Theoretical study of ArcB and its dimerization, interaction with anaerobic metabolites, and activation of ArcA.

The complex metabolism of Escherichia coli has been extensively studied, including its response to oxygen availability. The ArcA/B two-component system (TCS) is the key regulator for the transition between these two environmental conditions and has been thoroughly characterized using genetic and biochemical approaches. Still, to date, limited structural data is available. The breakthrough provided by AlphaFold2 in 2021 has brought a reliable tool to the scientific community for assessing the structural features of complex proteins. In this report, we analyzed the structural aspects of the ArcA/B TCS using AlphaFold2 models. The models are consistent with the experimentally determined structures of ArcB kinase. The predicted structure of the dimeric form of ArcB is consistent with the extensive genetic and biochemical data available regarding mechanistic signal perception and regulation. The predicted interaction of the dimeric form of ArcB with its cognate response regulator (ArcA) is also consistent with both the forward and reverse phosphotransfer mechanisms. The ArcB model was used to detect putative binding cavities to anaerobic metabolites, encouraging testing of these predictions experimentally. Finally, the highly accurate models of other ArcB homologs suggest that different experimental approaches are needed to determine signal perception in kinases lacking the PAS domain. Overall, ArcB is a kinase with features that need further testing, especially in determining its crystal structure under different conditions.

Trichomonas vaginalis triggers neutrophil extracellular traps reducing parasite integrity and growth.

Trichomoniasis-caused by the parasite Trichomonas vaginalis-is associated with a high inflammatory process that may contribute to the risk of suffering from other medical complications. Our study focused on the in vitro interaction of T. vaginalis with human neutrophils because these are the most abundant cells implicated in the characteristic inflammatory process of trichomoniasis. This study showed that T. vaginalis and its surface glycoconjugates (lipophosphoglycan and/or lipoglycan) induced the formation of human neutrophil extracellular traps (NETs). After the trichomonad-neutrophil interaction, parasite integrity was at 32.9%, and the subsequent parasite growth was at 35.2% compared to those of control trophozoites (100%) incubated under the same conditions without neutrophils. In the presence of an antibody against the TLR-4 receptor, DNase I or micrococcal nuclease (MNase), neutrophils reduced the DNA fibres of the NETs and the amount of extracellular DNA, allowing a higher subsequent growth of T. vaginalis, at 52% with the anti-TLR-4 antibody and 62.6% with the enzymes. These results indicated that T. vaginalis induced the formation of extracellular traps by human neutrophils and, because of the interaction with neutrophils and NETs, parasite integrity and growth decreased.

Reduced Trichomonas vaginalis viability in mice pretreated with parasite DNA.

Trichomonas vaginalis is an extracellular parasite that colonizes the human urogenital tract leading to trichomoniasis, the most common sexually-transmitted non-viral disease worldwide. The immune response plays a critical role in the host defense against this parasite. Trichomonas' DNA contains unmethylated CpG motifs (CpGDNA) that in other microorganisms act as modulators of the immune response. However, the molecular mechanisms responsible for CpGDNA immune modulation are still unclear. As macrophages participate in the first line of defense against infection, we investigated the type of immune response of murine macrophages to T. vaginalis DNA (TvDNA). We observed high expression of the proinflammatory cytokines IL-6 and IL-12p40 in macrophages stimulated with TvDNA. In contrast, the anti-inflammatory response, assessed by IL-10 and IL-13 mRNA expression was delayed. This suggests that the immune response induced by TvDNA is modulated through cytokine production, mediated partly by NADPH-oxidase activity, as TvDNA induced reactive species of oxygen production and a rounded morphology in macrophages indicative of an M1 phenotype. Furthermore, infected mice pretreated with TvDNA displayed persistent vulvar inflammation and decreased parasite viability consistent with higher proinflammatory cytokine levels during infection compared to untreated mice. Overall, our findings suggest that TvDNA pretreatment modulates the immune response favouring parasite elimination.

The component leaching from decellularized pericardial bioscaffolds and its implication in the macrophage response.

The extracellular matrix molecules remaining in bioscaffolds derived from decellularized xenogeneic tissues appear to be important for inducing cell functions conducting tissue regeneration. Here, we studied whether decellularization methods, that is, detergent Triton X-100 (TX) alone and TX combined with reversible alkaline swelling (STX), applied to bovine pericardial tissue, could affect the bioscaffold components. The in vitro macrophage response, subdermal biodegradation, and cell infiltration were also studied. The results indicate a lower leaching of fibronectin, but a higher leaching of laminin and sulfated glycosaminoglycans from tissues decellularized with STX and TX, respectively. The in vitro secretion of interleukin-6 and monocyte chemoattractant protein by RAW264.7 macrophages is promoted by decellularized bioscaffold leachates. A lower polymorphonuclear cell density is observed around decellularized bioscaffolds at 1-day implantation; concurrently showing a higher cell infiltration in STX- than in TX-implant. Cells infiltrated into TX-implant show a fibroblastic morphology at 7-day implantation, concurrently the capillary formation is observed at 14-day. Pericardial bioscaffolds suffer biodegradation more pronounced in STX- than in TX-implant. Both TX and STX decellularization methods favor a high leaching of basal lamina components, which presumably promotes a faster macrophage stimulation compared to nondecellularized tissue, and appear to be associated with an increased host cell infiltration in a rat subdermal implantation. Meanwhile, the connective tissue components leaching from TX decellularized bioscaffolds, unlike the STX ones, appear to be associated with an enhanced angiogenesis accompanied by an early-promoted fibroblastic cell transition. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2810-2822, 2016.

