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The development of inexpensive and portable point-of-care devices for measuring nutritional physiological parameters from blood (e.g., glucose, ketones) has accelerated our understanding and assessment of real-time variation in human health, but these have infrequently been tested or implemented in wild animals, especially in relation to other key biological or fitness-related traits. Here we used point-of-care devices to measure blood levels of glucose, ketones, uric acid, and triglycerides in free-ranging house finches (Haemorhous mexicanus)-a common songbird in North America that has been well-studied in the context of urbanization, nutrition, health, and sexual selection-during winter and examined (1) repeatability of these methods for evaluating blood levels in these wild passerines, (2) intercorrelations among these measurements within individuals, (3) how blood nutritional-physiology metrics related to a bird's body condition, habitat of origin (urban vs. suburban), poxvirus infection, and sex; and (4) if the expression of male sexually selected plumage coloration was linked to any of the nutritional-physiological metrics. All blood-nutritional parameters were repeatable. Also, there was significant positive covariation between concentrations of circulating triglycerides and glucose and triglycerides and uric acid. Urban finches had higher blood glucose concentrations than suburban finches, and pox-infected individuals had lower blood triglyceride concentrations than uninfected ones. Last, redder males had higher blood glucose, but lower uric acid levels. These results demonstrate that point-of-care devices can be useful, inexpensive ways of measuring real-time variation in the nutritional physiology of wild birds.
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Tentilhões , Passeriformes , Infecções por Poxviridae , Humanos , Masculino , Animais , Tentilhões/fisiologia , Urbanização , Ácido Úrico/metabolismo , Glicemia , Sistemas Automatizados de Assistência Junto ao Leito , Animais Selvagens , Ecossistema , Fenômenos Fisiológicos da Nutrição , Cetonas/metabolismo , TriglicerídeosRESUMO
Abstract This study aimed to assess the influence of smoking on the subgingival metatranscriptomic profile of young patients affected by stage III/IV and generalized periodontal disease. Methodology In total, six young patients, both smokers and non-smokers (n=3/group), who were affected by periodontitis were chosen. The STROBE (Strengthening the Reporting of Observational Studies in Epidemiology) guidelines for case-control reporting were followed. Periodontal clinical measurements and subgingival biofilm samples were collected. RNA was extracted from the biofilm and sequenced via Illumina HiSeq. Differential expression analysis used Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, and differentially expressed genes were identified using the Sleuth package in R, with a statistical cutoff of ≤0.05. Results This study found 3351 KEGGs in the subgingival biofilm of both groups. Smoking habits altered the functional behavior of subgingival biofilm, resulting in 304 differentially expressed KEGGs between groups. Moreover, seven pathways were modulated: glycan degradation, galactose metabolism, glycosaminoglycan degradation, oxidative phosphorylation, peptidoglycan biosynthesis, butanoate metabolism, and glycosphingolipid biosynthesis. Smoking also altered antibiotic resistance gene levels in subgingival biofilm by significantly overexpressing genes related to beta-lactamase, permeability, antibiotic efflux pumps, and antibiotic-resistant synthetases. Conclusion Due to the limitations of a small sample size, our data suggest that smoking may influence the functional behavior of subgingival biofilm, modifying pathways that negatively impact the behavior of subgingival biofilm, which may lead to a more virulent community.
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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.
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OBJECTIVE: To quantitatively and qualitatively analyze the proteomic profile of teeth with acute apical abscesses (AAA) compared with teeth with chronic apical periodontitis (CAP) and to correlate the expression of detected human proteins with their main biological functions. MATERIALS AND METHODS: Samples were obtained from root canals of 9 patients diagnosed with AAA and 9 with CAP. Samples were analyzed by reversed-phase liquid chromatography coupled to mass spectrometry. Label-free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in protein expression were calculated using the t-test (p < 0.05). RESULTS: In total, 246 human proteins were identified from all samples. Proteins exclusively found in the AAA group were mainly associated with the immunoinflammatory response and oxidative stress response. In the quantitative analysis, 17 proteins were upregulated (p < 0.05) in the AAA group, including alpha-1-acid glycoprotein, hemopexin, fibrinogen gamma chain, and immunoglobulin. Additionally, 61 proteins were downregulated (p < 0.05), comprising cathepsin G, moesin, gelsolin, and transketolase. Most of the proteins were from the extracellular matrix, cytoplasm, and nucleus. CONCLUSIONS: The common proteins between the groups were mainly associated with the immune response at both expression levels. Upregulated proteins mostly belonged to the acute-phase proteins, while the downregulated proteins were associated with DNA/RNA regulation and repair, and structural function. CLINICAL RELEVANCE: The host response is directly related to the development of apical abscesses. Thus, understanding the behavior of human proteins against the endodontic pathogens involved in this condition might contribute to the study of new approaches related to the treatment of this disease.
