Could the Urease of the Gut Bacterium Proteus mirabilis Play a Role in the Altered Gut-Brain Talk Associated with Parkinson's Disease?

Intestinal dysbiosis seems to play a role in neurodegenerative pathologies. Parkinson's disease (PD) patients have an altered gut microbiota. Moreover, mice treated orally with the gut microbe Proteus mirabilis developed Parkinson's-like symptoms. Here, the possible involvement of P. mirabilis urease (PMU) and its B subunit (PmUreß) in the pathogenesis of PD was assessed. Purified proteins were given to mice intraperitoneally (20 µg/animal/day) for one week. Behavioral tests were conducted, and brain homogenates of the treated animals were subjected to immunoassays. After treatment with PMU, the levels of TNF-α and IL-1ß were measured in Caco2 cells and cellular permeability was assayed in Hek 293. The proteins were incubated in vitro with α-synuclein and examined via transmission electron microscopy. Our results showed that PMU treatment induced depressive-like behavior in mice. No motor deficits were observed. The brain homogenates had an increased content of caspase-9, while the levels of α-synuclein and tyrosine hydroxylase decreased. PMU increased the pro-inflammatory cytokines and altered the cellular permeability in cultured cells. The urease, but not the PmUreß, altered the morphology of α-synuclein aggregates in vitro, forming fragmented aggregates. We concluded that PMU promotes pro-inflammatory effects in cultured cells. In vivo, PMU induces neuroinflammation and a depressive-like phenotype compatible with the first stages of PD development.

Helicobacter pylori Urease: Potential Contributions to Alzheimer's Disease.

Alzheimer's disease (AD) causes dementia and memory loss in the elderly. Deposits of beta-amyloid peptide and hyperphosphorylated tau protein are present in a brain with AD. A filtrate of Helicobacter pylori's culture was previously found to induce hyperphosphorylation of tau in vivo, suggesting that bacterial exotoxins could permeate the blood-brain barrier and directly induce tau's phosphorylation. H. pylori, which infects ~60% of the world population and causes gastritis and gastric cancer, produces a pro-inflammatory urease (HPU). Here, the neurotoxic potential of HPU was investigated in cultured cells and in rats. SH-SY5Y neuroblastoma cells exposed to HPU (50-300 nM) produced reactive oxygen species (ROS) and had an increased [Ca2+]i. HPU-treated BV-2 microglial cells produced ROS, cytokines IL-1ß and TNF-α, and showed reduced viability. Rats received daily i.p., HPU (5 µg) for 7 days. Hyperphosphorylation of tau at Ser199, Thr205 and Ser396 sites, with no alterations in total tau or GSK-3ß levels, and overexpression of Iba1, a marker of microglial activation, were seen in hippocampal homogenates. HPU was not detected in the brain homogenates. Behavioral tests were performed to assess cognitive impairments. Our findings support previous data suggesting an association between infection by H. pylori and tauopathies such as AD, possibly mediated by its urease.

Proteus mirabilis Urease: Unsuspected Non-Enzymatic Properties Relevant to Pathogenicity.

Infection by Proteus mirabilis causes urinary stones and catheter incrustation due to ammonia formed by urease (PMU), one of its virulence factors. Non-enzymatic properties, such as pro-inflammatory and neurotoxic activities, were previously reported for distinct ureases, including that of the gastric pathogen Helicobacter pylori. Here, PMU was assayed on isolated cells to evaluate its non-enzymatic properties. Purified PMU (nanomolar range) was tested in human (platelets, HEK293 and SH-SY5Y) cells, and in murine microglia (BV-2). PMU promoted platelet aggregation. It did not affect cellular viability and no ammonia was detected in the cultures' supernatants. PMU-treated HEK293 cells acquired a pro-inflammatory phenotype, producing reactive oxygen species (ROS) and cytokines IL-1ß and TNF-α. SH-SY5Y cells stimulated with PMU showed high levels of intracellular Ca2+ and ROS production, but unlike BV-2 cells, SH-SY5Y did not synthesize TNF-α and IL-1ß. Texas Red-labeled PMU was found in the cytoplasm and in the nucleus of all cell types. Bioinformatic analysis revealed two bipartite nuclear localization sequences in PMU. We have shown that PMU, besides urinary stone formation, can potentially contribute in other ways to pathogenesis. Our data suggest that PMU triggers pro-inflammatory effects and may affect cells beyond the renal system, indicating a possible role in extra-urinary diseases.

