Density-based lipoprotein depletion improves extracellular vesicle isolation and functional analysis.

BACKGROUND: Blood plasma is the main source of extracellular vesicles (EVs) in clinical studies aiming to identify biomarkers and to investigate pathophysiological processes, especially regarding EV roles in inflammation and thrombosis. However, EV isolation from plasma has faced the fundamental issue of lipoprotein contamination, representing an important bias since lipoproteins are highly abundant and modulate cell signaling, metabolism, and thromboinflammation. OBJECTIVES: Here, we aimed to isolate plasma EVs after depleting lipoproteins, thereby improving sample purity and EV thromboinflammatory analysis. METHODS: Density-based gradient ultracentrifugation (G-UC) was used for lipoprotein depletion before EV isolation from plasma through size-exclusion chromatography (SEC) or serial centrifugation (SC). Recovered EVs were analyzed by size, concentration, cellular source, ultrastructure, and bottom-up proteomics. RESULTS: G-UC efficiently separated lipoproteins from the plasma, allowing subsequent EV isolation through SEC or SC. Combined analysis from EV proteomics, cholesterol quantification, and apoB-100 detection confirmed the significant reduction in lipoproteins from isolated EVs. Proteomic analysis identified similar gene ontology and cellular components in EVs, regardless of lipoprotein depletion, which was consistent with similar EV cellular sources, size, and ultrastructure by flow cytometry and transmission electron microscopy. Importantly, lipoprotein depletion increased the detection of less abundant proteins in EV proteome and enhanced thromboinflammatory responses of platelets and monocytes stimulated in vitro with EV isolates. CONCLUSION: Combination of G-UC+SEC significantly reduced EV lipoprotein contamination without interfering in EV cellular source, gene ontology, and ultrastructure, allowing the recovery of highly pure EVs with potential implications for functional assays and proteomic and lipidomic analyses.

Analysis of thermal discomfort associated with synoptic conditions in the city of Pelotas, southernmost region of Brazil.

Here, we evaluated the influence of outdoor environmental conditions (synoptic weather conditions) on human thermal discomfort in the five macro-regions of Pelotas city, located in the southernmost region of Brazil. To do this, meteorological sensors (HOBO MX2301A) were installed outside the residences to measure the air temperature, dew point temperature, and relative humidity between 18 January and 20 August 2019. Two well-established simplified biometeorological indices were examined seasonally: (i) humidex for the summer months and (ii) effective temperature as a function of wind for the autumn and winter months. Our findings showed seasonal differences related to human thermal discomfort and outdoor environmental conditions. The thermal discomfort was highest in the afternoons during the summer months and at night during the winter months. The seasonal variation in human thermal discomfort was highly associated with the meteorological conditions. In summer, the presence of the South Atlantic Subtropical Anticyclone (SASA) contributed to heat stress. The SASA combined with the continent's low humidity contributed to the perceived sensation of thermal discomfort. In the winter, thermal discomfort was associated with the decrease in air humidity caused by high atmospheric pressure systems, which led to a decrease in both air temperature and air moisture content. Our findings suggest that a better understanding of the complex interplay between outdoor environmental factors and human thermal comfort is needed in order to mitigate the negative effects of thermal discomfort.

Global proteome of the saprophytic strain Leptospira biflexa and comparative analysis with pathogenic strain Leptospira interrogans uncover new pathogenesis mechanisms

Leptospira is a genus of bacteria that includes free-living saprophytic species found in water or soil, and pathogenic species, which are the etiologic agents of leptospirosis. Besides all the efforts, there are only a few proteins described as virulence factors in the pathogenic strain L. interrogans. This work aims to perform L. biflexa serovar Patoc1 strain Paris global proteome and to compare with the proteome database of pathogenic L. interrogans serovar Copenhageni strain Fiocruz L1–130. We identified a total of 2327 expressed proteins of L. biflexa by mass spectrometry. Using the Get Homologues software with the global proteome of L. biflexa and L. interrogans, we found orthologous proteins classified into conserved, low conserved, and specific proteins. Comparative bioinformatic analyses were performed to understand the biological functions of the proteins, subcellular localization, the presence of signal peptide, structural domains, and motifs using public softwares. These results lead to the selection of 182 low conserved within the saprophyte, and 176 specific proteins of L. interrogans. It is anticipated that these findings will indicate further studies to uncover virulence factors in the pathogenic strain. This work presents for the first time the global proteome of saprophytic strain L. biflexa serovar Patoc, strain Patoc1.