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Abscesso , Periodontite Periapical , Humanos , Periodontite Periapical/terapia , ProteômicaRESUMO
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.
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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.
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Leptospira interrogans , Leptospira , Leptospirose , Animais , Humanos , Leptospira interrogans/metabolismo , Proteínas da Membrana Bacteriana Externa/metabolismo , Adesinas Bacterianas , Anticorpos Antibacterianos , Mamíferos/metabolismoRESUMO
Dermatophytosis is the most common human skin infection worldwide caused by dermatophytes, such as Trichophyton interdigitale and Trichophyton rubrum. Itraconazole (ITZ) is one of the main antifungals used to treat these infections. However, especially for onychomycosis, the treatment requires long-term regimens, increasing the possibility of drug resistance. We evaluated the effects of ITZ in the physiology, virulence, and interaction of T. interdigitale with phagocytes and mice cutaneous infection. In a screening test, fungal growth in the presence of ITZ led to the spontaneous selection of less susceptible T. interdigitale and T. rubrum strains. Interestingly, this phenotype was permanent for some T. interdigitale strains. Then, we studied three T. interdigitale strains: one susceptible and two ITZ-adapted. The ITZ-adapted strains were also less susceptible to the cell wall and membrane stressors, suggesting a multidrug resistance (MDR) phenotype associated with the increased ERG11 and MDR3 expression. These strains also presented substantial alterations in ergosterol content, lipid peroxidation, biofilm, and extracellular matrix production. During interaction with macrophages, ITZ-adapted strains were less engulfed but increased the intracellular oxidative and nitrosative bursts. In addition, ITZ-adapted strains presented a reduced ability to grow in a murine model of dermatophytosis, although causing the same tissue damage as the parental strain. In conclusion, the T. interdigitale ITZ adaptation increases tolerance to antifungals and alters the interaction with macrophages and a mammalian host. We hypothesized that successive exposure to ITZ may influence the emergence of adapted strains and lead to the recalcitrance of dermatophytosis.
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Arthrodermataceae , Doenças dos Roedores , Tinha , Humanos , Camundongos , Animais , Itraconazol/farmacologia , Itraconazol/uso terapêutico , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Trichophyton , Tinha/microbiologia , Tinha/veterinária , Virulência , Testes de Sensibilidade Microbiana/veterinária , MamíferosRESUMO
Bacterial persisters represent a small subpopulation that tolerates high antibiotic concentrations without acquiring heritable resistance, and it may be generated by environmental factors. Here, we report the first antibiotic persistence mechanism in Streptococcus pneumoniae, which is induced by oxidative stress conditions and allows the pneumococcus to survive in the presence of fluoroquinolones. We demonstrated that fluoroquinolone persistence is prompted by both the impact of growth arrest and the oxidative stress response induced by H2O2 in bacterial cells. This process protected pneumococci against the deleterious effects of high ROS levels induced by fluoroquinolones. Importantly, S. pneumoniae develops persistence during infection, and is dependent on the oxidative stress status of the host cells, indicating that its transient intracellular life contributes to this mechanism. Furthermore, our findings suggest persistence may influence the outcome of antibiotic therapy and be part of a multistep mechanism in the evolution of fluoroquinolone resistance. IMPORTANCE In S. pneumoniae, different mechanisms that counteract antibiotic effects have been described, such as vancomycin tolerance, heteroresistance to penicillin and fluoroquinolone resistance, which critically affect the therapeutic efficacy. Antibiotic persistence is a type of antibiotic tolerance that allows a bacterial subpopulation to survive lethal antimicrobial concentrations. In this work, we used a host-cell infection model to reveal fluoroquinolone persistence in S. pneumoniae. This mechanism is induced by oxidative stress that the pneumococcus must overcome to survive in host cells. Many fluoroquinolones, such as levofloxacin and moxifloxacin, have a broad spectrum of activity against bacterial pathogens of community-acquired pneumonia, and they are used to treat pneumococcal diseases. However, the emergence of fluoroquinolone-resistant strains complicates antibiotic treatment of invasive infections. Consequently, antibiotic persistence in S. pneumoniae is clinically relevant due to prolonged exposure to fluoroquinolones likely favors the acquisition of mutations that generate antibiotic resistance in persisters. In addition, this work contributes to the knowledge of antibiotic persistence mechanisms in bacteria.
