RESUMO

Pathogenic species of Leptospira are etiologic agents of leptospirosis, an emerging zoonotic disease of worldwide extent and endemic in tropical regions. The growing number of identified leptospiral species sheds light to their genetic diversity and unique virulence mechanisms, many of them still remain unknown. Toxins and adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines with cross-protection and long-lasting effect against leptospirosis. For this aim, we used the shotgun phage display technique to unravel new proteins with adhesive properties. A shotgun library was constructed using fragmented genomic DNA from Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 and pG8SAET phagemid vector. Selection of phages bearing new possible cell-binding antigens was performed against VERO cells, using BRASIL biopanning methodology. Analysis of selected clones revealed the hypothetical protein LIC10778, a potentially exposed virulence factor that belongs to the virulence-modifying (VM) protein family (PF07598), composed of 13 members in the leptospiral strain Fiocruz L1-130. Prediction of LIC10778 tertiary structure indicates that the protein contains a cellular-binding domain (N-terminal portion) and an unknown domain of no assigned activity (C-terminal portion). The predicted N-terminal domain shared structural similarities with the cell-binding and internalization domain of toxins like Ricin and Abrin, as well as to the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin in Mycoplasma pneumoniae. Interestingly, recombinant portions of the N-terminal region of LIC10778 protein showed binding to laminin, collagens I and IV, vitronectin, and plasma and cell fibronectins using overlay blotting technique, especially regarding the binding site identified by phage display. These data validate our preliminary phage display biopanning and support the predicted three-dimensional models of LIC10778 protein and other members of PF07598 protein family, confirming the identification of the N-terminal cell-binding domains that are similar to ricin-like toxins. Moreover, fluorescent fused proteins also confirmed that N-terminal region of LIC10778 is capable of binding to VERO and A549 cell lines, further highlighting its virulence role during host-pathogen interaction in leptospirosis probably mediated by its C-terminal domain. Indeed, recent results in the literature confirmed this assumption by demonstrating the cytotoxicity of a closely related PF07598 member.

RESUMO

Pathogenic species of Leptospira are etiologic agents of leptospirosis, an emerging zoonotic disease of worldwide extent and endemic in tropical regions. The growing number of identified leptospiral species sheds light to their genetic diversity and unique virulence mechanisms, many of them still remain unknown. Toxins and adhesins are important virulence factors in several pathogens, constituting promising antigens for the development of vaccines with cross-protection and long-lasting effect against leptospirosis. For this aim, we used the shotgun phage display technique to unravel new proteins with adhesive properties. A shotgun library was constructed using fragmented genomic DNA from Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 and pG8SAET phagemid vector. Selection of phages bearing new possible cell-binding antigens was performed against VERO cells, using BRASIL biopanning methodology. Analysis of selected clones revealed the hypothetical protein LIC10778, a potentially exposed virulence factor that belongs to the virulence-modifying (VM) protein family (PF07598), composed of 13 members in the leptospiral strain Fiocruz L1-130. Prediction of LIC10778 tertiary structure indicates that the protein contains a cellular-binding domain (N-terminal portion) and an unknown domain of no assigned activity (C-terminal portion). The predicted N-terminal domain shared structural similarities with the cell-binding and internalization domain of toxins like Ricin and Abrin, as well as to the Community-Acquired Respiratory Distress Syndrome (CARDS) toxin in Mycoplasma pneumoniae. Interestingly, recombinant portions of the N-terminal region of LIC10778 protein showed binding to laminin, collagens I and IV, vitronectin, and plasma and cell fibronectins using overlay blotting technique, especially regarding the binding site identified by phage display. These data validate our preliminary phage display biopanning and support the predicted three-dimensional models of LIC10778 protein and other members of PF07598 protein family, confirming the identification of the N-terminal cell-binding domains that are similar to ricin-like toxins. Moreover, fluorescent fused proteins also confirmed that N-terminal region of LIC10778 is capable of binding to VERO and A549 cell lines, further highlighting its virulence role during host-pathogen interaction in leptospirosis probably mediated by its C-terminal domain. Indeed, recent results in the literature confirmed this assumption by demonstrating the cytotoxicity of a closely related PF07598 member.