LipL41 and LigA/LigB Gene Silencing on a LipL32 Knockout Leptospira interrogans Reveals the Impact of Multiple Mutations on Virulence.

Leptospirosis is a global zoonosis caused by pathogenic bacteria of the genus Leptospira. The application of the CRISPR/Cas9 system has facilitated the generation of mutants and subsequent evaluation of phenotypes. Since DNA breaks induced by RNA-guided Cas9 nuclease are lethal to Leptospira, different methodologies were implemented to overcome this limitation. Initially, CRISPR interference (CRISPRi) was employed to create knockdown mutants, utilizing a catalytically inactive Cas9 (dCas9). Subsequently, the co-expression of CRISPR/Cas9 and a DNA repair system from Mycobacterium smegmatis enabled the generation of scarless knockout mutants. We eliminated plasmids from the lipL32 knockout L. interrogans strain and further achieved multiple gene mutations via gene silencing in this knockout background. Strains lacking both LipL41 and LipL32 and LigA, LigB, and LipL32, were evaluated. The absence of proteins LipL32 and LipL41 had no effect on leptospiral virulence. On the other hand, mutants lacking LigA, LigB, and LipL32 were unable to cause acute disease. The expanded apparatus for genetic manipulation of pathogenic leptospires via the CRISPR/Cas9 system has allowed the evaluation of multiple mutations upon leptospiral virulence. This work shows that LipL32 and LipL41 are not required for acute disease and consolidates LigA and LigB proteins as virulence factors.

Capybara Oil Improves Renal Pathophysiology and Inflammation in Obese Mice.

Obesity is an inflammatory disease associated with secondary diseases such as kidney disease, which can cause lipotoxicity, inflammation and loss of organ function. Polyunsaturated fatty acids act in the production of lipid mediators and have anti-inflammatory characteristics. In this work, the objective was to evaluate renal histopathology in obese mice and the effects of treatment with capybara oil (CO) (5000 mg/kg/day for 4 weeks). Parameters such as body mass, lipid profile, systolic blood pressure, urinary creatinine and protein excretion, structure and ultrastructure of the renal cortex, fibrosis, tissue inflammation and oxidative stress were analyzed. CO treatment in obese mice showed improvement in the lipid profile and reduction in systolic blood pressure levels, in addition to beneficial remodeling of the renal cortex. Our data demonstrated that CO decreased inflammation, oxidative stress and renal fibrosis, as evidenced by quantifying the expression of TNF-α, IL-10, CAT, SOD, α-SMA and TGF-ß. Although treatment with CO did not show improvement in renal function, ultrastructural analysis showed that the treatment was effective in restoring podocytes and pedicels, with restructuring of the glomerular filtration barrier. These results demonstrate, for the first time, that treatment with CO is effective in reducing kidney damage, being considered a promising treatment for obesity.

Euterpe oleracea Mart. (Açai) seed extract improves physical performance in old rats by restoring vascular function and oxidative status and activating mitochondrial muscle biogenesis.

OBJECTIVES: Alterations in cardiovascular and skeletal muscle function are hallmarks of ageing that lead to exercise intolerance. We aimed to examine whether the treatment with Euterpe oleracea Mart. seed extract (ASE) associated with exercise training improves aerobic exercise performance by promoting healthy ageing in the elderly. METHODS: Male Wistar rats were divided into five groups: Young (3 months), Old (18 months), Old+ASE (ASE 200 mg/kg/day), Old+Training (exercise training 30 min/day; 5 days/week) and Old+Training+ASE, for 4 weeks. KEY FINDINGS: ASE treatment increased the exercise time and the running distance concerning the initial maximal treadmill stress test (MTST) in the Old+Training+ASE group. Exercise training or ASE treatment restored the aorta oxidative damage and antioxidant defence. It reduced the acetylcholine (ACh)-induced vasodilation in the aorta of old animals to the same values as the young and improved hypertension. Only the association of both strategies restored the ACh-induced vasodilation in mesentery arteries. Remarkably, exercise training associated with ASE increased the antioxidant defence, nitrite levels and expression of the mitochondrial SIRT-1, PGC1α in soleus muscle homogenates. CONCLUSIONS: ASE treatment associated with exercise training contributes to better exercise performance and tolerance in ageing by improving vascular function, oxidative stress and activating the muscle SIRT-1/PGC-1α pathway.

