Evidence for glycosylation on a DNA-binding protein of Salmonella enterica

BACKGROUND: All organisms living under aerobic atmosphere have powerful mechanisms that confer their macromolecules protection against oxygen reactive species. Microorganisms have developed biomolecule-protecting systems in response to starvation and/or oxidative stress, such as DNA biocrystallization with Dps (DNA-binding protein from starved cells). Dps is a protein that is produced in large amounts when the bacterial cell faces harm, which results in DNA protection. In this work, we evaluated the glycosylation in the Dps extracted from Salmonella enterica serovar Typhimurium. This Dps was purified from the crude extract as an 18-kDa protein, by means of affinity chromatography on an immobilized jacalin column. RESULTS: The N-terminal sequencing of the jacalin-bound protein revealed 100% identity with the Dps of S. enterica serovar Typhimurium. Methyl-alpha-galactopyranoside inhibited the binding of Dps to jacalin in an enzyme-linked lectin assay, suggesting that the carbohydrate recognition domain (CRD) of jacalin is involved in the interaction with Dps. Furthermore, monosaccharide compositional analysis showed that Dps contained mannose, glucose, and an unknown sugar residue. Finally, jacalin-binding Dps was detected in larger amounts during the bacterial earlier growth periods, whereas high detection of total Dps was verified throughout the bacterial growth period. CONCLUSION: Taken together, these results indicate that Dps undergoes post-translational modifications in the pre- and early stationary phases of bacterial growth. There is also evidence that a small mannose-containing oligosaccharide is linked to this bacterial protein

El receptor de aerobactina IutA, una proteína aislada en columna de agarosa, no es esencial para la infección por Escherichia coli uropatógena

Apenas alguns relatos na literatura demonstram que lectinas são importantes nos processos de colonização e infecção por Escherichia coli. A falta de compreensão clara dos mecanismos envolvendo lectinas, no processo de colonização por E. coli, motivou a realização deste estudo para se identificar a presença de outras lectinas não descritas em E. coli. Neste trabalho, isolou-se uma proteína de 75kDa de E. coli em coluna de Sepharose, correspondente ao receptor de aerobactina férrica (IutA). A associação de IutA com virulência de cepas de E. coli é controversa, principalmente em E. coli uropatogênica (UPEC), o que levou a se avaliar a presença do gene iutA em UPECs isoladas de pacientes com infecção urinária. O gene \ud estava presente em 38% dos isolados, sugerindo fraca associação com virulência. Devido à existência de redundância nos sistemas de captura de ferro, sugere-se, aqui, que IutA possa ser vantajosa, mas não essencial para UPEC.Although many proteins have been described involved in Escherichia coli colonization and infection, only few reports have shown lectins as important components in these processes. Because the mechanisms underlying E. coli colonization process involving lectins are not fully understood, we sought to identify the presence of other non-described lectins in E. coli. Here, we isolated a 75-kDa protein from E. coli on Sepharose column and identified it as ferric aerobactin receptor (IutA). Since IutA is controversially associated with virulence of some E. coli strains, mainly in uropathogenic E. coli (UPEC), we evaluated the presence of iutA gene in UPEC isolated from patients with urinary infection. This gene was present in only 38% of the isolates, suggesting a weak association with virulence. Because there is a redundancy in the siderophore-mediated uptake systems, we suggest that IutA can be advantageous but not essential for UPEC.La falta de una clara comprensión de los mecanismos de participación de las lectinas en el proceso de colonización por Escherichia coli, nos motivó a identificar la presencia de otras lectinas que no han sido descritas en Escherichia coli. En este estudio, se aisló una proteína de 75kDa de Escherichia coli en una columna de Sepharosa, correspondiente al receptor de aerobactina (IutA). La asociación de IutA con cepas virulentas de Escherichia coli es controvertido, especialmente en Escherichia coli uropatógena (UPEC), lo que nos llevó a evaluar la presencia del gen iutA en UPECs aisladas de pacientes con infección urinaria. El gen estaba presente en 38% de los aislamientos, lo que sugiere una débil asociación con la virulencia. Debido a la existencia de redundancia en los sistemas de captura de hierro, se sugiere que IutA puede ser una ventaja, sin embargo no es esencial para la UPEC.FAPESP [06/04482-6

Expression of Hsp60 and its cell location in Paracoccidioides brasiliensis

Paracoccidioides species cause paracoccidioidomycosis (PCM), a systemic mycosis highly prevalent in Brazil. Therapy of PCM has some issues that make studies for new therapeutic and vaccine targets relevant, such as the P. brasiliensis 60-kDa-heat-shock protein (PbHsp60), an immunogenic antigen that induces protection in experimental mice infection. Here, we investigated the relative expression of mRNA for PbHsp60 in P. brasiliensis in the different morphotypes of P. brasiliensis and in morphological transition phases. In addition, antibodies to rPbHsp60 were produced and used to analyze the location of PbHsp60 in yeast and hyphae by electron microscopy. The analyses showed a substantial increase in the relative amounts of HSP60 mRNA in yeast when compared to mycelium and an intermediate expression in transitional forms. Regarding the cell location, immunoelectron microscopy analysis revealed that PbHsp60 is within the cell wall. These observations suggest that this protein may be involved in the maintenance of the cell wall integrity and the interaction with the host for colonization, infection and pathogenesis

