Potent hydroxamate-derived compounds arrest endodyogeny of Toxoplasma gondii tachyzoites.

Toxoplasmosis is a zoonosis that is a worldwide health problem, commonly affecting fetal development and immunodeficient patients. Treatment is carried out with a combination of pyrimethamine and sulfadiazine, which can cause cytopenia and intolerance and does not lead to a parasitological cure of the infection. Lysine deacetylases (KDACs), which remove an acetyl group from lysine residues in histone and non-histone proteins are found in the Toxoplasma gondii genome. Previous work showed the hydroxamate-type KDAC inhibitors Tubastatin A (TST) and Vorinostat (Suberoylanilide Hydroxamic Acid, SAHA) were effective against T. gondii. In the present study, the effects of three hydroxamates (KV-24, KV-30, KV-46), which were originally designed to inhibit human KDAC6, showed different effects against T. gondii. These compounds contain a heterocyclic cap group and a benzyl linker bearing the hydroxamic acid group in para-position. All compounds showed selective activity against T. gondii proliferation, inhibiting tachyzoite proliferation with IC50 values in a nanomolar range after 48h treatment. Microscopy analyses showed that after treatment, tachyzoites presented mislocalization of the apicoplast, disorganization of the inner membrane complex, and arrest in the completion of new daughter cells. The number of dividing cells with incomplete endodyogeny increased significantly after treatment, indicating the compounds can interfere in the late steps of cell division. The results obtained in this work that these new hydroxamates should be considered for future in vivo tests and the development of new compounds for treating toxoplasmosis.

Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis.

BACKGROUND: Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE: The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS: B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS: TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS: Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.

Anthelmintic effect of herbicidal dinitroanilines on the nematode model Caenorhabditis elegans.

Dinitroanilines are known herbicides that impair the polymerization of microtubules. This study investigated the effects of oryzalin and trifluralin on the viability, morphology, and ultrastructure of different life stages of Caenorhabditis elegans. Both drugs reduced the survival of the adult population in 50% after three days of treatment with concentrations of approximately 30 µM and 57 µM, respectively. The development of new adults was monitored for seven days and treatment with both drugs also showed a decrease in the adult population. 25 µM Oryzalin or trifluralin inhibited the hatching of eggs by nearly 100%. Both drugs showed remarkable larvicidal activity at 25 µM against the larvae at first and second stages (L1-L2) and at third and fourth stages (L3-L4) after 24 h. Treatment with dinitroanilines led to incomplete egg embryo development. The oryzalin and trifluralin treatments caused the detachment of cuticular layers of adults and larvae and the formation of a large number of intracellular membrane whirls and lipid bodies in the hypodermal cells and non-contractile muscles of adults. Both drugs also provoked the bagging process, which generated lesions in the uterus of the adults. In addition, trifluralin caused the detachment of certain areas of the cuticle adjacent to the hypodermis in a large number of nematodes. Our results suggest that dinitroanilines are a potentially new alternative for anthelmintic chemotherapy.

Caenorhabditis elegans as a model for the screening of anthelminthic compounds: ultrastructural study of the effects of albendazole.

This study investigated the effects of albendazole on the viability, morphology and ultrastructure of different life stages of Caenorhabditis elegans. The albendazole EC50 value after seven days of treatment was 18.43 µM. This concentration was very efficient against all the stages. Light and electron microscopy analysis showed damage to the body wall of the adults and larvae. An intense desquamation of the cuticle of larvae and of the surface of the eggs was observed, preventing their hatching and development. The main ultrastructural damage detected was the degeneration of the mitochondria in the noncontractile muscle of the body wall, which appeared as large vacuoles. This study reaffirmed the use of C. elegans as a screening system for compounds with potential anthelmintic activity and showed the effects of albendazole on the different life stages of these worms.

Toxoplasma gondii: the effect of fluconazole combined with sulfadiazine and pyrimethamine against acute toxoplasmosis in murine model.