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Fluoroquinolonas , Streptococcus pneumoniae , Streptococcus pneumoniae/genética , Fluoroquinolonas/farmacologia , Peróxido de Hidrogênio/farmacologia , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Levofloxacino/farmacologia , Bactérias , Testes de Sensibilidade MicrobianaRESUMO
Eugenia brejoensis Mazine (Myrtaceae) is source of an essential oil (EbEO) with anti-infective activities against Staphylococcus aureus. This study evaluated the antimicrobial and anti-inflammatory potentials of EbEO in S. aureus-infected skin wounds. The excisional lesions (64 mm2) were induced on Swiss mice back (6 to 8-week-old) that were allocated into 3 groups (n = 12): 1) non-infected wounds (CON); 2) wounds infected with S. aureus ATCC 6538 (Sa); 3) S. aureus-infected wounds and treated with EbEO (Sa + EbEO). The infected groups received approximately 104 CFU/wound. The animals were treated with EbEO (10 µg/wound/day) or vehicle from the 1-day post-infection (dpi) until the 10th dpi. The clinical parameters (wound area, presence of exudate, edema intensity, etc.) were daily analyzed. The levels of inflammatory mediators (cytokines, nitric oxide, VEGF) and bacterial load were measured at the cutaneous tissue at 4th dpi and 10th dpi. Topical application of EbEO accelerated wound contraction with an average contraction of 83.48 ± 11.27 % of the lesion area until 6th dpi. In this period, the rates of lesion contraction were 54.28 ± 5.57% and 34.5 ± 2.67% for CON and Sa groups, respectively. The positive effects of EbEO on wound contraction were associated with significantly (p < 0.05) reduction on bacterial load and the release of inflammatory mediators (IL-6, IL-17A, TNF-α, NO and VEGF). Taken together, these data confirm the antimicrobial potential of EbEO and provide insights into its anti-inflammatory effects, making this essential oil an interesting candidate for the development of new therapeutic alternatives for infected cutaneous wounds.
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AIM: This study aimed to quantitatively and qualitatively determine the proteomic profile of apical periodontitis (AP) in type 2 diabetes mellitus (T2DM) patients in comparison with systemically noncompromised patients and to correlate the protein expression of both groups with their biological functions. METHODOLOGY: The sample consisted of 18 patients with asymptomatic AP divided into two groups according to the presence of T2DM: diabetic group-patients with T2DM (n = 9) and control group-systemically healthy patients (n = 9). After sample collection, the root canal samples were prepared for proteomic analysis using reverse-phase liquid chromatography-mass spectrometry. Label-free quantitative proteomic analysis was performed by Protein Lynx Global Service software. Differences in protein expression between groups were calculated using t-test (p < .05). Biological functions were analysed using the Homo sapiens UniProt database. RESULTS: A total of 727 human proteins were identified in all samples. Among them, 124 proteins common to both groups were quantified, out of which 65 proteins from the diabetic group showed significant differences compared with the control: 43 upregulated (p < .05) and 22 downregulated (p < .05) proteins. No significant differences in protein expression were seen for the remaining 59 proteins (p > .05). Most proteins with differences in expression were related to immune/inflammatory response. Neutrophil gelatinase-associated lipocalin, Plastin-2, Lactotransferrin and 13 isoforms of immunoglobulins were upregulated. In contrast, Protein S100-A8, Protein S100-A9, Histone H2B, Neutrophil defensin 1, Neutrophil defensin 3 and Prolactin-inducible protein were downregulated. CONCLUSIONS: Quantitative differences were demonstrated in the expression of proteins common to diabetic and control groups, mainly related to immune response, oxidative stress, apoptosis and proteolysis. These findings revealed biological pathways that provide the basis to support clinical findings on the relationship between AP and T2DM.
