Identification of pathogenic and nonpathogenic Leptospira species of Brazilian isolates by Matrix Assisted Laser Desorption/Ionization and Time Flight mass spectrometry

ABSTRACT Matrix Assisted Laser Desorption/Ionization and Time of Flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the identification of bacteria through the detection and analysis of their proteins or fragments derived from ribosomes. Slight sequence variations in conserved ribosomal proteins distinguish microorganisms at the subspecies and strain levels. Characterization of Leptospira spp. by 16S RNA sequencing is costly and time-consuming, and recent studies have shown that closely related species (e.g., Leptospira interrogans and Leptospira kirschneri) may not be discriminated using this technology. Herein, we report an in-house Leptospira reference spectra database using Leptospira reference strains that were validated with a collection of well-identified Brazilian isolates kept in the Bacterial Zoonosis Laboratory at the Veterinary Preventive Medicine and Animal Health Department at Sao Paulo University. In addition, L. interrogans and L. kirschneri were differentiated using an in-depth mass spectrometry analysis with ClinProTools™ software. In conclusion, our in-house reference spectra database has the necessary accuracy to differentiate pathogenic and non-pathogenic species and to distinguish L. interrogans and L. kirschneri.

Características sociodemográficas y clínicas de pacientes con infección por Leptospira spp. atendidos en cuatro centros hospitalarios de Medellín, Colombia, 2008-2013 / Sociodemographic and clinical characteristics of patients infected with Leptospira spp. treated at four hospitals in Medellín, Colombia, 2008-2013

Resumen Introducción: La leptospirosis continúa siendo un problema significativo de salud en regiones tropicales, incluidos los países de Latinoamérica, donde es 100 veces más frecuente que en otras regiones del mundo. En los cuadros graves de la enfermedad, su mortalidad alcanza el 10 %. Su diagnóstico es un reto debido a que las manifestaciones clínicas en la fase inicial son inespecíficas y a la poca disponibilidad de pruebas diagnósticas. Objetivo: Describir las características sociodemográficas y clínicas, y el desenlace de la enfermedad en pacientes hospitalizados con leptospirosis. Materiales y métodos: Es un estudio retrospectivo que incluyó pacientes atendidos en cuatro instituciones de Medellín, entre enero de 2009 y diciembre de 2013, con un cuadro clínico sugestivo e IgM positiva para Leptospira spp. Resultados: Se incluyeron 119 pacientes, 80 % hombres y 58 % de procedencia rural. La duración promedio de los síntomas fue de 9,6 días (DE=9,6). El 89 % de los pacientes presentó fiebre; el 62 %, ictericia; el 74 %, mialgias; el 46 %, diarrea; el 41 %, hepatomegalia; el 13 %, esplenomegalia, y 13 %, enrojecimiento de los ojos. En 54 % de los pacientes hubo deterioro de la función renal, en 32 %, compromiso pulmonar y, en 13 %, falla hepática. El 16 % de los pacientes requirió atención en la unidad de cuidados intensivos, el 12 %, asistencia respiratoria mecánica, y el 11 %, administración de vasopresores. En 38,6 % de ellos la enfermedad cursó con síndrome de Weil y el 5 % falleció. La duración promedio de la hospitalización fue de 11 días (DE=9,6). Conclusiones:. La leptospirosis en esta población tuvo manifestaciones clínicas y complicaciones similares a las reportadas en la literatura científica. Se observó una mortalidad general relativamente baja comparada con las estadísticas mundiales.

Abstract Introduction: Leptospirosis remains a significant health problem in tropical regions including Latin America, where its presentation is 100 times higher than that observed in other regions of the world. Mortality reaches 10% in severe cases. Its diagnosis is challenging because clinical manifestations during the initial phase are non-specific and because of limited availability of diagnostic tests Objective: To describe the demographic and clinical characteristics and the outcomes in hospitalized patients with leptospirosis. Materials and methods: This retrospective study included patients treated at four institutions in Medellín between January, 2009, and December, 2013, with a compatible clinical picture and a positive IgM for Leptospira spp. Results: We included 119 patients, 80% male, and 58% of rural origin. The mean duration of symptoms was 9.6 days (SD=9.6). Eighty nine per cent of patients had fever; 62%, jaundice; 74%, myalgia; 46%, diarrhea; 41%, hepatomegaly; 13%, splenomegaly, and 13%, conjunctival injection. Fifty four per cent of patients had impaired renal function; 32%, pulmonary compromise, and 13%, liver failure. Sixteen per cent required admission to the ICU; 12%, mechanical ventilation, and 11%, vasopressor therapy. Weil's syndrome occurred in 38.6% and 5% died. The average hospital stay was 11 days (SD=9.6). Conclusions: In this population, the clinical manifestations and complications of leptospirosis were similar to those reported in the literature. We observed a relatively low overall mortality in relation to global statistics.