T Helper 1–Inducing Adjuvant Protects against Experimental Paracoccidioidomycosis

Immunostimulatory therapy is a promising approach to improving the treatment of systemic fungal infections such as paracoccidioidomycosis (PCM), whose drug therapy is usually prolonged and associated with toxic side effects and relapses. The current study was undertaken to determine if the injection of a T helper (Th) 1–stimulating adjuvant in P. brasiliensis–infected mice could have a beneficial effect on the course of experimental PCM. For this purpose, mice were infected and treated with complete Freund's adjuvant (CFA), a well-established Th1 experimental inductor, or incomplete Freund's adjuvant (IFA - control group) on day 20 postinfection. Four weeks after treatment, the CFA-treated mice presented a mild infection in the lungs characterized by absence of epithelioid cell granulomas and yeast cells, whereas the control mice presented multiple sites of focal epithelioid granulomas with lymphomonocytic halos circumscribing a high number of viable and nonviable yeast cells. In addition, CFA administration induced a 2.4 log reduction (>99%) in the fungal burden when compared to the control group, and led to an improvement of immune response, reversing the immunosuppression observed in the control group. The immunotherapy with Th1-inducing adjuvant, approved to be used in humans, might be a valuable tool in the treatment of PCM and potentially useful to improve the clinical cure rate in humans

Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice

Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection

Involvement of Dihydrolipoyl Dehydrogenase in the Phagocytosis and Killing of Paracoccidioides brasiliensis by Macrophages

Paracoccidioides brasiliensis and Paracoccidioides lutzii are fungi causing paracoccidioidomycosis (PCM), an autochthonous systemic mycosis found in Latin America. These microorganisms contain a multitude of molecules that may be associated with the complex interaction of the fungus with the host. Here, we identify the enzyme dihydrolipoyl dehydrogenase (DLD) as an exoantigen from P. brasiliensis (Pb18_Dld) by mass spectrometry. Interestingly, the DLD gene expression in yeast form showed higher expression levels than those in mycelial form and transitional phases. Pb18_Dld gene was cloned, and the recombinant protein (rPb18_Dld) was expressed and purified for subsequent studies and production of antibodies. Immunogold labeling and transmission electron microscopy revealed that the Pb18_Dld is also localized in mitochondria and cytoplasm of P. brasiliensis. Moreover, when macrophages were stimulated with rPb18Dld, there was an increase in the phagocytic and microbicidal activity of these cells, as compared with non-stimulated cells. These findings suggest that Pb18_Dld can be involved in the pathogen-host interaction, opening possibilities for studies of this protein in PCM

Low-dose plasmid DNA treatment increases plasma vasopressin and regulates blood pressure in experimental endotoxemia

Abstract\ud \ud Background\ud Although plasmid DNA encoding an antigen from pathogens or tumor cells has been widely studied as vaccine, the use of plasmid vector (without insert) as therapeutic agent requires further investigation.\ud \ud \ud Results\ud Here, we showed that plasmid DNA (pcDNA3) at low doses inhibits the production of IL-6 and TNF-α by lipopolysaccharide (LPS)-stimulated macrophage cell line J774. These findings led us to evaluate whether plasmid DNA could act as an anti-inflammatory agent in a Wistar rat endotoxemia model. Rats injected simultaneously with 1.5 mg/kg of LPS and 10 or 20 μg of plasmid DNA had a remarkable attenuation of mean arterial blood pressure (MAP) drop at 2 hours after treatment when compared with rats injected with LPS only. The beneficial effect of the plasmid DNA on MAP was associated with decreased expression of IL-6 in liver and increased concentration of plasma vasopressin (AVP), a known vasoconstrictor that has been investigated in hemorrhagic shock management. No difference was observed in relation to nitric oxide (NO) production.\ud \ud \ud Conclusion\ud Our results demonstrate for the first time that plasmid DNA vector at low doses presents anti-inflammatory property and constitutes a novel approach with therapeutic potential in inflammatory diseases

Protection against Paracoccidioides brasiliensis infection conferred by the prophylactic administration of native and recombinant ArtinM

We determined the prophylactic effect of both the d-mannose-binding lectin ArtinM extracted from the seeds of Artocarpus integrifolia (jackfruit) and its recombinant counterpart during the course of experimental paracoccidioidomycosis induced in BALB/c mice. Four experimental protocols of prophylaxis were employed to evaluate the most protective regimen of ArtinM administration. It was demonstrated that the best effect was obtained by administration of two ArtinM doses on days 10 and 3 before the challenge with Paracoccidioides brasiliensis. By following this protocol, the lungs of mice that received native or recombinant ArtinM exhibited reduced fungal burden and granuloma incidence. In addition, the protocol augmented contents of IL-12, IFN-gamma, TNF-alpha and NO. On the other hand, the control group consisting of untreated infected mice had higher pulmonary levels of IL-4 and IL-10. In conclusion, prophylaxis with ArtinM significantly reproduces the effect of its therapeutic administration, i.e, it confers resistance to P. brasiliensis infection in mouse models by promoting IL-12 production and favours Th1-immunity.FAPESP[00/09333-2]FAPESP[06/60642-2]FAPESP[02/12725-5]FAPESP[05/00303-7]CNPq[350418/00-4]CAPE