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Diabetes Mellitus Tipo 2 , Periodontite Periapical , Estudos Transversais , Defensinas , Cavidade Pulpar , Diabetes Mellitus Tipo 2/complicações , Humanos , ProteômicaRESUMO
Aspergillus fumigatus is a ubiquitous and saprophytic filamentous fungus and the main etiologic agent of aspergillosis. Infections caused by A. fumigatus culminate in a strong inflammatory response that can evolve into respiratory failure and may be lethal in immunocompromised individuals. In the last decades, it has been demonstrated that extracellular vesicles (EVs) elicit a notable biological response in immune cells. EVs carry a variety of biomolecules, therefore are considered potential antigen delivery vehicles. The role of EVs as a strategy for modulating an effective response against infections caused by A. fumigatus remains unexplored. Here we investigate the use of EVs derived from A. fumigatus as an immunization tool to induce a more robust immune response to A. fumigatus pulmonary infection. In order to investigate that, male C57BL/6 mice were immunized with two doses of EVs and infected with A. fumigatus. Pre-exposure of mice to EVs was able to induce the production of specific IgG serum for fungal antigens. Besides that, the immunization with EVs reduced the neutrophilic infiltrate into the alveoli, as well as the extravasation of total proteins and the production of proinflammatory mediators IL-1ß, IL-6, and CXCL-1. In addition, immunization prevented extensive lung tissue damage and also improved phagocytosis and fungus clearance. Noteworthy, immunization with EVs, associated with subclinical doses of Amphotericin B (AmB) treatment, rescued 50% of mice infected with A. fumigatus from lethal fungal pneumonia. Therefore, the present study shows a new role for A. fumigatus EVs as host inflammatory response modulators, suggesting their use as immunizing agents.
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Aspergilose , Vesículas Extracelulares , Aspergilose Pulmonar , Animais , Aspergillus fumigatus , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
RESUMEN La actual pandemia de COVID-19 fue inducida por la emergencia de un coronavirus en un animal reservorio. De esta manera, es de gran importancia conocer como ocurre la evolución de estos agentes virales en la naturaleza. En este artículo, son presentados los principales mecanismos asociados a la evolución de los coronavirus considerando las especies de animales que actúan como reservorios o huéspedes evolutivos, los mecanismos genéticos virales arrollados en la generación de variantes virales y la contribución de las acciones humanas que puedan generar nuevos coronavirus recombinantes con potencial pandémico. Considerando los puntos discutidos en este artículo, concluimos que la generación de nuevos coronavirus podrá ser evitada con la implementación de políticas públicas que propongan acciones de salud única y así solo habrá salud humana habiendo salud ambiental y salud animal.
ABSTRACT The current COVID-19 pandemic was induced by the emergence of a coronavirus from an animal as a reservoir. Thus, it is of great importance to know how the evolution of these viral agents occurs in the nature. In this article, the main mechanisms associated with the evolution of coronaviruses were presented, indicating the animal species that act as reservoirs or evolutionary hosts, the viral genetic mechanisms involved in the generation of viral variants, the contribution of human actions to generate recombinant coronaviruses with pandemic potential. From the points discussed in the article, we conclude that the generation of new coronaviruses can be avoided with the implementation of public policies that propose health actions and thus there will only be human health if there is environmental health and animal health.
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Candida albicans is one of the major pathogens found in superficial and invasive infections. This fungus expresses several virulence factors and fitness attributes that are essential to the pathogenesis. In our previous study using a murine model of serial systemic candidiasis, virulence of the recovered C. albicans was enhanced and several virulence factors were also modified after five successive passages through mice (P1-P5). In this study, we aimed to correlate the different fungal morphologies, as well as the filamentation, invasion, and stress resistance abilities, of the cells recovered after passing through this model of infection with our previous findings regarding virulence. We obtained two colony morphology types from the recovered cells, differing in their peripheral filamentation. The morphotype 1, which presented zero to five filaments in the colony edge, was higher in P2, while morphotype 2, which presented more than five filaments in the colony edge, was predominant from P3 to P5. In general, morphotype 1 showed similar levels regarding filamentation in serum, invasion of agar and cells, and resistance to osmotic, oxidative, and thermal stress in all passages analyzed. The morphotype 2, however, exhibited an enhancement in these abilities over the passages. We observed an accordance with the increased virulence over the passages obtained in our previous study and the increased adaptability profile of morphotype 2. Therefore, we suggest that the behavior observed previously in the pathogenesis and virulence could be attributed, at least in part, to the greater presence and ability of morphotype 2.