Identification of the transcription factor Znc1p, which regulates the yeast-to-hypha transition in the dimorphic yeast Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica is used as a model to study fungal differentiation because it grows as yeast-like cells or forms hyphal cells in response to changes in environmental conditions. Here, we report the isolation and characterization of a gene, ZNC1, involved in the dimorphic transition in Y. lipolytica. The ZNC1 gene encodes a 782 amino acid protein that contains a Zn(II)2C6 fungal-type zinc finger DNA-binding domain and a leucine zipper domain. ZNC1 transcription is elevated during yeast growth and decreases during the formation of mycelium. Cells in which ZNC1 has been deleted show increased hyphal cell formation. Znc1p-GFP localizes to the nucleus, but mutations within the leucine zipper domain of Znc1p, and to a lesser extent within the Zn(II)2C6 domain, result in a mislocalization of Znc1p to the cytoplasm. Microarrays comparing gene expression between znc1::URA3 and wild-type cells during both exponential growth and the induction of the yeast-to-hypha transition revealed 1,214 genes whose expression was changed by 2-fold or more under at least one of the conditions analyzed. Our results suggest that Znc1p acts as a transcription factor repressing hyphal cell formation and functions as part of a complex network regulating mycelial growth in Y. lipolytica.

Indirect determination of nitric oxide production by reduction of nitrate with a freeze-thawing-resistant nitrate reductase from Escherichia coli MC1061.

Preparation of a nitrate reductase lysate of Escherichia coli MC1061 to measure nitrate and nitrite in biologic fluids is described. To obtain the crude bacterial lysate containing nitrate reductase activity, E. coli MC1061 was subjected to 16-20 freeze-thawing cycles, from -70 to 60 degrees C, until nitrite reductase activity was < or = 25%. Nitrate reductase activity was detected mainly in the crude preparation. To validate the nitrate reduction procedure, standard nitrate solutions (1.6-100 microM) were incubated with the nitrate reductase preparation for 3 h at 37 degrees C, and nitrite was estimated by the Griess reaction in a microassay. Nitrate solutions were reduced to nitrite in a range of 60-70%. Importantly, no cofactors were necessary to perform nitrate reduction. The biological samples were first reduced with the nitrate reductase preparation. After centrifugation, samples were deproteinized with either methanol/ether or zinc sulfate and nitrite was quantified. The utility of the nitrate reductase preparation was assessed by nitrate+nitrite determination in serum of animals infected with the protozoan Entamoeba histolytica or the bacteria E. coli and in the supernatant of cultured lipopolysaccharide-stimulated RAW 264.7 mouse macrophages. Our results indicate that the nitrate reductase-containing lysate provides a convenient tool for the reduction of nitrate to determine nitrate+nitrite in biological fluids by spectrophotometric methods.

Iron chelators modulate the fusogenic properties of Salmonella-containing phagosomes.

In macrophages, the divalent cations transporter Nramp1 is recruited from the lysosomal compartment to the membrane of phagosomes formed in these cells. Nramp1 mutations cause susceptibility to infection with intracellular pathogens such as Salmonella and Mycobacterium. Intracellular survival of Salmonella involves segregation in an endomembrane compartment (Salmonella-containing vacuole, SCV) that remains negative for the mannose-6-phosphate receptor (M6PR) and that is inaccessible to the endocytic pathway. Expression of Nramp1 at the membrane of SCVs stimulates both acquisition of M6PR and accessibility to newly formed endosomes. The possible role of Nramp1-mediated iron transport on SCV maturation was investigated with membrane-permeant iron chelators. Pretreatment of primary macrophages from Nramp1 mutant mice or of RAW264.7 macrophages (from BALBc mice bearing an Nramp1(D169)-deficient allele) with either desferrioxamine or salicylaldehyde isocotinoyl hydrazone restored recruitment of M6PR and delivery of the fluid phase marker rhodamine dextran to SCVs to levels similar to those seen in macrophages expressing WT Nramp1. The effect was specific and dose-dependent and could be abrogated by preincubation with excess iron. These data suggest that Nramp1-mediated deprivation of iron and possibly of other divalent metals in macrophages antagonizes the ability of Salmonella to alter phagosome maturation.