Overcoming problems to produce the recombinant protein LipL21 of Leptospira interrogans.

The production of leptospiral recombinant proteins in the soluble form and in high yield from Escherichia coli is still a challenge. This work presents the cloning, expression and purification of the outer membrane protein of Leptospira interrogans, LipL21, which is considered an interesting target for vaccine and diagnostics development. The expression profile and yield of LipL21 was compared after cloning in the vectors pAE, pET28a and pET-SUMO, and it was observed that LipL21 was expressed in a low amount with pAE vector. By using the pET-28a vector, protein expression was increased, but the majority of the product was obtained as inclusion bodies. As a highlight, using a pET-SUMO vector was shown to overcome the problems of low expression and solubility of the lipoprotein LipL21.

Structural and biochemical characterization of Leptospira interrogans Lsa45 reveals a penicillin-binding protein with esterase activity.

Leptospirosis is a bacterial disease that affects humans and animals and is caused by Leptospira. The recommended treatment for leptospirosis is antibiotic therapy, which should be given early in the course of the disease. Despite the use of these antibiotics, their role during the course of the disease is still not completely clear because of the lack of effective clinical trials, particularly for severe cases of the disease. Here, we present the characterization of L. interrogans Lsa45 protein by gel filtration, protein crystallography, SAXS, fluorescence and enzymatic assays. The oligomeric studies revealed that Lsa45 is monomeric in solution. The crystal structure of Lsa45 revealed the presence of two subdomains: a large α/ß subdomain and a small α-helical subdomain. The large subdomain contains the amino acids Ser122, Lys125, and Tyr217, which correspond to the catalytic triad that is essential for ß-lactamase or serine hydrolase activity in similar enzymes. Additionally, we also confirmed the bifunctional promiscuity of Lsa45, in hydrolyzing both the 4-nitrophenyl acetate (p-NPA) and nitrocefin ß-lactam antibiotic. Therefore, this study provides novel insights into the structure and function of enzymes from L. interrogans, which furthers our understanding of this bacterium and the development of new therapies for the prevention and treatment of leptospirosis.

Angle class II/2nd division: is there an alignment? / Classe II/2ª divisão de Angle: existe alinhamento?

Introduction: Interdisciplinary investigative study of the stomatognathic-cervical complex, necessary to understand the structure and biomechanics of this system in Angle Class I and II / 2nd Division participants. Objective: To evaluate alignment and position of cranial cervical structures on radiographs and their functional relationship with the stomatognathic system. Material and method: Trans Oral and Profile radiographs were submitted to biomechanical analysis, considered the linear and angular measurement of Atlas and Axis through the application included in the radiogram software. Result: We observed a significant difference in the angular measurements of vertical alignment between the Skull, Axis, and the Mandible (p = <0.001), and in the left joint between the Atlas and the Axis (AE: p = 0.011; SEA: p = 0.042). Among the linear measures of the distances between the Atlas and the Axis, the AOD distance presented statistics quite close to the level of significance (p = 0.0502), but above. There was no statistically significant difference in the other measures. Conclusion: In this study, the alignment between the Atlas and Axis cervical vertebrae and the mandible and angles of the atlanto-occipital joints are altered in Class II / 2nd Division participants. There is no difference in the size and distance of the Atlas and the Axis between the Classes.

Introdução: Estudo interdisciplinar investigativo do complexo cérvico-estomatognático, necessário para compreender a estrutura e a biomecânica desse sistema em participantes Classe I e II/2ª Divisão de Angle. Objetivo: Avaliar o alinhamento e a posição das estruturas crânio cervicais nas radiografias de participantes Classe I e II/2ª Divisão de Angle e sua relação funcional com o sistema estomatognático. Material e método: As imagens digitais Trans Oral e Perfil de participantes com maloclusão Classe I e II/2ª Divisão de Angle foram submetidas à medição linear e angular do crânio, mandíbula, Atlas e do Áxis através do software Advantage Workstation 4.6 (AW4.6 ext. 04). Resultado: Houve diferença significativa nas medidas angulares de alinhamento vertical entre o Crânio, Áxis e a Mandíbula (p = <0,001), e da articulação esquerda entre o Atlas e o Áxis (AE: p = 0,011; AAE: p = 0,042). Das medidas lineares das distâncias entre o Atlas e o Áxis, a distância AOD apresentou estatística bastante próxima do nível de significância (p=0,0502), porém acima. Não houve diferença significativamente estatística nas demais medidas avaliadas. Conclusão: Neste estudo, o alinhamento entre as vértebras cervicais Atlas e Áxis e a mandíbula e os ângulos das articulações atlanto occipitais se mostraram alterados nos participantes Classe II/2ª Divisão de Angle. Não há diferença significativa no tamanho e distância do Atlas e do Áxis entre as Classes.

