Surface proteomics and label-free quantification of Leptospira interrogans serovar Pomona

Leptospirosis is a re-emerging zoonosis with a global distribution. Surface-exposed outer membrane proteins (SE-OMPs) are crucial for bacterial–host interactions. SE-OMPs locate and expose their epitope on cell surface where is easily accessed by host molecules. This study aimed to screen for surface-exposed proteins and their abundance profile of pathogenic Leptospira interrogans serovar Pomona. Two complementary approaches, surface biotinylation and surface proteolytic shaving, followed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) were employed to identify SE-OMPs of intact leptospires. For quantitative comparison, in-depth label-free analysis of SE-OMPs obtained from each method was performed using MaxQuant. The total number of proteins identified was 1,001 and 238 for surface biotinylation and proteinase K shaving, respectively. Among these, 39 were previously known SE-OMPs and 68 were predicted to be localized on the leptospiral surface. Based on MaxQuant analysis for relative quantification, six known SE-OMPs including EF- Tu, LipL21, LipL41, LipL46, Loa22, and OmpL36, and one predicted SE-OMPs, LipL71 were found in the 20 most abundant proteins, in which LipL41 was the highest abundant SE-OMP. Moreover, uncharacterized LIC14011 protein (LIP3228 ortholog in serovar Pomona) was identified as a novel predicted surface βb-OMP. High-abundance leptospiral SE-OMPs identified in this study may play roles in virulence and infection and are potential targets for development of vaccine or diagnostic tests for leptospirosis.

Draft Genome Sequences of Four Different Strains Belonging to Leptospira interrogans Serovar Pomona Isolated from Mammals in the Island of Sardinia, Italy

Leptospirosis is a zoonotic disease of global importance caused by a pathogenic group of bacteria belonging to the genus Leptospira . Here, we report four draft genome sequences of Leptospira interrogans serovar Pomona isolated on Sardinia (Italy) from four different species of mammals (i.e., dolphin, wild boar, cow, and fox).

Cytotoxicity of the 42 kDa SMase C sphingomyelinase secreted by Leptospira interrogans serovar Pomona on Vero cells

Sphingomyelinases produced by the pathogenic members of the genus Leptospira are implicated in the haemorrhagic manifestations seen in the severe form of leptospirosis. With multiple sphingomyelinase genes present in the genome of pathogenic Leptospira , much remains to be understood about these molecules. They include factors regulating their expression, post-translational modifications, and release of the biologically active forms of these molecules. In this study, serovar Pomona was chosen as it is reported to express high levels of sphingomyelinase that explained the haemolytic activity seen in experimental animals infected with this pathogen. Here, we demonstrate the cytotoxicity of a 42 kDa sphingomyelinase secreted by Leptospira interrogans serovar Pomona strain Pomona upon infecting Vero cells. This sphingomyelinase detected using specific anti-sphingomyelinase antibodies, exhibited haemolytic and sphingomyelinase activities that caused host-cell damage evident from the confocal images and scanning electron micrographs. The implications of these findings and the detection of a 42 kDa sphingomyelinase in the urine of human patients with leptospirosis in our earlier study is discussed with an emphasis on the potential of these sphingomyelinases as candidate markers for the early diagnosis of leptospirosis.

Reduced Renal Colonization and Enhanced Protection by Leptospiral Factor H Binding Proteins as a Multisubunit Vaccine Against Leptospirosis in Hamsters

Subunit vaccines conferring complete protection against leptospirosis are not currently available. The interactions of factor H binding proteins (FHBPs) on pathogenic leptospires and host factor H are crucial for immune evasion by inhibition of complement-mediated killing. The inhibition of these interactions may be a potential strategy to clear leptospires in the host. This study aimed to evaluate a multisubunit vaccine composed of four known leptospiral FHBPs: LigA domain 7–13 (LigAc), LenA, LcpA, and Lsa23, for its protective efficacy in hamsters. The mono and multisubunit vaccines formulated with LMQ adjuvant, a combination of neutral liposome, monophosphoryl lipid A, and Quillaja saponaria fraction 21, induced high and comparable specific antibody (IgG) production against individual antigens. Hamsters immunized with the multisubunit vaccine showed 60% survival following the challenge by 20 LD50 of Leptospira interrogans serovar Pomona. No significant difference in survival rate and pathological findings of target organs was observed after vaccinations with multisubunit or mono-LigAc vaccines. However, the multisubunit vaccine significantly reduced leptospiral burden in surviving hamsters in comparison with the monosubunit vaccines. Therefore, the multisubunit vaccine conferred partial protection and reduced renal colonization against virulence Leptospira infection in hamsters. Our multisubunit formulation could represent a promising vaccine against leptospirosis.