Toxoplasma gondii is an important opportunistic pathogen for immunocompromised patients and responsible for toxoplasmic encephalitis, which is often lethal. Treatment for this infection is limited to a restricted therapeutic arsenal. In this work we tested the combination of fluconazole with the current treatment for acute toxoplasmosis on the murine model in vivo. Different experimental groups were treated with combinations of sulfadiazine plus pyrimethamine with fluconazole and pyrimethamine with fluconazole. Fluconazole is an important antifungal triazole used against others CNS related opportunistic pathogens such as Cryptococcus neoformans and Candida spp. The combinations of fluconazole plus sulfadiazine and pyrimethamine or fluconazole plus pyrimethamine were remarkably effective against T. gondii in vivo. The 10-day treatment with 10mg/kg/day of fluconazole combined with 40/1mg/kg/day sulfadiazine and pyrimethamine resulted in 93% survival of CF1 mice acutely infected with the highly virulent T. gondii RH strain, versus 36% of mice treated with just sulfadiazine and pyrimethamine. Combinations of fluconazole with lower doses of sulfadiazine and pyrimethamine or with just pyrimethamine were also efficient in reducing the mortality of mice compared with the treatment without fluconazole. The results obtained are promising for the treatment of human toxoplasmosis and point to the need to extend these studies to other murine models.

The organization of the wall filaments and characterization of the matrix structures of Toxoplasma gondii cyst form.

The encystation process is a key step in Toxoplasma gondii life cycle, allowing the parasite to escape from the host immune system and the transmission among the hosts. A detailed characterization of the formation and structure of the cyst stage is essential for a better knowledge of toxoplasmosis. Here we isolated cysts from mice brains and analysed the cyst wall structure and cyst matrix organization using different electron microscopy techniques. Images obtained showed that the cyst wall presented a filamentous aspect, with circular openings on its surface. The filaments were organized in two layers: a compact one, facing the exterior of the whole cyst and a more loosen one, facing the matrix. Within the cyst wall, we observed tubules and a large number of vesicles. The cyst matrix presented vesicles of different sizes and tubules, which were organized in a network connecting the bradyzoites to each other and to the cyst wall. Large vesicles, with a granular material in their lumen of glycidic nature were observed. Similar vesicles were also found associated with the posterior pole of the bradyzoites and in proximity to the cyst wall.

HDAC inhibitors Tubastatin A and SAHA affect parasite cell division and are potential anti-Toxoplasma gondii chemotherapeutics.

The redirectioning of drugs in the pharmaceutical market is a well-known practice to identify new therapies for parasitic diseases. The histone deacetylase inhibitors Tubastatin A (TST) and Suberoylanilide Hydroxamic Acid (SAHA), firstly developed for cancer treatment, are effective against protozoa parasites. In this work, we aimed to demonstrate the activity of these drugs as potential agents against Toxoplasma gondii, the causative agent of toxoplasmosis. TST and SAHA were active against different genotypes of Toxoplasma gondii, such as, RH (type I), EGS (I/III) and ME49 (type II) strains. The IC50 values for the RH strain were 19 ± 1 nM and 520 ± 386 nM for TST and 41 ± 3 nM and 67 ± 36 nM for SAHA, for 24 and 48 h, respectively. Both compounds were highly selective for T. gondii and their anti-proliferative effect was irreversible for 8 days. The calculated selectivity indexes (39 for TST and 30 for SAHA) make them lead compounds for the future development of anti-Toxoplasma molecules. Western blotting showed TST led to a significant increase of the nuclear histone H4 and a decrease of H3 acetylation levels. Treatment with 1 µM TST and 0.1 µM SAHA for 48 h decreased the amount of global α-tubulin. Fluorescence and electron microscopy showed that both drugs affected the endodyogeny process impairing the budding of daughter cells. The drugs led to the formation of large, rounded masses of damaged parasites with several centrosomes randomly dispersed and incorrect apicoplast division and positioning. TST-treated parasites showed a rupture of the mitochondrial membrane potential and led to a failure of the IMC assembling of new daughter cells. SAHA and TST possibly inhibit HDAC3 and other cytoplasmic or organelle targeted HDACs involved in the modification of proteins other than histones.

Dichloroacetate and Pyruvate Metabolism: Pyruvate Dehydrogenase Kinases as Targets Worth Investigating for Effective Therapy of Toxoplasmosis.