Case Report: Placental Maternal Vascular Malperfusion Affecting Late Fetal Development and Multiorgan Infection Caused by SARS-CoV-2 in Patient With PAI-1 4G/5G Polymorphism.

Background: Pregnant women are susceptible to the novel coronavirus (SARS-CoV-2), and the consequences for the fetus are still uncertain. Here, we present a case of a pregnant woman with subclinical hypothyroidism and a plasminogen activator inhibitor type 1 (PAI-1) 4G/5G polymorphism who was infected with SARS-CoV-2 at the end of the third trimester of pregnancy, with unexpected evolution of death of the newborn 4 days postpartum. Methods: Nested PCR was performed to detect the virus, followed by ssDNA sequencing. Results: Transplacental transmission of SARS-CoV-2 can cause placental inflammation, ischemia, and neonatal viremia, with complications such as preterm labor and damage to the placental barrier in patients with PAI-1 4G/5G polymorphism. Conclusion: We showed a newborn with several damages potentially caused due to the PAI-1 polymorphisms carried by the mother infected with SARS-CoV-2 during pregnancy.

Evaluation of drug repositioning by molecular docking of pharmaceutical resources available in the Brazilian healthcare system against SARS-CoV-2.

In 2020 SARS-CoV-2 reached pandemic status, reaching Brazil in mid-February. As of now, no specific drugs for treating the disease are available. In this work, the possibility of interaction between SARS-CoV-2 viral proteins (open and closed spike protein, isolate spike protein RBD, NSP 10, NSP 16, main protease, and RdRp polymerase) and multiple molecules is addressed through the repositioning of drugs available for the treatment of other diseases that are approved by the FDA and covered by SUS, the Brazilian Public Health System. Three different docking software were used, followed by a unification of the results by independent evaluation. Afterwards, the chemical interactions of the compounds with the targets were inspected via molecular dynamics and analyzed. The results point to a potential effectiveness of Penciclovir, Ribavirin, and Zanamivir, from a set of 48 potential candidates. They may also be multi-target drugs, showing high affinity with more than one viral protein. Further in vitro and in vivo validation is required to assess the suitability of repositioning the proposed drugs for COVID-19.

Structure-Function Insights of Jaburetox and Soyuretox: Novel Intrinsically Disordered Polypeptides Derived from Plant Ureases.

Intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs) do not have a stable 3D structure but still have important biological activities. Jaburetox is a recombinant peptide derived from the jack bean (Canavalia ensiformis) urease and presents entomotoxic and antimicrobial actions. The structure of Jaburetox was elucidated using nuclear magnetic resonance which reveals it is an IDP with small amounts of secondary structure. Different approaches have demonstrated that Jaburetox acquires certain folding upon interaction with lipid membranes, a characteristic commonly found in other IDPs and usually important for their biological functions. Soyuretox, a recombinant peptide derived from the soybean (Glycine max) ubiquitous urease and homologous to Jaburetox, was also characterized for its biological activities and structural properties. Soyuretox is also an IDP, presenting more secondary structure in comparison with Jaburetox and similar entomotoxic and fungitoxic effects. Moreover, Soyuretox was found to be nontoxic to zebra fish, while Jaburetox was innocuous to mice and rats. This profile of toxicity affecting detrimental species without damaging mammals or the environment qualified them to be used in biotechnological applications. Both peptides were employed to develop transgenic crops and these plants were active against insects and nematodes, unveiling their immense potentiality for field applications.

Jaburetox, a urease-derived peptide: Effects on enzymatic pathways of the cockroach Nauphoeta cinerea.