Expression and purification of the non-tagged LipL32 of pathogenic Leptospira

Leptospirosis is a reemerging infectious disease and the most disseminated zoonosis worldwide. A leptospiral surface protein, LipL32, only occurs in pathogenic Leptospira, and is the most abundant protein on the bacterial surface, being described as an important factor in host immunogenic response and also in bacterial infection. We describe here an alternative and simple purification protocol for non-tagged recombinant LipL32. The recombinant LipL32(21-272) was expressed in Escherichia coli without His-tag or any other tag used to facilitate recombinant protein purification. The recombinant protein was expressed in the soluble form, and the purification was based on ion exchange (anionic and cationic) and hydrophobic interactions. The final purification yielded 3 mg soluble LipL32(21-272) per liter of the induced culture. Antiserum produced against the recombinant protein was effective to detect native LipL32 from cell extracts of several Leptospira serovars. The purified recombinant LipL32(21-272) produced by this protocol can be used for structural, biochemical and functional studies and avoids the risk of possible interactions and interferences of the tags commonly used as well as the time consuming and almost always inefficient methods to cleave these tags when a tag-free LipL32 is needed. Non-tagged LipL32 may represent an alternative antigen for biochemical studies, for serodiagnosis and for the development of a vaccine against leptospirosis.

Proteome and immunome of pathogenic Leptospira spp. revealed by 2DE and 2DE-immunoblotting with immune serum.

In this study, proteomes of two pathogenic Leptospira spp., namely L. interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni and L. borgpetersenii, serogroup Tarassovi, serovar Tarassovi, were revealed by using two dimensional gel electrophoresis (2DE)-based-proteomics. Bacterial cells were disrupted in a lysis buffer containing 30 mM Tris, 2 M thiourea, 7 M urea, 4% CHAPS, 2% IPG buffer pH 3-10 and protease inhibitors and then subjected to sonication in order to solubilize as much as possible the bacterial proteins. The 2DE-separated components of both Leptospira homogenates were blotted individually onto membranes and antigenic components (immunomes) were revealed by probing the blots with immune serum of a mouse readily immunized with the homogenate of L. interrogans, serogroup Icterohaemorrhagiae, serovar Copenhageni. The immunogenic proteins of the two pathogenic Leptospira spp. could be grouped into 10 groups. These are: 1) proteins involved in the bacterial transcription and translation including beta subunit transcription anti-termination protein of DNA polymerase III, elongation factors Tu and Ts, and tRNA (guanine-N1)-methyltransferase; 2) proteins functioning as enzymes for metabolisms and nutrient acquisition including acetyl-Co-A acetyltransferase, putative glutamine synthetase, glyceraldehyde-3-phospahte dehydrogenase, NifU-like protein, 3-oxoacyl-(acyl-carrier-protein) reductase, oxidoreductase, sphingomyelinase C precursor, spermidine synthase, beta subunit of succinyl-CoA synthetase, and succinate dehydrogenase iron-sulfur subunit; 3) proteins/enzymes necessary for energy and electron transfer, i.e. electron transfer flavoprotein, and proton-translocating transhydrogenase; 4) enzymes for degradation of misfolded proteins, i.e. ATP-dependent Clp protease; 5) molecular chaperone, i.e. 60 kDa chaperonin; 6) signal transduction system, i.e. response regulator; 7) protein involved in immune evasion in host, i.e. peroxiredoxin; 8) cell structure proteins including MreB (cytoskeletal) and flagellin/ periplasmic flagellin; 9) lipoproteins/outer membrane proteins: LipL32, LipL41, LipL45 and OmpL1; and 10) various hypothetical proteins. Many immunogenic proteins are common to both Leptospira spp. These proteins not only are the diagnostic targets but also have potential as candidates of a broad spectrum leptospirosis vaccine especially the surface exposed components which should be vulnerable to the host immune effector factors.