Toxoplasmosis, a protozoan infection caused by Toxoplasma gondii, is estimated to affect around 2.5 billion people worldwide. Nevertheless, the side effects of drugs combined with the long period of therapy usually result in discontinuation of the treatment. New therapies should be developed by exploring peculiarities of the parasite's metabolic pathways, similarly to what has been well described in cancer cell metabolism. An example is the switch in the metabolism of cancer that blocks the conversion of pyruvate into acetyl coenzyme A in mitochondria. In this context, dichloroacetate (DCA) is an anticancer drug that reverts the tumor proliferation by inhibiting the enzymes responsible for this switch: the pyruvate dehydrogenase kinases (PDKs). DCA has also been used in the treatment of certain symptoms of malaria; however, there is no evidence of how this drug affects apicomplexan species. In this paper, we studied the metabolism of T. gondii and demonstrate that DCA also inhibits T. gondii's in vitro infection with no toxic effects on host cells. DCA caused an increase in the activity of pyruvate dehydrogenase followed by an unbalanced mitochondrial activity. We also observed morphological alterations frequently in mitochondria and in a few apicoplasts, essential organelles for parasite survival. To date, the kinases that potentially regulate the activity of pyruvate metabolism in both organelles have never been described. Here, we confirmed the presence in the genome of two putative kinases (T. gondii PDK [TgPDK] and T. gondii branched-chain α-keto acid dehydrogenase kinase [TgBCKDK]), verified their cellular localization in the mitochondrion, and provided in silico data suggesting that they are potential targets of DCA.IMPORTANCE Currently, the drugs used for toxoplasmosis have severe toxicity to human cells, and the treatment still lacks effective and safer alternatives. The search for novel drug targets is timely. We report here that the treatment of T. gondii with an anticancer drug, dichloroacetate (DCA), was effective in decreasing in vitro infection without toxicity to human cells. It is known that PDK is the main target of DCA in mammals, and this inactivation increases the conversion of pyruvate into acetyl coenzyme A and reverts the proliferation of tumor cells. Moreover, we verified the mitochondrial localization of two kinases that possibly regulate the activity of pyruvate metabolism in T. gondii, which has never been studied. DCA increased pyruvate dehydrogenase (PDH) activity in T. gondii, followed by an unbalanced mitochondrial activity, in a manner similar to what was previously observed in cancer cells. Thus, we propose the conserved kinases as potential regulators of pyruvate metabolism and interesting targets for new therapies.

Toxoplasma gondii: fluconazole and itraconazole activity against toxoplasmosis in a murine model.

Toxoplasma gondii is an important opportunistic pathogen affecting immunocompromised patients with AIDS. Toxoplasmic encephalitis is responsible for high morbidity and mortality. In this study, we investigated the activity of the antifungals fluconazole (FLZ) and itraconazole (ITZ) against T. gondii in mice infected with the Me49 strain. As previously reported for ITZ, FLZ also demonstrated a selective effect against T. gondii in vitro; the IC(50) values obtained for FLZ were 8.9 microM and 3.1 microM after 24h and 48 h of treatment, respectively. A 10-day treatment of mice with orally or intraperitoneally administered 20mg/kg/day FLZ showed a significant survival difference compared to untreated mice. The administration of 20mg/kg/day ITZ significantly reduced the brain cyst burden compared to untreated mice but did not exert significant protection against death. The results obtained in this work are rather promising as ITZ and FLZ are safe and low-cost drugs available on the market.

The life-cycle of Toxoplasma gondii reviewed using animations.

Toxoplasma gondii is a protozoan parasite that is the causative agent of toxoplasmosis, an infection with high prevalence worldwide. Most of the infected individuals are either asymptomatic or have mild symptoms, but T. gondii can cause severe neurologic damage and even death of the fetus when acquired during pregnancy. It is also a serious condition in immunodeficient patients. The life-cycle of T. gondii is complex, with more than one infective form and several transmission pathways. In two animated videos, we describe the main aspects of this cycle, raising questions about poorly or unknown issues of T. gondii biology. Original plates, based on electron microscope observations, are also available for teachers, students and researchers. The main goal of this review is to provide a source of learning on the fundamental aspects of T. gondii biology to students and teachers contributing for better knowledge and control on this important parasite, and unique cell model. In addition, drawings and videos point to still unclear aspects of T. gondii lytic cycle that may stimulate further studies.

Toxoplasma gondii: further studies on the subpellicular network.

The association of the pellicle with cytoskeletal elements in Toxoplasma gondii allows this parasite to maintain its mechanical integrity and makes possible its gliding motility and cell invasion. The inner membrane complex (IMC) resembles the flattened membrane sacs observed in free-living protozoa and these sacs have been found to associate with cytoskeletal proteins such as articulins. We used immunofluorescence microscopy to characterise the presence and distribution of plateins, a sub-family of articulins, in T. gondii tachyzoites. A dispersed labelling of the whole protozoan body was observed. Electron microscopy of detergent-extracted cells revealed the presence of a network of 10 nm filaments distributed throughout the parasite. These filaments were labelled with anti-platein antibodies. Screening the sequenced T. gondii genome, we obtained the sequence of an IMC predicted protein with 25% identity and 42% similarity to the platein isoform alpha 1 present in Euplotes aediculatus, but with 42% identity and 55% similarity to that found in Euglena gracilis, suggesting strong resemblance to articulins.