LipL41 and LigA/LigB gene silencing on a LipL32 knockout Leptospira interrogans reveals the impact of multiple mutations on virulence

Leptospirosis is a global zoonosis caused by pathogenic bacteria of the genus Leptospira. The application of the CRISPR/Cas9 system has facilitated the generation of mutants and subsequent evaluation of phenotypes. Since DNA breaks induced by RNA-guided Cas9 nuclease are lethal to Leptospira, different methodologies were implemented to overcome this limitation. Initially, CRISPR interference (CRISPRi) was employed to create knockdown mutants, utilizing a catalytically inactive Cas9 (dCas9). Subsequently, the co-expression of CRISPR/Cas9 and a DNA repair system from Mycobacterium smegmatis enabled the generation of scarless knockout mutants. We eliminated plasmids from the lipL32 knockout L. interrogans strain and further achieved multiple gene mutations via gene silencing in this knockout background. Strains lacking both LipL41 and LipL32 and LigA, LigB, and LipL32, were evaluated. The absence of proteins LipL32 and LipL41 had no effect on leptospiral virulence. On the other hand, mutants lacking LigA, LigB, and LipL32 were unable to cause acute disease. The expanded apparatus for genetic manipulation of pathogenic leptospires via the CRISPR/Cas9 system has allowed the evaluation of multiple mutations upon leptospiral virulence. This work shows that LipL32 and LipL41 are not required for acute disease and consolidates LigA and LigB proteins as virulence factors.

Evaluation of Leptospira interrogans knockdown mutants for LipL32, LipL41, LipL21, and OmpL1 proteins

Introduction: Leptospirosis is a worldwide zoonosis caused by pathogenic and virulent species of the genus Leptospira, whose pathophysiology and virulence factors remain widely unexplored. Recently, the application of CRISPR interference (CRISPRi) has allowed the specific and rapid gene silencing of major leptospiral proteins, favoring the elucidation of their role in bacterial basic biology, host-pathogen interaction and virulence. Episomally expressed dead Cas9 from the Streptococcus pyogenes CRISPR/Cas system (dCas9) and single-guide RNA recognize and block transcription of the target gene by base pairing, dictated by the sequence contained in the 5′ 20-nt sequence of the sgRNA. Methods: In this work, we tailored plasmids for silencing the major proteins of L. interrogans serovar Copenhageni strain Fiocruz L1-130, namely LipL32, LipL41, LipL21 and OmpL1. Double- and triple-gene silencing by in tandem sgRNA cassettes were also achieved, despite plasmid instability. Results: OmpL1 silencing resulted in a lethal phenotype, in both L. interrogans and saprophyte L. biflexa, suggesting its essential role in leptospiral biology. Mutants were confirmed and evaluated regarding interaction with host molecules, including extracellular matrix (ECM) and plasma components, and despite the dominant abundance of the studied proteins in the leptospiral membrane, protein silencing mostly resulted in unaltered interactions, either because they intrinsically display low affinity to the molecules assayed or by a compensation mechanism, where other proteins could be upregulated to fill the niche left by protein silencing, a feature previously described for the LipL32 mutant. Evaluation of the mutants in the hamster model confirms the augmented virulence of the LipL32 mutant, as hinted previously. The essential role of LipL21 in acute disease was demonstrated, since the LipL21 knockdown mutants were avirulent in the animal model, and even though mutants could still colonize the kidneys, they were found in markedly lower numbers in the animals' liver. Taking advantage of higher bacterial burden in LipL32 mutant-infected organs, protein silencing was demonstrated in vivo directly in leptospires present in organ homogenates. Discussion: CRISPRi is now a well-established, attractive genetic tool that can be applied for exploring leptospiral virulence factors, leading to the rational for designing more effective subunit or even chimeric recombinant vaccines.