RESUMO

SARS-CoV-2 Nucleocapsid (N) is the most abundant viral protein expressed in host samples and is an important antigen for diagnosis. N is a 45 kDa protein that does not present disulfide bonds. Intending to avoid non-specific binding of SARS-CoV-2 N to antibodies from patients who previously had different coronaviruses, a 35 kDa fragment of N was expressed without a conserved motif in E. coli as inclusion bodies (N122-419-IB). Culture media and IB washing conditions were chosen to obtain N122-419-IB with high yield (370 mg/L bacterial culture) and protein purity (90%). High pressure solubilizes protein aggregates by weakening hydrophobic and ionic interactions and alkaline pH promotes solubilization by electrostatic repulsion. The association of pH 9.0 and 2.4 kbar promoted efficient solubilization of N122-419-IB without loss of native-like tertiary structure that N presents in IB. N122-419 was refolded with a yield of 85% (326 mg/L culture) and 95% purity. The refolding process takes only 2 hours and the protein is ready for use after pH adjustment, avoiding the necessity of dialysis or purification. Antibody binding of COVID-19-positive patients sera to N122-419 was confirmed by Western blotting. ELISA using N122-419 is effective in distinguishing between sera presenting antibodies against SARS-CoV-2 from those who do not. To the best of our knowledge, the proposed condition for IB solubilization is one of the mildest described. It is possible that the refolding process can be extended to a wide range of proteins with high yields and purity, even those that are sensible to very alkaline pH.

RESUMO

BACKGROUND: Leptospirosis is a zoonotic disease caused by infection with spirochetes from Leptospira genus. It has been classified into at least 17 pathogenic species, with more than 250 serologic variants. This wide distribution may be a result of leptospiral ability to colonize the renal tubules of mammalian hosts, including humans, wildlife, and many domesticated animals. Previous studies showed that the expression of proteins belonging to the microbial heat shock protein (HSP) family is upregulated during infection and also during various stress stimuli. Several proteins of this family are known to have important roles in the infectious processes in other bacteria, but the role of HSPs in Leptospira spp. is poorly understood. In this study, we have evaluated the capacity of the protein GroEL, a member of HSP family, of interacting with host proteins and of stimulating the production of cytokines by macrophages. RESULTS: The binding experiments demonstrated that the recombinant GroEL protein showed interaction with several host components in a dose-dependent manner. It was also observed that GroEL is a surface protein, and it is secreted extracellularly. Moreover, two cytokines (tumor necrosis factor-α and interleukin-6) were produced when macrophages cells were stimulated with this protein. CONCLUSIONS: Our findings showed that GroEL protein may contribute to the adhesion of leptospires to host tissues and stimulate the production of proinflammatory cytokines during infection. These features might indicate an important role of GroEL in the pathogen-host interaction in the leptospirosis.

Assuntos

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Introduction: Brazilian borreliosis (BB) disease is an infectious disease transmitted by ticks that mimics Lyme disease (LD) from the Northern Hemisphere. The BB clinical picture is characterized by a pathognomonic skin lesion (migratory erythema) and joint, neurological, cardiac and psychiatric symptoms. Innate and Th1/Th17 adaptive immunity seem to play an important role in the pathogenesis of Lyme disease. Objective: The aim of this study was to characterize the role of innate and Th1/Th17 adaptive immunity in BB patients with acute (<3 months) and convalescent (>3 months) disease. Methods: Fifty BB patients (28 with acute and 22 with convalescent disease) without treatment and 30 healthy subjects were evaluated. Levels of 20 cytokines or chemokines associated with innate and Th1/Th17 adaptive immunity were analyzed using Luminex (Millipore Corp., Billerica, MA). Results: Overall, BB patients had increased levels of IL-8 (6.29 vs 2.12 p = 0.002) and MIP-1α/CCL3 (5.20 vs 2.06, p = 0.030), associated with innate immunity, and MIP3B/CCL19 (Th1; 297.86 vs 212.41, p = 0.031) and IL-17A (Th17; 3.11 vs 2.20, p = 0.037), associated with adaptive immunity, compared with the levels of healthy controls. When comparing acute BB vs. convalescent BB subjects vs. healthy controls, IL-1β, IL-8 and MIP-1α/CCL3 (innate mediators) levels were highest in patients in the acute phase of disease (p < 0.05). TNF-α was associated with disseminated symptoms and with humoral reactivity against Borrelia burgdorferi. IL-10 was significantly correlated with IL-6 (r = 0.59, p = 0.003), IL-8 (r = 0.51, p < 0.001), MIP-1α/CCL3 (r = 0.42, p < 0.001) and MIP-3β/CCL19 (r = 0.40, p = 0.002) in all BB patients. Conclusions: This is the first study describing that innate and Th1/Th17 adaptive immunity play a crucial role in BB disease. Furthermore, innate mediators are particularly important in acute BB disease, and TNF-α is associated with evolution of BB symptoms.