Assay to Access Anthelmintic Activity of Small Molecule Drugs Using Caenohabidtis elegans as a Model.

This protocol proposes to use the nematode Caenorhabditis elegans as a model to screen and study the anthelmintic activity of natural and synthetic compounds and to observe their effects on the morphology and the ultrastructure of the helminths. Furthermore, C. elegans can be used to investigate the anthelmintic activity in embryonated eggs, larval stages and in the adults' survival. As most current anthelmintics are not effective against all nematode life stages, this protocol can contribute to the identification of new alternatives to helminthic infections (Sant' Anna et al., 2016 ).

A Glycosylphosphatidylinositol-Anchored Carbonic Anhydrase-Related Protein of Toxoplasma gondii Is Important for Rhoptry Biogenesis and Virulence.

Carbonic anhydrase-related proteins (CARPs) have previously been described as catalytically inactive proteins closely related to α-carbonic anhydrases (α-CAs). These CARPs are found in animals (both vertebrates and invertebrates) and viruses as either independent proteins or domains of other proteins. We report here the identification of a new CARP (TgCA_RP) in the unicellular organism Toxoplasma gondii that is related to the recently described η-class CA found in Plasmodium falciparum. TgCA_RP is posttranslationally modified at its C terminus with a glycosylphosphatidylinositol anchor that is important for its localization in intracellular tachyzoites. The protein localizes throughout the rhoptry bulbs of mature tachyzoites and to the outer membrane of nascent rhoptries in dividing tachyzoites, as demonstrated by immunofluorescence and immunoelectron microscopy using specific antibodies. T. gondii mutant tachyzoites lacking TgCA_RP display a growth and invasion phenotype in vitro and have atypical rhoptry morphology. The mutants also exhibit reduced virulence in a mouse model. Our results show that TgCA_RP plays an important role in the biogenesis of rhoptries. IMPORTANCEToxoplasma gondii is an intracellular pathogen that infects humans and animals. The pathogenesis of T. gondii is linked to its lytic cycle, which starts when tachyzoites invade host cells and secrete proteins from specialized organelles. Once inside the host cell, the parasite creates a parasitophorous vacuole (PV) where it divides. Rhoptries are specialized secretory organelles that contain proteins, many of which are secreted during invasion. These proteins have important roles not only during the initial interaction between parasite and host but also in the formation of the PV and in the modification of the host cell. We report here the identification of a new T. gondii carbonic anhydrase-related protein (TgCA_RP), which localizes to rhoptries of mature tachyzoites. TgCA_RP is important for the morphology of rhoptries and for invasion and growth of parasites. TgCA_RP is also critical for parasite virulence. We propose that TgCA_RP plays a role in the biogenesis of rhoptries.

Intravacuolar network may act as a mechanical support for Toxoplasma gondii inside the parasitophorous vacuole.

The intravacuolar network inside the parasitophorous vacuole of Toxoplasma gondii consists of an intricate system of membrane-limited tubules of uncertain role in parasite development. We propose that it is an important structural support to the maintenance of the parasites in the characteristic rosette arrangement of parasites inside the vacuole, rather than being associated with the nutrient acquisition from the host cell, as previously suggested. We based our assumptions on observations made by field emission scanning electron microscopy of an epithelial cell line (LLCMK2) infected at various time intervals. Scraping the surface of infected monolayers with Scotch tape exposed the inner organization of the parasitophorous vacuole. Ultrathin sections and freeze-fracture replicas of analogous samples were correlated with field emission observations and added new data on tubular membranes and general organization of the parasitophorous vacuole.

Antiproliferative synergism of azasterols and antifolates against Toxoplasma gondii.

The antiproliferative effects of two azasterols, 22,26-azasterol (20-piperidin-2-yl-5alpha-pregnan-3beta-20(R,S)-diol, AZA) and 24,25(R,S)-epiminolanosterol (EIL), in combination with sulphadiazine (SDZ) and pyrimethamine (PYR) were studied against Toxoplasma gondii tachyzoites growing in cultured mammalian cells. We had previously shown that AZA and EIL, two known inhibitors of Delta24(25)sterol methyl transferase in fungi and protozoa, have a potent and selective anti-T. gondii activity, although no 24-alkyl sterols have been detected in this parasite. We now report that when these sterol analogues were used in combination with the conventional SDZ/PYR treatment, potent synergistic effects were observed, ranging from 10- to 100-fold reductions of the IC50 values in the presence of sub-optimal doses of azasterols. When exposed to these drug combinations, intracellular T. gondii parasites displayed diverse subcellular alterations, including mitochondrial swelling, the arrest of the endodiogeny process with fragmented nuclei and subsequent cell lysis. These results suggest a potential new approach for the treatment of toxoplasmosis, which could significantly lower the required levels of antifolates and thus their adverse side effects.