Role of 3-phosphoinositides in the maturation of Salmonella-containing vacuoles within host cells.

Salmonella typhimurium invades mammalian cells and replicates within a vacuole that protects it from the host's microbicidal weapons. The Salmonella-containing vacuole (SCV) undergoes a remodelling akin to that of the host cell's endocytic pathway, but SCV progression is arrested prior to fusion with lysosomes. We studied the role of phosphatidylinositol 3-kinase (PI3-K) in SCV maturation within HeLa cells. Phosphatidylinositol 3-phosphate (PI3P), monitored in situ using fluorescent conjugates of FYVE or PX domains, was found to accumulate transiently on the SCV. Wortmannin prevented PI3P accumulation and the recruitment of EEA1 but did not affect the association of Rab5 with the SCV. Importantly, inhibition of PI3-K also impaired fusion of the SCV with vesicles containing LAMP-1. Rab7, which is thought to be required for association of LAMP-1 with the SCV, still associated with SCV in wortmannin-treated cells. We have therefore concluded that a 3-phosphoinositide-dependent step exists following recruitment of Rab7 to the SCV. The data also imply that 3-phosphoinositide-dependent effectors of Rab5 are not an absolute requirement for recruitment of Rab7. Despite failure to acquire LAMP-1, the SCV persists and allows effective replication of Salmonella within wortmannin-treated host cells. These findings imply that PI3-K is involved in the development of the SCV but is not essential for intracellular survival and proliferation of Salmonella.

Nramp1 modifies the fusion of Salmonella typhimurium-containing vacuoles with cellular endomembranes in macrophages.

Salmonella survive and replicate within mammalian cells by becoming secluded within specialized membrane-bound vacuoles inaccessible to the host defense mechanisms. Delayed acidification of the vacuole and its incomplete fusion with lysosomes have been implicated in intracellular Salmonella survival. Nramp1 confers to macrophages resistance to a variety of intracellular pathogens, including Salmonella, but its precise mode of action is not understood. We investigated whether Nramp1 affects the maturation and acidification of Salmonella-containing vacuoles (SCV). A mouse-derived macrophage line (RAW/Nramp1(-)) devoid of Nramp1 and therefore susceptible to infection was compared with isogenic clones stably transfected with Nramp1 (RAW/Nramp1(+)). Intravacuolar pH, measured in situ, was similar in Nramp1-expressing and -deficient cells. SCV acquired LAMP1 and fused with preloaded fluid-phase markers in both cell types. In contrast, although few vacuoles in RAW/Nramp1(-) acquired mannose 6-phosphate receptor, many more contained M6PR in RAW/Nramp1(+) cells. Shortly after closure, SCV in RAW/Nramp1(-) became inaccessible to extracellular markers, suggesting inability to fuse with newly formed endosomes. Expression of Nramp1 markedly increased the access to extracellularly added markers. We propose that Nramp1 counteracts the ability of Salmonella to become secluded in a compartment that limits access of bactericidal agents, allowing the normal degradative pathway of the macrophage to proceed.

Multiple GTP-binding proteins in sea urchin sperm: Evidence for Gs and small G-proteins.

Sea urchin sperm plasma membranes isolated from heads and flagella were used to examine the presence of Gs (stimulatory guanine nucleotide-binding regulatory protein) and small G-proteins. Flagellar plasma membranes incubated with [32 P]NAD and cholera toxin (CTX) displayed radiolabeling in a protein of 48 kDa, which was reactive by immunoblotting with a specific antibody against mammalian Gs. CTX-catalyzed [32 P]ADP-ribosylation in conjunction with immunoprecipitation with anti-Gs, followed by electrophoresis and autoradiography, revealed one band of 48 kDa. Head plasma membranes, in contrast, did not show substrates for ADP-ribosylation by CTX. In flagellar and head plasma membranes pertussis toxin (PTX) ADP-ribosylated the same protein described previously in membranes from whole sperm; the extent of ADP-ribosylation by PTX was higher in flagellar than in head membranes. Small G-proteins were investigated by [32 P]GTP-blotting. Both head and flagellar plasma membranes showed three radiolabeled bands of 28, 25 and 24 kDa. Unlabeled GTP and GDP, but not other nucleotides, interfered with the [α-32 P]GTP-binding in a concentration-dependent manner. A monoclonal antibody against human Ras p21 recognized a single protein of 21 kDa only in flagellar membranes. Thus, sea urchin sperm contain a membrane protein that shares characteristics with mammalian Gs and four small G-proteins, including Ras. Gs, Gi and Ras are enriched in flagellar membranes while the other small G-proteins do not display a preferential distribution along the sea urchin sperm plasma membrane. The role of these G-proteins in sea urchin sperm is presently under investigation.