Draft Genome Sequence of Brazilian Leptospira interrogans Serovar Pomona Strain GR5, Isolated from Apparently Healthy Gilt

ABSTRACT Leptospira interrogans serovar Pomona is one of the most important serovars associated with worldwide porcine leptospirosis, and its infection is characterized by high antibody titers and the establishment of a renal carrier state. Here, we present the draft genome sequence of Leptospira interrogans serovar Pomona strain GR5 isolated from apparently healthy gilt in Brazil.

Determinación de anticuerpos contra patógenos virales y bacterianos seleccionados en la población de cerdos silvestres (Sus scrofa) de la Reserva Natural Bahía Samborombón, Argentina

Los cerdos silvestres (Sus scrofa) descienden de cruzamientos entre cerdos domésticos liberados durante la colonización con jabalíes salvajes euroasiáticos, liberados con propósitos cinegéticos. Son invasivos y su coexistencia con especies domésticas implica riesgos sanitarios. Argentina es considerada libre de fiebre aftosa (FA), peste porcina clásica (PPC) y africana (PPA) y síndrome reproductivo y respiratorio porcino (PRRS). La enfermedad de Aujeszky (EA) y la leptospirosis son endémicas en ciertas áreas del país. El objetivo fue evaluar la presencia de ciertas enfermedades zoonóticas y/o de importancia para la producción animal y la conservación de la biodiversidad en cerdos silvestres de la Bahía de Samborombón. Se capturaron 118 animales. Se tomaron muestras de suero, tonsilas, músculo, intestino delgado, linfonódulos, entre otras. Se estudió la presencia de anticuerpos contra Brucella spp., coronavirus respiratorio porcino, virus de la estomatitis vesicular, de la FA, de la gastroenteritis transmisible porcina (TGEV), de la PPC, PPA, EA, PRRS y Leptospira spp. Se realizaron análisis bacteriológicos para Mycobacterium spp. Los resultados ratificaron la ausencia de las enfermedades exóticas e indicaron que 36 % de los animales presentó anticuerpos contra Leptospira interrogans serovar pomona y 62,5 % contra el virus de la EA. Estos resultados remarcan la importancia del monitoreo de la interfase productiva/silvestre en función de la salud pública, producción animal y conservación de la biodiversidad.

Characterization of Leptospira interrogans serovar Pomona isolated from swine in Brazil

Introduction: Leptospira interrogans swine infection is a cause of serious economic loss and a potential human health hazard. In Brazil, the most common serovars associated with swine infections are Pomona, Icterohaemorrhagie and Tarassovi. Cross-reactions among serovars and the failure of infected animals to seroconvert may complicate the interpretation of serological tests. Molecular methods with better discriminatory powers are useful tools for swine leptospirosis characterization and diagnosis. Methodology: This study evaluated nine L. interrogans isolates from the States of Sao Paulo and Minas Gerais during different time periods. Isolates from diseased and apparently healthy swine were characterized by microscopic agglutination tests with polyclonal antibodies and were genotyped by VNTR, PFGE and MLST techniques. Broth microdilution was used to determine the minimal inhibitory concentration of the antimicrobials of veterinary interest. Results: The strains were identified as L. interrogans serogroup Pomona serovar Pomona Genotype A, while MLST grouped all of the isolates in sequence type 37. The PFGE analysis resulted in two pulsotypes with more than 70% similarity, distinguishing serovar Pomona isolates from the serovar Kennewicki reference strain. All of the isolates presented low MIC values to penicillin, ampicillin, ceftiofur and tulathromycin. High MIC values for fluoroquinolones, tiamulin, gentamicin, tetracyclines, neomycin, tilmicosin and sulfas were also observed. Conclusions: All molecular techniques were concordant in L. interrogans serovar Pomona identification. This serovar may have a different antibiotic susceptibility profile than previously reported for Leptospira isolates.