (Fiocruz L1-130). Results: Molecular typing revealed that the isolate corresponded to the pathogenic species L. santarosai, which was recovered from hamsters´ kidneys and lungs and detected by lipL32 PCR from day 3 post-infection in these organs. There was a marked increase of C-reactive protein in animals at day 5 post-infection (3.25 mg/dl; normal value: 0.3 mg/dl) with decreases by day 18 (2.60 mg/dl: normal value: 0.8 mg/dl). Biomarkers of urea showed changes consistent with possible renal acute failure (day 5 post-infection: 49.01 mg/dl and day 18 post-infection: 53.71 mg/dl). Histopathological changes included interstitial pneumonia with varying degrees of hemorrhage and interstitial nephritis. Conclusion: The pathogenic species L. santarosai was identified in Colombia. Its pathogenicity as determined by tropism to lung and kidney was comparable to that of L. interrogans Fiocruz L1-130, well known for its virulence and pulmonar tropism. The biological aspects studied here had never before been evaluated in an autochthonous isolate.

Leptospira interrogans induces uterine inflammatory responses and abnormal expression of extracellular matrix proteins in dogs.

Leptospira interrogans (L. interrogans), a worldwide zoonosis, infect humans and animals. In dogs, four syndromes caused by leptospirosis have been identified: icteric, hemorrhagic, uremic (Stuttgart disease) and reproductive (abortion and premature or weak pups), and also it caused inflammation. Extracellular matrix (ECM) is a complex mixture of matrix molecules that is crucial to the reproduction. Both inflammatory response and ECM are closed relative to reproductive. The aim of this study was to clarify how L. interrogans affected the uterus of dogs, by focusing on the inflammatory responses, and ECM expression in dogs uterine tissue infected by L. interrogans. In the present study, 27 dogs were divided into 3 groups, intrauterine infusion with L. interrogans, to make uterine infection, sterile EMJH, and normal saline as a control, respectively. The uteruses were removed by surgical operation in 10, 20, and 30 days, respectively. The methods of histopathological analysis, ELISA, Western blot and qPCR were used. The results showed that L. interrogans induced significantly inflammatory responses, which were characterized by inflammatory cellular infiltration and high expression levels of tumor necrosis factor α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in uterine tissue of these dogs. Furthermore, L. interrogans strongly down-regulated the expression of ECM (collagens (CL) IV, fibronectins (FN) and laminins (LN)) in mRNA and protein levels. These data indicated that strongly inflammatory responses, and abnormal regulation of ECM might contribute to the proliferation of dogs infected by L. interrogans.

Identification of Leptospira interrogans phospholipase C as a novel virulence factor responsible for intracellular free calcium ion elevation during macrophage death.

BACKGROUND: Leptospira-induced macrophage death has been confirmed to play a crucial role in pathogenesis of leptospirosis, a worldwide zoonotic infectious disease. Intracellular free Ca(2+) concentration ([Ca(2+)]i) elevation induced by infection can cause cell death, but [Ca(2+)]i changes and high [Ca(2+)]i-induced death of macrophages due to infection of Leptospira have not been previously reported. METHODOLOGY/PRINCIPAL FINDINGS: We first used a Ca(2+)-specific fluorescence probe to confirm that the infection of L. interrogans strain Lai triggered a significant increase of [Ca(2+)]i in mouse J774A.1 or human THP-1 macrophages. Laser confocal microscopic examination showed that the [Ca(2+)]i elevation was caused by both extracellular Ca(2+) influx through the purinergic receptor, P2X7, and Ca(2+) release from the endoplasmic reticulum, as seen by suppression of [Ca(2+)]i elevation when receptor-gated calcium channels were blocked or P2X7 was depleted. The LB361 gene product of the spirochete exhibited phosphatidylinositol phospholipase C (L-PI-PLC) activity to hydrolyze phosphatidylinositol-4,5-bisphosphate (PIP2) into inositol-1,4,5-trisphosphate (IP3), which in turn induces intracellular Ca(2+) release from endoplasmic reticulum, with the Km of 199 µM and Kcat of 8.566E-5 S(-1). Secretion of L-PI-PLC from the spirochete into supernatants of leptospire-macrophage co-cultures and cytosol of infected macrophages was also observed by Western Blot assay. Lower [Ca(2+)]i elevation was induced by infection with a LB361-deficient leptospiral mutant, whereas transfection of the LB361 gene caused a mild increase in [Ca(2+)]i. Moreover, PI-PLCs (PI-PLC-ß3 and PI-PLC-γ1) of the two macrophages were activated by phosphorylation during infection. Flow cytometric detection demonstrated that high [Ca(2+)]i increases induced apoptosis and necrosis of macrophages, while mild [Ca(2+)]i elevation only caused apoptosis. CONCLUSIONS/SIGNIFICANCE: This study demonstrated that L. interrogans infection induced [Ca(2+)]i elevation through extracellular Ca(2+) influx and intracellular Ca(2+) release cause macrophage apoptosis and necrosis, and the LB361 gene product was shown to be a novel PI-PLC of L. interrogans responsible for the [Ca(2+)]i elevation.