LIC12254 is a leptospiral protein that interacts with integrins via the RGD motif

Pathogenic leptospires can bind to receptors on mammalian cells such as cadherins and integrins. Leptospira effectively adheres to cells, overcomes host barriers and spreads into the bloodstream, reaching internal target organs such as the lungs, liver and kidneys. Several microorganisms produce proteins that act as ligands of integrins through the RGD motif. Here, we characterized a leptospiral RGD-containing protein encoded by the gene lic12254. In silico analysis of pathogenic, intermediate and saprophytic species showed that LIC12254 is highly conserved among pathogenic species, and is unique in presenting the RGD motif. The LIC12254-coding sequence is greatly expressed in the virulent Leptospira interrogans L1-130 strain compared with the culture-attenuated L. interrogans M20 strain. We also showed that the recombinant protein rLIC12254 binds to αVβ8 and α8 human integrins most likely via the RGD motif. These interactions are dose-dependent and saturable, a typical property of receptor–ligand interactions. The binding of the recombinant protein lacking this motif—rLIC12254 ΔRAA—to αVβ8 was almost totally abolished, while that with the α8 human integrin was decreased by 65%. Taken together, these results suggest that this putative outer membrane protein interacts with integrins via the RGD domain and may play a key role in leptospirosis pathogenesis.

Overcoming problems to produce the recombinant protein LipL21 of Leptospira interrogans

The production of leptospiral recombinant proteins in the soluble form and in high yield from Escherichia coli is still a challenge. This work presents the cloning, expression and purification of the outer membrane protein of Leptospira interrogans, LipL21, which is considered an interesting target for vaccine and diagnostics development. The expression profile and yield of LipL21 was compared after cloning in the vectors pAE, pET28a and pET-SUMO, and it was observed that LipL21 was expressed in a low amount with pAE vector. By using the pET-28a vector, protein expression was increased, but the majority of the product was obtained as inclusion bodies. As a highlight, using a pET-SUMO vector was shown to overcome the problems of low expression and solubility of the lipoprotein LipL21.

Structural and biochemical characterization of Leptospira interrogans Lsa45 reveals a penicillin-binding protein with esterase activity

Leptospirosis is a bacterial disease that affects humans and animals and is caused by Leptospira. The recommended treatment for leptospirosis is antibiotic therapy, which should be given early in the course of the disease. Despite the use of these antibiotics, their role during the course of the disease is still not completely clear because of the lack of effective clinical trials, particularly for severe cases of the disease. Here, we present the characterization of L. interrogans Lsa45 protein by gel filtration, protein crystallography, SAXS, fluorescence and enzymatic assays. The oligomeric studies revealed that Lsa45 is monomeric in solution. The crystal structure of Lsa45 revealed the presence of two subdomains: a large α/β subdomain and a small α-helical subdomain. The large subdomain contains the amino acids Ser122, Lys125, and Tyr217, which correspond to the catalytic triad that is essential for β-lactamase or serine hydrolase activity in similar enzymes. Additionally, we also confirmed the bifunctional promiscuity of Lsa45, in hydrolyzing both the 4-nitrophenyl acetate (p-NPA) and nitrocefin β-lactam antibiotic. Therefore, this study provides novel insights into the structure and function of enzymes from L. interrogans, which furthers our understanding of this bacterium and the development of new therapies for the prevention and treatment of leptospirosis.

Host Cell Binding Mediated by Leptospira interrogans Adhesins.

Leptospirosis is a neglected infectious disease with global impact on both humans and animals. The increase in urban development without sanitation planning is one of the main reasons for the disease spreading. The symptoms are similar to those of flu-like diseases, such as dengue, yellow fever, and malaria, which can result in a misleading clinical diagnosis. The characterization of host-pathogen interactions is important in the development of new vaccines, treatments, and diagnostics. However, the pathogenesis of leptospirosis is not well understood, and many gaps remain to be addressed. Here, we aimed to determine if Leptospira strains, virulent, culture-attenuated, and saprophytic, and the major outer membrane proteins OmpL37, OmpL1, LipL21, LipL41, and LipL46 are able to adhere to different endothelial, epithelial and fibroblast cell lines in vitro. We showed that virulent leptospires robustly bind to all cells compared to the culture-attenuated and saprophytic lines. The recombinant proteins exhibited certain adhesion, but only OmpL1 and LipL41 were able to bind to several cell lines, either in monolayer or in cell suspension. Blocking OmpL1 with polyclonal antibodies caused a decrease in bacterial binding to cells, contrasting with an increase observed when anti-LipL41 antibodies were used. The adhesion of OmpL1 to HMEC-1 and EA.hy926 was inhibited when cells were pre-incubated with collagen IV, suggesting that both compete for the same cell receptor. We present here for the first time the interaction of five leptospiral outer membrane proteins with several cell lines, and we conclude that LipL41 and OmpL1 may have an impact on leptospiral adhesion to mammalian cells and may mediate the colonization process in leptospiral pathogenesis.