Ciprofloxacin Derivatives Affect Parasite Cell Division and Increase the Survival of Mice Infected with Toxoplasma gondii.

Toxoplasmosis, caused by the protozoan Toxoplasma gondii, is a worldwide disease whose clinical manifestations include encephalitis and congenital malformations in newborns. Previously, we described the synthesis of new ethyl-ester derivatives of the antibiotic ciprofloxacin with ~40-fold increased activity against T. gondii in vitro, compared with the original compound. Cipro derivatives are expected to target the parasite's DNA gyrase complex in the apicoplast. The activity of these compounds in vivo, as well as their mode of action, remained thus far uncharacterized. Here, we examined the activity of the Cipro derivatives in vivo, in a model of acute murine toxoplasmosis. In addition, we investigated the cellular effects T. gondii tachyzoites in vitro, by immunofluorescence and transmission electron microscopy (TEM). When compared with Cipro treatment, 7-day treatments with Cipro derivatives increased mouse survival significantly, with 13-25% of mice surviving for up to 60 days post-infection (vs. complete lethality 10 days post-infection, with Cipro treatment). Light microscopy examination early (6 and 24h) post-infection revealed that 6-h treatments with Cipro derivatives inhibited the initial event of parasite cell division inside host cells, in an irreversible manner. By TEM and immunofluorescence, the main cellular effects observed after treatment with Cipro derivatives and Cipro were cell scission inhibition--with the appearance of 'tethered' parasites--malformation of the inner membrane complex, and apicoplast enlargement and missegregation. Interestingly, tethered daughter cells resulting from Cipro derivatives, and also Cipro, treatment did not show MORN1 cap or centrocone localization. The biological activity of Cipro derivatives against C. parvum, an apicomplexan species that lacks the apicoplast, is, approximately, 50 fold lower than that in T. gondii tachyzoites, supporting that these compounds targets the apicoplast. Our results show that Cipro derivatives improved the survival of mice acutely infected with T. gondii and inhibited parasite replication early in the first cycle of infection in vitro, highlighting their therapeutic potential for the treatment of toxoplasmosis.

Cultured embryonic retina systems as a model for the study of underlying mechanisms of Toxoplasma gondii infection.

PURPOSE: Toxoplasma gondii, the most common cause of retinochoroiditis in humans, is an obligate intracellular protozoan parasite that depends on te host cell's microenvironment to proliferate. Because congenital infection is associated with a higher risk of ocular involvement than a postnatally acquired infection, this study was conducted to investigate the ability of Toxoplasma gondii to infect retinal tissue during development, when cellular environmental changes normally occur. METHODS: Retinas from 5- to 9-day-old chick embryos were used. Stationary cultures were prepared in 24-well cell culture dishes and maintained at 37 degrees C in DMEM plus 5% fetal bovine serum for 2 to 6 days. Then the wells were infected with 4 x 10(5) tachyzoites. Retina explants and aggregate cell cultures were maintained in DMEM under rotation at 37 degrees C. T. gondii proliferation was measured using [(3)H]-thymidine incorporation after 72 hours. Ornithine and arginine decarboxylase (ODC and ADC) activities were determined by measuring CO(2) production from [1-(14)C]-ornithine and [1-(14)C]-arginine, respectively. RESULTS: The proliferation of tachyzoites was high in dense, stationary cultures expressing elevated ODC and ADC activity. The addition of ODC or ADC inhibitors reduced T. gondii proliferation by approximately 20% to 40%. As for cultured retina cells, retina explants also allowed T. gondii proliferation whenever ODC activity was high. CONCLUSIONS: The data suggest a direct correlation between retinal polyamine biosynthesis and the proliferation of T. gondii, in agreement with the observation that individuals infected congenitally have a greater risk of development of toxoplasmic retinochoroiditis.

Selective anti-Toxoplasma gondii activities of azasterols.