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

OBJECTIVE: To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. METHODS: OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. RESULTS: The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01). CONCLUSION: Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Risk factors and predictors of severe leptospirosis in New Caledonia.

BACKGROUND: Leptospirosis is a major public health concern in New Caledonia (NC) and in other tropical countries. Severe manifestations of the disease are estimated to occur in 5-15% of all human infections worldwide and factors associated with these forms are poorly understood. Our objectives were to identify risk factors and predictors of severe forms of leptospirosis in adults. METHODS AND FINDINGS: We conducted a retrospective case-control study of inpatients with laboratory-confirmed leptospirosis who were admitted to two public hospitals in NC in 2008-2011. Cases were patients with fatal or severe leptospirosis, as determined by clinical criteria. This approach was meant to be pragmatic and to reflect the routine medical management of patients. Controls were defined as patients hospitalized for milder leptospirosis. Risk and prognostic factors were identified by multivariate logistic regression. Among the 176 patients enrolled in the study, 71 had criteria of severity including 10 deaths (Case Fatality Rate = 14.1%). Three risk factors were independently associated with severe leptospirosis: current cigarette smoking (OR = 2.94 [CI 1.45-5.96]); delays >2 days between the onset of symptoms and the initiation of antibiotherapy (OR = 2.78 [CI 1.31-5.91]); and Leptospira interrogans serogroup Icterohaemorrhagiae as the infecting strain (OR = 2.79 [CI 1.26-6.18]). The following post-admission laboratory results correlated with poor prognoses: platelet count ≤50,000/µL (OR = 6.36 [CI 1.79-22.62]), serum creatinine >200 mM (OR = 5.86 [CI 1.61-21.27]), serum lactate >2.5 mM (OR = 5.14 [CI 1.57-16.87]), serum amylase >250 UI/L (OR = 4.66 [CI 1.39-15.69]) and leptospiremia >1000 leptospires/mL (OR = 4.31 [CI 1.17-15.92]). CONCLUSIONS: To assess the risk of developing severe leptospirosis, our study illustrates the benefit for clinicians to have: i) the identification of the infective strain, ii) a critical threshold of qPCR-determined leptospiremia and iii) early laboratory results. In New Caledonia, preventative measures should focus on early presumptive antibacterial therapy and on rodent (reservoir of Icterohaemorrhagiae serogroup) control.

The mammalian cell entry (Mce) protein of pathogenic Leptospira species is responsible for RGD motif-dependent infection of cells and animals.

Mammalian cell entry proteins (Mces) contribute to Mycobacterium tuberculosis virulence. A mce homologue has been identified in the Leptospira interrogans genome, but its function was unknown. We showed that the mce gene is expressed only by pathogenic Leptospira strains tested. Leptospiral mce mRNA and Mce protein levels increased during infection of macrophages. The ability to infect macrophages was significantly lower in a strain with the mce gene deleted (mce(-) ). Complementation of the mce gene restored the ability of the mutant strain (mce(com) ) to adhere to and invade cells. Importantly, the mce gene knock-in strain (mce(+) ) derived from L. biflexa acquired the ability to infect cells, and the mce(+/ΔRAA) knock-in strain (in which the RGD motif was replaced by RAA) was unable to infect cells. The mce(-) mutant was also dramatically less efficient in infecting hamsters than the wild-type L. interrogans strain, and fewer leptospires of the mutant were found in peripheral blood monocytes and the urine from infected animals. The recombinant Mce protein showed a high binding affinity to the integrins α5ß1 and α(V) ß3. Blockade of the two integrins or the Mce protein decreased leptospiral adherence and invasiveness. The results showed that Mce is an RGD-motif-dependent virulence factor in pathogenic Leptospira species.