Identification of Leptospiral Protein Antigens Recognized by WC1+ γδ T Cell Subsets as Target for Development of Recombinant Vaccines.

Pathogenic Leptospira species cause leptospirosis, a neglected zoonotic disease recognized as a global public health problem. It is also the cause of the most common cattle infection that results in major economic losses due to reproductive problems. γδ T cells play a role in the protective immune response in livestock species against Leptospira, while human γδ T cells also respond to Leptospira. Thus, activation of γδ T cells has emerged as a potential component in the optimization of vaccine strategies. Bovine γδ T cells proliferate and produce gamma interferon (IFN-γ) in response to vaccination with inactivated leptospires, and this response is mediated by a specific subpopulation of the WC1-bearing γδ T cells. WC1 molecules are members of the group B scavenger receptor cysteine-rich (SRCR) superfamily and are composed of multiple SRCR domains, of which particular extracellular domains act as ligands for Leptospira. Since WC1 molecules function as both pattern recognition receptors and γδ TCR coreceptors, the WC1 system has been proposed as a novel target to engage γδ T cells. Here, we demonstrate the involvement of leptospiral protein antigens in the activation of WC1+ γδ T cells and identify two leptospiral outer membrane proteins able to interact directly with them. Interestingly, we show that the protein-specific γδ T cell response is composed of WC1.1+ and WC1.2+ subsets, although a greater number of WC1.1+ γδ T cells respond. Identification of protein antigens will enhance our understanding of the role γδ T cells play in the leptospiral immune response and in recombinant vaccine development.

Host cell binding mediated by Leptospira interrogans adhesins

Leptospirosis is a neglected infectious disease with global impact on both humans and animals. The increase in urban development without sanitation planning is one of the main reasons for the disease spreading. The symptoms are similar to those of flu-like diseases, such as dengue, yellow fever, and malaria, which can result in a misleading clinical diagnosis. The characterization of host–pathogen interactions is important in the development of new vaccines, treatments, and diagnostics. However, the pathogenesis of leptospirosis is not well understood, and many gaps remain to be addressed. Here, we aimed to determine if Leptospira strains, virulent, culture-attenuated, and saprophytic, and the major outer membrane proteins OmpL37, OmpL1, LipL21, LipL41, and LipL46 are able to adhere to different endothelial, epithelial and fibroblast cell lines in vitro. We showed that virulent leptospires robustly bind to all cells compared to the culture-attenuated and saprophytic lines. The recombinant proteins exhibited certain adhesion, but only OmpL1 and LipL41 were able to bind to several cell lines, either in monolayer or in cell suspension. Blocking OmpL1 with polyclonal antibodies caused a decrease in bacterial binding to cells, contrasting with an increase observed when anti-LipL41 antibodies were used. The adhesion of OmpL1 to HMEC-1 and EA.hy926 was inhibited when cells were pre-incubated with collagen IV, suggesting that both compete for the same cell receptor. We present here for the first time the interaction of five leptospiral outer membrane proteins with several cell lines, and we conclude that LipL41 and OmpL1 may have an impact on leptospiral adhesion to mammalian cells and may mediate the colonization process in leptospiral pathogenesis.

A new recombinant multiepitope chimeric protein of Leptospira interrogans is a promising marker for the serodiagnosis of leptospirosis

The zoonotic disease leptospirosis is caused by pathogenic species of the genus Leptospira and was recently included in the list of Neglected Diseases by the World Health Organization. Leptospirosis burden is estimated to have over a million human cases and cause 60 thousand deaths annually, in addition to its economic impact and veterinary concern. The microscopic agglutination test (MAT), recommended by the World Health Organization, exhibits reduced sensitivity at the beginning of the disease, in addition to being technically difficult. New recombinant antigens are being pursued for rapid and specific serodiagnostic tests, especially in the initial phase of the disease, and chimeric multiepitope proteins are a strategy with a great potential to be implemented in serology. Based on previous subproteomic results, we designed a synthetic construct comprising 10 conserved leptospiral surface antigens, and the recombinant protein was purified and evaluated regarding its diagnostic potential. The protein termed rChi2 was recognized by antibodies in serum from patients both at the onset (MAT−) and in the convalescent (MAT+) phase in 75 and 82% of responders, respectively. In addition, rChi2 immunization in hamsters elicited a strong humoral response, and anti-rChi2 antibodies recognized several immobilized intact Leptospira species, validating its potential as an early, broad, and cross-reactive diagnostic test.