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Candida albicans , Candidíase , Animais , Candidíase/microbiologia , Proteínas Fúngicas , Camundongos , Virulência , Fatores de VirulênciaRESUMO
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.
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BACKGROUND Trypanosoma cruzi shows an exuberant genetic diversity. Currently, seven phylogenetic lineages, called discrete typing units (DTUs), are recognised: TcI-TcVI and Tcbat. Despite advances in studies on T. cruzi and its populations, there is no consensus regarding its heterogeneity. OBJECTIVES This study aimed to perform molecular characterisation of T. cruzi strains, isolated in the state of São Paulo, to identify the DTUs involved and evaluate their genetic diversity. METHODS T. cruzi strains were isolated from biological samples of chronic chagasic patients, marsupials and triatomines through culture techniques and subjected to molecular characterisation using the fluorescent fragment length barcoding (FFLB) technique. Subsequently, the results were correlated with complementary information to enable better discrimination between the identified DTUs. FINDINGS It was possible to identify TcI in two humans and two triatomines; TcII/VI in 19 humans, two marsupials and one triatomine; and TcIII in one human host, an individual that also presented a result for TcI, which indicated the possibility of a mixed infection. Regarding the strains characterised by the TcII/VI profile, the correlation with complementary information allowed to suggest that, in general, these parasite populations indeed correspond to the TcII genotype. MAIN CONCLUSIONS The TcII/VI profile, associated with domestic cycles and patients with chronic Chagas disease, was the most prevalent among the identified DTUs. Furthermore, the correlation of the study results with complementary information made it possible to suggest that TcII is the predominant lineage of this work.
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Hookworm is one type of soil-transmitted helminth, which could exert an anti-inflammatory effect in human or animal host, which provides a beneficial possibility for the discovery of inflammatory-related disease interventions. The identification of hookworm-derived anti-inflammatory molecules is urgently needed for future translational research. The emergence of metabolomics has become a powerful approach to comprehensively characterize metabolic alterations in recent times. Herein, excretory and secretory products (ESPs) were collected from cultured adult worm, while small intestinal contents were obtained from Nippostrongylus brasiliensis (N. brasiliensis, Nb)-infected mice. Through ultra-high-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS) platform, metabolomics analysis was used to explore the identification of anti-inflammatory molecules. Out of 45 differential metabolites that were discovered from ESPs, 10 of them showed potential anti-inflammatory properties, which could be subclassed into amino acids, furanocoumarins, linear diarylheptanoids, gamma butyrolactones, and alpha-keto acids. In terms of intestinal contents that were derived from N. brasiliensis-infected mice, 14 out of 301 differential metabolites were discovered to demonstrate anti-inflammatory effects, with possible subclassification into amino acids, benzylisoquinolines, quaternary ammonium salts, pyrimidines, pregnane steroids, purines, biphenyls, and glycerophosphocholines. Furthermore, nine of the differential metabolites appeared both in ESPs and infected intestinal contents, wherein four were proven to show anti-inflammation properties, namely, L-glutamine, glutamine (Gln), pyruvate, and alanine-Gln (Ala-Gln). In summary, we have provided a method for the identification and analysis of parasite-derived molecules with potential anti-inflammatory properties in the present study. This array of anti-inflammatory metabolites could provide clues for future evaluation and translational study of these anti-inflammatory molecules.