Jaburetox is a recombinant peptide derived from one of the Canavalia ensiformis urease isoforms. This peptide induces several toxic effects on insects of different orders, including interference on muscle contractility in cockroaches, modulation of UDP-N-acetylglucosamine pyrophosphorylase (UAP) and nitric oxide synthase (NOS) activities in the central nervous system of triatomines, as well as activation of the immune system in Rhodnius prolixus. When injected, the peptide is lethal for R. prolixus and Triatoma infestans. Here, we evaluated Jaburetox toxicity to Nauphoeta cinerea cockroaches, exploring the effects on the central nervous system through the activities of UAP, NOS, acid phosphatases (ACP), and acetylcholinesterase (AChE). The results indicated that N. cinerea is not susceptible to the lethal effect of the peptide. Moreover, both in vivo and in vitro treatments with Jaburetox inhibited NOS activity, without modifying the protein levels. No alterations on ACP activity were observed. In addition, the enzyme activity of UAP only had its activity affected at 18 hr after injection. The peptide increased the AChE activity, suggesting a mechanism involved in overcoming the toxic effects. In conclusion, our findings indicate that Jaburetox affects the nitrinergic signaling as well as the AChE and UAP activities and establishes N. cinerea as a Jaburetox-resistant model for future comparative studies.

The role of extracellular nucleic acids in the immune system modulation of Rhodnius prolixus (Hemiptera: Reduviidae).

Extracellular traps (ETs) are extracellular nucleic acids associated with cytoplasmic proteins that may aid in the capture and killing of pathogens. To date, only a few insects were shown to display this kind of immune response. Jaburetox, a peptide derived from jack bean urease, showed toxic effects in Rhodnius prolixus, affecting its immune response. The present study aims to evaluate the role of extracellular nucleic acids in R. prolixus' immune response, using Jaburetox as a model entomotoxin. The insects were treated with extracellular nucleic acids and/or Jaburetox, and the cellular and humoral responses were assessed. We also evaluated the release of extracellular nucleic acids induced by toxins, and performed immunocompetence assays using pathogenic bacteria. Our results demonstrated that extracellular nucleic acids can modulate the insect immune responses, either alone or associated with the toxin. Although RNA and DNA induced a cellular immune response, only DNA was able to neutralize the Jaburetox-induced aggregation of hemocytes. Likewise, the activation of the humoral response was different for RNA and DNA. Nevertheless, it was observed that both, extracellular DNA and RNA, immunocompensated the Jaburetox effects on insect defenses upon the challenge of a pathogenic bacterium. The toxin was not able to alter cellular viability, in spite of inducing an increase in the reactive species of oxygen formation. In conclusion, we have demonstrated a protective role for extracellular nucleic acids in R. prolixus´ immune response to toxins and pathogenic bacteria.

Jaburetox-induced toxic effects on the hemocytes of Rhodnius prolixus (Hemiptera: Reduviidae).

Jaburetox is a recombinant peptide derived from a Canavalia ensiformis urease that presents toxic effects upon several species of insects, phytopathogenic fungi and yeasts of medical importance. So far, no toxicity of Jaburetox to mammals has been shown. Previous reports have identified biochemical targets of this toxic peptide in insect models, although its mechanism of action is not completely understood. In this work, we aimed to characterize the effects of Jaburetox in hemolymphatic insect cells. For this purpose, the model insect and Chagas' disease vector Rhodnius prolixus was used. In vivo and in vitro experiments indicated that Jaburetox interacts with a subset of hemocytes and it can be found in various subcellular compartments. In insects injected with Jaburetox there was an increase in the gene expression of the enzymes UDP-N-acetylglucosamine pyrophosphorylase (UAP), chitin synthase and nitric oxide synthase (NOS). Nevertheless, the expression of NOS protein, the enzyme activities of UAP and acid phosphatase (a possible link between UAP and NOS) as well as the phosphorylation state of proteins remained unchanged upon the in vivo Jaburetox treatment. Nitric oxide (NO) imaging using fluorescent probes showed that Jaburetox augmented NO production in the hemocyte aggregates when compared to controls. Even though Jaburetox activated the hemocytes, as demonstrated by wheat germ agglutinin binding assays, the peptide did not lead to an increase of their phagocytic behavior. Taken together, these findings contribute to our understanding of toxic effects of Jaburetox, a peptide with biotechnological applications and a prospective tool for rational insect control.

Jaburetox affects gene expression and enzyme activities in Rhodnius prolixus, a Chagas' disease vector.