RESUMO

Background: Leptospirosis is a zoonotic disease caused by infection with spirochetes from Leptospira genus. It has been classified into at least 17 pathogenic species, with more than 250 serologic variants. This wide distribution may be a result of leptospiral ability to colonize the renal tubules of mammalian hosts, including humans, wildlife, and many domesticated animals. Previous studies showed that the expression of proteins belonging to the microbial heat shock protein (HSP) family is upregulated during infection and also during various stress stimuli. Several proteins of this family are known to have important roles in the infectious processes in other bacteria, but the role of HSPs in Leptospira spp. is poorly understood. In this study, we have evaluated the capacity of the protein GroEL, a member of HSP family, of interacting with host proteins and of stimulating the production of cytokines by macrophages. Results: The binding experiments demonstrated that the recombinant GroEL protein showed interaction with several host components in a dose-dependent manner. It was also observed that GroEL is a surface protein, and it is secreted extracellularly. Moreover, two cytokines (tumor necrosis factor-α and interleukin-6) were produced when macrophages cells were stimulated with this protein. Conclusions: Our findings showed that GroEL protein may contribute to the adhesion of leptospires to host tissues and stimulate the production of proinflammatory cytokines during infection. These features might indicate an important role of GroEL in the pathogen-host interaction in the leptospirosis.

RESUMO

In March 2013 it was reported by the World Health Organization (WHO) the first cases of human infections with avian influenza virus A (H7N9). From 2013 to December 2019, 1568 cases have been reported with 616 deaths. H7N9 infection has been associated with high morbidity and mortality rates, and vaccination is currently the most effective way to prevent infections and consequently flu-related severe illness. Developing and producing vaccines against pandemic influenza viruses is the main strategy for a response to a possible pandemic. This study aims to present the production of three industrial lots under current Good Manufacturing Practices (cGMP) of the active antigen used to produce the pandemic influenza vaccine candidate against A(H7N9). These batches were characterized and evaluated for quality standards and tested for immunogenicity in mice. The average yield was 173.50 ± 7.88 μg/mL of hemagglutinin and all the preparations met all the required specifications. The formulated H7N9 vaccine is poorly immunogenic and needs to be adjuvanted with an oil in water emulsion adjuvant (IB160) to achieve a best immune response, in a prime and in a boost scheme. These data are important for initial production planning and preparedness in the case of a H7N9 pandemic.

RESUMO

Leptospirosis is an acute infection caused by pathogenic species of the genus Leptospira, which affects humans and animals in all world. In severe forms of the disease, kidneys, liver and lungs are the main affected organs, resulting in acute kidney injury, jaundice and pulmonary hemorrhage. Previous post-mortem studies have shown that lesions are not limited to these organs. Cardiac and striated muscle injuries have already been reported, but the pathophysiology of cardiac and skeletal lesions in leptospirosis is not fully understood. It has been suggested that the tissue damage observed in leptospirosis could be directly mediated by leptospires or by their toxic cellular components. LipL32 and Lp25 are leptospira membrane proteins with unknown functions, that are present only in pathogenic strains of Leptospira spp. Both proteins induce skeletal muscle lesions similar to those observed when normal guinea pigs are inoculated with leptospires. Through immunohistochemistry, this study showed the presence of LipL32 and Lp25 proteins on muscle cell membranes and in the underlying cytoplasm of skeletal muscles, as well as focal lesions in cardiac tissues of fatal cases of leptospirosis. Altogether, these results reinforce that both proteins can be important factors in the pathogenesis of leptospirosis.

Assuntos

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

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Enteropathogenic Escherichia coli (EPEC) are important agents of acute diarrhea in children living in developing countries. A severe dysfunction of the intestinal epithelial barrier occurs during EPEC infection, leading to diarrhea and inflammation as consequences. EPEC main virulence factors include the adhesins intimin and bundle-forming pilus (BFP), as well as several effector proteins translocated to the enterocyte by the type-three secretion system. The initial interaction of EPEC with the host cell and the role of effector proteins in this process are well known. However, the role of the EPEC virulence factors in macrophage activation is not fully understood. Hence, we analyzed the ability of intimin and bundle-forming pilus (BfpA) to activate the innate response mediated by macrophages, where the production of the proinflammatory cytokines TNF-α, IL-1, IL-6 and IL-12, as well as the anti-inflammatory cytokine IL-10 and chemokine MCP-1, were evaluated. Our results showed that recombinant intimin and BfpA activate macrophages in a dose-dependent manner, and the stimulated cells produced TNF-α, IL-12, IL-6, IL-10 and MCP-1, but not IL-1β. No synergistic effect was observed in the production of pro-inflammatory cytokines by combining BfpA and intimin, although production of IL-10, an anti-inflammatory mediator, was potentiated at a higher dose. The effect observed was largely attributed to these proteins, as the treatment of proteins with polymyxin B did not alter the production of TNF-α. Thus, herein we showed that intimin and BfpA can activate the innate immune response, inducing the production of pro- and anti-inflammatory cytokines, as well as chemokines, playing additional role as inflammatory molecules in the early steps of EPEC infection.