A novel breakthrough in Leptospira spp. mutagenesis: knockout by combination of CRISPR/Cas9 and non-homologous end-joining systems

Leptospirosis is of general concern as it is a widespread zoonotic disease caused by pathogenic species of the genus Leptospira, although this genus also includes free-living saprophytic strains. Understanding the pathophysiology of leptospirosis is still in its infancy even after several years of its discovery, because of the lack of effective genetic tools. The use of the Streptococcus pyogenes CRISPR/Cas9 system and its variations have pushed the leptospirosis research forward, relying on the simplicity of the technique. However, the lethality of double-strand breaks (DSB) induced by the RNA-guided Cas9 enzyme has limited the generation of knockout mutants. In this work, we demonstrated sustained cell viability after concurrent expression of CRISPR/Cas9 and Mycobacterium tuberculosis non-homologous end-joining components in a single-plasmid strategy in L. biflexa. Scarless mutations resulting in null phenotypes could be observed in most of the colonies recovered, with deletions in the junctional site ranging from 3 to almost 400 bp. After plasmid curing by in vitro passages in a medium without antibiotic, selected marker-free and targeted mutants could be recovered. Knockout mutants for LipL32 protein in the pathogen L. interrogans could be obtained using M. smegmatis NHEJ machinery, with deletions ranging from 10 to 345 bp. In conclusion, we now have a powerful genetic tool for generating scarless and markerless knockout mutants for both saprophytic and pathogenic strains of Leptospira.

Evaluation of LipL32 and LigA/LigB knockdown mutants in Leptospira interrogans serovar copenhageni: impacts to proteome and virulence

Leptospirosis is a worldwide zoonosis caused by pathogenic species of the genus Leptospira. The recent application of CRISPR interference (CRISPRi) to Leptospira facilitates targeted gene silencing and provides a new tool to investigate pathogenic mechanisms of leptospirosis. CRISPRi relies on the expression of a catalytically “dead” Cas9 (dCas9) and a single-guide RNA (sgRNA). Previously, our group generated a LipL32 and a double LigA/LigB (LigAB) mutant, which, in the current study, are characterized by whole-cell proteomics in comparison with control leptospires harboring plasmid expressing dCas9 alone. Comparison of control and LigAB mutant leptospires identified 46 significantly differentially expressed (DE) proteins, including 27 proteins that were less abundant and 19 proteins that were more abundant in the LigAB mutant compared with the control. Comparison of the control and LipL32 mutant leptospires identified 243 DE proteins, of which 84 proteins were more abundant and 159 were less abundant in the LipL32 mutant strain. Significantly increased amounts of known virulence impactors and surface membrane receptors, including LipL45, LipL31, LigB, and LipL41, were identified. The virulence of LipL32 and LigAB mutants were evaluated in the hamster model of leptospirosis; the LigAB mutant was unable to cause acute disease although mutant leptospires could still be recovered from target organs, albeit at a significantly lower bacterial burden (<850 and <16-fold in liver and kidney, respectively, in comparison with control), indicating attenuation of virulence and a shift to chronic bacterial persistence. Notably, the LipL32 mutant displayed augmented virulence as evidenced by early onset of clinical symptoms and increased numbers of circulating foamy macrophages. Validation of LipL32 and LigAB mutants recovered from liver and kidney in the presence or absence of antibiotic selection revealed high plasmid stability and, by extension, gene silencing in vivo. Collectively, this work emphasizes the advantages and feasibility of using CRISPRi technology to evaluate and characterize virulence factors of leptospires and their respective host–pathogen interactions in animal models of leptospirosis. Importantly, it also provides insight into the requirements of LigA and LigB for acute disease and explores the impact of silencing expression of lipL32, which resulted in substantial changes in amounts of outer membrane proteins.