We report potent and selective inhibitory effects of 22,26-azasterol and 24,25-(R,S)-epiminolanosterol, known inhibitors of Delta24(25)-sterol methyltranferase (SMT) in fungi and protozoa, on the proliferation of Toxoplasma gondii in LLCMK2 cells. These compounds produced a dose-dependent reduction in parasite proliferation. 22,26-azasterol had an IC50 of 5.3 microM after 24 h and 4.5 microM after 48 h, while for 24,25-(R,S)-epiminolanosterol the IC50 values were 1 microM after 24 h and 0.12 microM after 48 h. The rapid reduction of parasite load suggested these compounds have selective cytotoxic effects against T. gondii. However, we were unable to detect 24-alkyl sterols in purified T. gondii tachyzoites using highly sensitive gas-liquid chromatography/mass spectrometry methods, a fact which indicated that the anti-proliferative effects of these azasterols were not mediated by inhibition of SMT. Transmission electron microscopy showed that the mitochondrion was the major target of drugs. Ultrastructural effects on plasma membrane, apicoplast and the formation of autophagosomal structures were also observed.

Inactivation of a fibronectin-binding TonB-dependent protein increases adhesion properties of Bacteroides fragilis.

Bacteroides fragilis is the Gram-negative strictly anaerobic bacterium most frequently isolated from clinical infections, including intra-abdominal abscess and bacteraemia. A number of factors can contribute to its virulence, including the expression of adhesins. Some of them are already characterized and can recognize and bind to extracellular matrix components, such as fibronectin. One of the molecules responsible for fibronectin-binding is an outer-membrane protein previously described by our group, which belongs to the TonB-dependent family. The aim of the present work was to characterize this protein. Initially, it was confirmed by fluorescence and electron microscopy that the fibronectin-binding molecules were located in the bacterial surface, but the distribution of these molecules on the surface was not uniform. To further evaluate the role of this protein, the gene bf1991, responsible for encoding this protein, was inactivated by a suicide vector and the mutant strains generated were used in several experiments to verify possible phenotypical alterations. In adherence assays with fibronectin immobilized on latex beads an increased adhesion was observed with the mutant strains compared with the wild-type strain. Western blot analysis in the mutant strain revealed the absence of the 120 kDa TonB-dependent outer-membrane protein and an alteration in the expression of an unknown 30 kDa protein. Killing assays using peritoneal macrophages were performed to evaluate the role of this protein as a virulence attribute and it was observed that the mutant strains were more efficiently internalized than the wild-type strains, with more internalization in the samples covered with fibronectin than in the samples not covered with it.

Identification of the alpha-enolase P46 in the extracellular membrane vesicles of Bacteroides fragilis

BACKGROUND Members of the Bacteroides fragilis group are the most important components of the normal human gut microbiome, but are also major opportunistic pathogens that are responsible for significant mortality, especially in the case of bacteraemia and other severe infections, such as intra-abdominal abscesses. Up to now, several virulence factors have been described that might explain the involvement of B. fragilis in these infections. The secretion of extracellular membrane vesicles (EMVs) has been proposed to play a role in pathogenesis and symbiosis in gram-negative bacteria, by releasing soluble proteins and other molecules. In B. fragilis, these vesicles are known to have haemagglutination and sialidosis activities, and also contain a capsular polysaccharide (PSA), although their involvement in virulence is still not clear. OBJECTIVE The aim of this study was to identify proteins in the EMV of the 638R B. fragilis strain by mass spectrometry, and also to assess for the presence of Bfp60, a surface plasminogen (Plg) activator, previously shown in B. fragilis to be responsible for the conversion of inactive Plg to active plasmin, which can also bind to laminin-1. METHODS B. fragilis was cultured in a minimum defined media and EMVs were obtained by differential centrifugation, ultracentrifugation, and filtration. The purified EMVs were observed by both transmission electron microscopy (TEM) and immunoelectron microscopy (IM). To identify EMV constituent proteins, EMVs were separated by 1D SDS-PAGE and proteomic analysis of proteins sized 35 kDa to approximately 65 kDa was performed using mass spectrometry (MALDI-TOF MS). FINDINGS TEM micrographs proved the presence of spherical vesicles and IM confirmed the presence of Bfp60 protein on their surface. Mass spectrometry identified 23 proteins with high confidence. One of the proteins from the B. fragilis EMVs was identified as an enolase P46 with a possible lyase activity. MAIN CONCLUSIONS Although the Bfp60 protein was not detected by proteomics, α-enolase P46 was found to be present in the EMVs of B. fragilis. The P46 protein has been previously described to be present in the outer membrane of B. fragilis as an iron-regulated protein.