Mycoplasma genitalium (Mg) is a mollicute that causes a range of human urogenital infections. A hallmark of these bacteria is their ability to establish chronic infections that can persist despite completion of appropriate antibiotic therapies and intact and functional immune systems. Intimate adherence and surface colonization of mycoplasmas to host cells are important pathogenic features. However, their facultative intracellular nature is poorly understood, partly due to difficulties in developing and standardizing cellular interaction model systems. Here, we characterize growth and invasion properties of two Mg strains (G37 and 1019V). Mg G37 is a high-passage laboratory strain, while Mg 1019V is a low-passage isolate recovered from the cervix. The two strains diverge partially in gene sequences for adherence-related proteins and exhibit subtle variations in their axenic growth. However, with both strains and consistent with our previous studies, a subset of adherent Mg organisms invade host cells and exhibit perinuclear targeting. Remarkably, intranuclear localization of Mg proteins is observed, which occurred as early as 30 min after infection. Mg strains deficient in adherence were markedly reduced in their ability to invade and associate with perinuclear and nuclear sites.
Bacterial toxins possess specific mechanisms of binding and uptake by mammalian cells. Mycoplasma pneumoniae CARDS (Community Acquired Respiratory Distress Syndrome) toxin is a 68 kDa protein, which demonstrates high binding affinity to human surfactant protein-A and exhibits specific biological activities including mono-ADP ribosylation and vacuolization. These properties lead to inflammatory processes in the airway and a range of cytopathologies including ciliostasis, loss of tissue integrity and injury, and cell death. However, the process by which CARDS toxin enters target cells is unknown. In this study, we show that CARDS toxin binds to mammalian cell surfaces and is internalized rapidly in a dose and time-dependent manner using a clathrin-mediated pathway, as indicated by inhibition of toxin internalization by monodansylcadaverine but not by methyl-β-cyclodextrin or filipin. Furthermore, the internalization of CARDS toxin was markedly inhibited in clathrin-depleted cells.
In this study, we identified and characterized the enzymatic properties of MG_186, a calcium-dependent Mycoplasma genitalium nuclease. MG_186 displays the hallmarks of nucleases, as indicated by its amino acid sequence similarity to other nucleases. We cloned, UGA corrected, expressed, purified, and demonstrated that recombinant MG_186 (rMG_186) exhibits nuclease activity similar to that of typical sugar-nonspecific endonucleases and exonucleases. Biochemical characterization indicated that Ca 2؉ alone enhances its activity, which was inhibited by divalent cations, such as Zn 2؉ and Mn 2؉ . Chelating agents EGTA and EDTA also inhibited nuclease activity. Mycoplasma membrane fractionation and Triton X-114 phase separation showed that MG_186 was a membrane-associated lipoprotein, and electron microscopy revealed its surface membrane location. Incubation of purified human endometrial cell nuclei with rMG_186 resulted in DNA degradation and morphological changes typical of apoptosis. Further, immunofluorescence analysis of rMG_186-treated nuclei indicated that morphological changes were linked to the disintegration of lamin and the internalization of rMG_186. Since M. genitalium has the capacity to invade eukaryotic cells and localize to the perinuclear and nuclear region of parasitized target cells, MG_186 has the potential to provide M. genitalium, which possesses the smallest genome of any self-replicating cell, with the ability to degrade host nucleic acids both as a source of nucleotide precursors for growth and for pathogenic purposes.Mycoplasma genitalium was first identified as a urogenital tract pathogen in men and subsequently implicated in a range of women pathologies, including pelvic inflammatory diseases, cervicitis, endometritis, salpingitis, and tubal factor infertility (5, 37, 40). In addition to its urogenital niche, M. genitalium has been detected in synovial and respiratory tract specimens (3, 39). M. genitalium DNA sequencing revealed a reduced genome size of 580 kb and a low GC content, along with 482 protein-encoding genes, of which 76 were categorized as hypothetical proteins (18). The streamlined genome of M. genitalium results in gene deficits that dramatically limit its biosynthetic capabilities, leading to a complete dependence on the host for metabolic precursors, such as nucleotides, amino acids, fatty acids, and sterols.Since M. genitalium, like most mollicutes, is unable to synthesize de novo purine and pyrimidine bases (27), it must scavenge nucleotides from the host in order to replicate and persist. Only Mycoplasma penetrans has an orotate-related pathway for converting carbamoyl-phosphate to uridine-5Ј-monophosphate (34). The importance of nucleases in the life cycle of mycoplasmas is reinforced by their detection in at least 20 Mycoplasma species (26). Purification of membrane-associated Ca 2ϩ /Mg 2ϩ -dependent M. penetrans and Mycoplasma hyorhinis nucleases and their relation to mycoplasma survival and pathogenesis have been reported (7,8,29,30). Also, a membrane nuclease gene, ...
SUMMARY Community-acquired respiratory distress syndrome (CARDS) toxin from Mycoplasma pneumoniae is a 591 amino acid virulence factor with ADP-ribosyltransferase (ADPRT) and vacuolating activities. It is expressed at low levels during in vitro growth and at high levels during colonization of the lung. Exposure of experimental animals to purified recombinant CARDS toxin alone is sufficient to recapitulate the cytopathology and inflammatory responses associated with M. pneumoniae infection in humans and animals. Here, by molecular modeling, serial truncations and site-directed mutagenesis, we show that the N-terminal region is essential for ADP-ribosylating activity. Also, by systematic truncation and limited proteolysis experiments we identified a portion of the C-terminal region that mediates toxin binding to mammalian cell surfaces and subsequent internalization. In addition, the C-terminal region alone induces vacuolization in a manner similar to full-length toxin. Together, these data suggest that CARDS toxin has a unique architecture with functionally separable N-terminal and C-terminal domains.
There are 160,000 cancer patients worldwide treated with particle radiotherapy (RT). With the advent of proton, and high (H) charge (Z) and energy (E) HZE ionizing particle RT, the cardiovascular diseases risk estimates are uncertain. In addition, future deep space exploratory-type missions will expose humans to unknown but low doses of particle irradiation (IR). We examined molecular responses using transcriptome profiling in left ventricular murine cardiomyocytes isolated from mice that were exposed to 90 cGy, 1 GeV proton ((1)H) and 15 cGy, 1 GeV/nucleon iron ((56)Fe) over 28 days after exposure. Unsupervised clustering analysis of gene expression segregated samples according to the IR response and time after exposure, with (56)Fe-IR showing the greatest level of gene modulation. (1)H-IR showed little differential transcript modulation. Network analysis categorized the major differentially expressed genes into cell cycle, oxidative responses, and transcriptional regulation functional groups. Transcriptional networks identified key nodes regulating expression. Validation of the signal transduction network by protein analysis and gel shift assay showed that particle IR clearly regulates a long-lived signaling mechanism for ERK1/2, p38 MAPK signaling and identified NFATc4, GATA4, STAT3, and NF-κB as regulators of the response at specific time points. These data suggest that the molecular responses and gene expression to (56)Fe-IR in cardiomyocytes are unique and long-lasting. Our study may have significant implications for the efforts of National Aeronautics and Space Administration to develop heart disease risk estimates for astronauts and for patients receiving conventional and particle RT via identification of specific HZE-IR molecular markers.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.