Jaburetox, a recombinant peptide of ∼11kDa derived from one of the Canavalia ensiformis (Jack Bean) urease isoforms, is toxic and lethal to insects belonging to different orders when administered orally or via injection. Previous findings indicated that Jaburetox acts on insects in a complex fashion, inhibiting diuresis and the transmembrane potential of Malpighian tubules, interfering with muscle contractility and affecting the immune system. In vitro, Jaburetox forms ionic channels and alters permeability of artificial lipid membranes. Moreover, recent data suggested that the central nervous system (CNS) is a target organ for ureases and Jaburetox. In this work, we employed biochemical, molecular and cellular approaches to explore the mode of action of Jaburetox using Rhodnius prolixus, one of the main Chagas' disease vectors, as experimental model. In vitro incubations with fluorescently labeled Jaburetox indicated a high affinity of the peptide for the CNS but not for salivary glands (SG). The in vitro treatment of CNS or SG homogenates with Jaburetox partially inhibited the activity of nitric oxide synthase (NOS), thus disrupting nitrinergic signaling. This inhibitory effect was also observed in vivo (by feeding) for CNS but not for SG, implying differential modulation of NOS in these organs. The inhibition of NOS activity did not correlate to a decrease in expression of its mRNA, as assessed by qPCR. UDP-N-acetylglucosamine pyrophosphorylase (UAP), a key enzyme in chitin synthesis and glycosylation pathways and a known target of Jaburetox in insect CNS, was also affected in SG, with activation of the enzyme seen after both in vivo or in vitro treatments with the peptide. Unexpectedly, incubation of Jaburetox with a recombinant R. prolixus UAP had no effect on its activity, implying that the enzyme's modulation by the peptide requires the participation of other factor(s) present in CNS or SG homogenates. Feeding Jaburetox to R. prolixus decreased the mRNA levels of UAP and chitin synthase, indicating a complex regulation exerted by the peptide on these enzymes. No changes were observed upon Jaburetox treatment in vivo and in vitro on the activity of the enzyme acid phosphatase, a possible link between UAP and NOS. Here we have demonstrated for the first time that the Jaburetox induces changes in gene expression and that SG are another target for the toxic action of the peptide. Taken together, these findings contribute to a better understanding of the mechanism of action of Jaburetox as well as to the knowledge on basic aspects of the biochemistry and neurophysiology of insects, and might help in the development of optimized strategies for insect control.

Humoral and cellular immune responses induced by the urease-derived peptide Jaburetox in the model organism Rhodnius prolixus.

BACKGROUND: Although the entomotoxicity of plant ureases has been reported almost 20 years ago, their insecticidal mechanism of action is still not well understood. Jaburetox is a recombinant peptide derived from one of the isoforms of Canavalia ensiformis (Jack Bean) urease that presents biotechnological interest since it is toxic to insects of different orders. Previous studies of our group using the Chagas disease vector and model insect Rhodnius prolixus showed that the treatment with Jack Bean Urease (JBU) led to hemocyte aggregation and hemolymph darkening, among other effects. In this work, we employed cell biology and biochemical approaches to investigate whether Jaburetox would induce not only cellular but also humoral immune responses in this species. RESULTS: The findings indicated that nanomolar doses of Jaburetox triggered cation-dependent, in vitro aggregation of hemocytes of fifth-instar nymphs and adults. The use of specific eicosanoid synthesis inhibitors revealed that the cellular immune response required cyclooxygenase products since indomethacin prevented the Jaburetox-dependent aggregation whereas baicalein and esculetin (inhibitors of the lipoxygenases pathway) did not. Cultured hemocytes incubated with Jaburetox for 24 h showed cytoskeleton disorganization, chromatin condensation and were positive for activated caspase 3, an apoptosis marker, although their phagocytic activity remained unchanged. Finally, in vivo treatments by injection of Jaburetox induced both a cellular response, as observed by hemocyte aggregation, and a humoral response, as seen by the increase of spontaneous phenoloxidase activity, a key enzyme involved in melanization and defense. On the other hand, the humoral response elicited by Jaburetox injections did not lead to an increment of antibacterial or lysozyme activities. Jaburetox injections also impaired the clearance of the pathogenic bacteria Staphylococcus aureus from the hemolymph leading to increased mortality, indicating a possible immunosuppression induced by treatment with the peptide. CONCLUSIONS: In our experimental conditions and as part of its toxic action, Jaburetox activates some responses of the immune system of R. prolixus both in vivo and in vitro, although this induction does not protect the insects against posterior bacterial infections. Taken together, these findings contribute to the general knowledge of insect immunity and shed light on Jaburetox's mechanism of action.