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Metabolômica , Nippostrongylus , Animais , Anti-Inflamatórios , Espectrometria de Massas , Camundongos , ProteômicaRESUMO
Leptospirosis is a widespread zoonosis caused by pathogenic Leptospira spp. It is considered a neglected infectious disease of human and veterinary concern. Our group has been investigating proteins annotated as hypothetical, predicted to be located on the leptospiral surface. Because of their location, these proteins may have the ability to interact with various host components, which could allow establishment of the infection. These proteins act as adherence factors by binding to host receptor molecules, such as the extracellular matrix (ECM) components laminin and glycosaminoglycans to help bacterial colonization. Leptospira also interacts with the host fibrinolytic system, which has been demonstrated to be a powerful tool for invasion mechanisms. The interaction with fibrinogen and thrombin has been shown to reduce fibrin clot formation. Additionally, the degradation of coagulation cascade components by secreted proteases or by acquired surface plasmin could also play a role in reducing clot formation, hence facilitating dissemination during infection. Interaction with host complement system regulators also plays a role in helping bacteria to evade the immune system, facilitating invasion. Interaction of Leptospira to cell receptors, such as cadherins, can contribute to investigate molecules that participate in virulence. To achieve a better understanding of the host-pathogen interaction, leptospiral mutagenesis tools have been developed and explored. This work presents several proteins that mediate binding to components of the ECM, plasma, components of the complement system and cells, to gather research achievements that can be helpful in better understanding the mechanisms of leptospiral-host interactions and discuss genetic manipulation for Leptospira spp. aimed at protein function validation.
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Leptospira interrogans , Leptospira , Leptospirose , Interações Hospedeiro-Patógeno , Humanos , Motivação , Ligação ProteicaRESUMO
Early host-pathogen interactions drive the host response and shape the outcome of natural infections caused by intracellular microorganisms. These interactions involve a number of immune and non-immune cells and tissues, along with an assortment of host and pathogen-derived molecules. Our current knowledge has been predominantly derived from research on the relationships between the pathogens and the invaded host cell(s), limiting our understanding of how microbes elicit and modulate immunological responses at the organismal level. In this study, we explored the early host determinants of healing and non-healing responses in human cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) panamensis. We performed a comparative transcriptomic profiling of peripheral blood mononuclear cells from healthy donors (PBMCs, n=3) exposed to promastigotes isolated from patients with chronic (CHR, n=3) or self-healing (SH, n=3) CL, and compared these to human macrophage responses. Transcriptomes of L. V. panamensis-infected PBMCs showed enrichment of functional gene categories derived from innate as well as adaptive immune cells signatures, demonstrating that Leishmania modulates adaptive immune cell functions as early as after 24h post interaction with PBMCs from previously unexposed healthy individuals. Among differentially expressed PBMC genes, four broad categories were commonly modulated by SH and CHR strains: cell cycle/proliferation/differentiation, metabolism of macromolecules, immune signaling and vesicle trafficking/transport; the first two were predominantly downregulated, and the latter upregulated in SH and CHR as compared to uninfected samples. Type I IFN signaling genes were uniquely up-regulated in PBMCs infected with CHR strains, while genes involved in the immunological synapse were uniquely downregulated in SH infections. Similarly, pro-inflammatory response genes were upregulated in isolated macrophages infected with CHR strains. Our data demonstrate that early responses during Leishmania infection extend beyond innate cell and/or phagocytic host cell functions, opening new frontiers in our understanding of the triggers and drivers of human CL.
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Leishmania guyanensis , Leishmania , Leishmaniose Cutânea , Humanos , Leucócitos , Leucócitos MononuclearesRESUMO
This work evaluated the immunomodulatory and anti-infective effects of Cratylia mollis lectin (Cramoll) in a model of wound infection induced by S. aureus. Swiss mice were divided into 3 groups (n = 12/group): non-inoculated (Control group); inoculated with S. aureus (Sa group); inoculated with S. aureus and treated with Cramoll (Sa + Cramoll group). In each animal, one lesion (64 mm2) was induced on the back and contaminated with S. aureus (~4.0 × 106 CFU/wound). The treatment with Cramoll (5 µg/animal/day) started 1-day post-infection (dpi) and extended for 10 days. Clinical parameters (wound size, inflammatory aspects, etc.) were daily recorded; while cytokines levels, bacterial load and histological aspects were determined in the cutaneous tissue at 4th dpi or 11th dpi. The mice infected with S. aureus exhibited a delay in wound contraction and the highest inflammatory scores. These effects were impaired by the treatment with Cramoll which reduced the release of key inflammatory mediators (TNF-α, NO, VEGF) and the bacterial load at wound tissue. Histological evaluations showed a restauration of skin structures in the animals treated with Cramoll. Taken together, these results provide more insights about the healing and immunomodulatory properties of Cramoll and suggest this lectin as a lead compound for treatment of wound infection.