RESUMEN
PURPOSE OF REVIEW: We wished to overview recent data on a subset of epigenetic changes elicited by intracellular bacteria in human cells. Reprogramming the gene expression pattern of various host cells may facilitate bacterial growth, survival, and spread. RECENT FINDINGS: DNA-(cytosine C5)-methyltransferases of Mycoplasma hyorhinis targeting cytosine-phosphate-guanine (CpG) dinucleotides and a Mycobacterium tuberculosis methyltransferase targeting non-CpG sites methylated the host cell DNA and altered the pattern of gene expression. Gene silencing by CpG methylation and histone deacetylation, mediated by cellular enzymes, also occurred in M. tuberculosis-infected macrophages. M. tuberculosis elicited cell type-specific epigenetic changes: it caused increased DNA methylation in macrophages, but induced demethylation, deposition of euchromatic histone marks and activation of immune-related genes in dendritic cells. A secreted transposase of Acinetobacter baumannii silenced a cellular gene, whereas Mycobacterium leprae altered the epigenotype, phenotype, and fate of infected Schwann cells. The 'keystone pathogen' oral bacterium Porphyromonas gingivalis induced local DNA methylation and increased the level of histone acetylation in host cells. These epigenetic changes at the biofilm-gingiva interface may contribute to the development of periodontitis. SUMMARY: Epigenetic regulators produced by intracellular bacteria alter the epigenotype and gene expression pattern of host cells and play an important role in pathogenesis.
Asunto(s)
Bacterias/patogenicidad , Epigénesis Genética , Regulación Bacteriana de la Expresión Génica/genética , Acinetobacter baumannii/enzimología , Acinetobacter baumannii/patogenicidad , Bacterias/enzimología , Metilación de ADN , Células Dendríticas/metabolismo , Células Dendríticas/microbiología , Humanos , Macrófagos/metabolismo , Macrófagos/microbiología , Mycobacterium leprae/enzimología , Mycobacterium leprae/patogenicidad , Mycobacterium tuberculosis/enzimología , Mycobacterium tuberculosis/patogenicidad , Mycoplasma hyorhinis/enzimología , Mycoplasma hyorhinis/patogenicidad , Porphyromonas gingivalis/enzimología , Porphyromonas gingivalis/patogenicidad , Células de Schwann/metabolismo , Células de Schwann/microbiologíaRESUMEN
Microbial proteases degrade a variety of host proteins(1-3). However, it has remained largely unknown why microorganisms have evolved to acquire such proteases and how the host responds to microbially degraded products. Here, we have found that immunoglobulins disrupted by microbial pathogens are specifically detected by leukocyte immunoglobulin-like receptor A2 (LILRA2), an orphan activating receptor expressed on human myeloid cells. Proteases from Mycoplasma hyorhinis, Legionella pneumophila, Streptococcus pneumonia and Candida albicans cleaved the N-terminus of immunoglobulins. Identification of the immunoglobulin-cleaving protease from L. pneumophila revealed that the protease is conserved across some bacteria including Vibrio spp. and Pseudomonas aeruginosa. These microbially cleaved immunoglobulins but not normal immunoglobulins stimulated human neutrophils via LILRA2. In addition, stimulation of primary monocytes via LILRA2 inhibited the growth of L. pneumophila. When mice were infected with L. pneumophila, immunoglobulins were cleaved and recognized by LILRA2. More importantly, cleaved immunoglobulins were detected in patients with bacterial infections and stimulated LILRA2-expressing cells. Our findings demonstrate that LILRA2 is a type of innate immune receptor in the host immune system that detects immunoglobulin abnormalities caused by microbial pathogens.
Asunto(s)
Bacterias/enzimología , Inmunoglobulinas/metabolismo , Péptido Hidrolasas/metabolismo , Receptores Inmunológicos/inmunología , Animales , Bacterias/metabolismo , Citocinas/metabolismo , Humanos , Inmunidad Innata , Inmunoglobulinas/farmacología , Legionella pneumophila/efectos de los fármacos , Legionella pneumophila/enzimología , Legionella pneumophila/crecimiento & desarrollo , Legionella pneumophila/inmunología , Enfermedad de los Legionarios , Ratones , Monocitos/efectos de los fármacos , Monocitos/microbiología , Mycoplasma hyorhinis/enzimología , Neutrófilos/efectos de los fármacos , Receptores Inmunológicos/metabolismo , Streptococcus pneumoniae/enzimologíaRESUMEN
Mycoplasma hyorhinis is commonly found in the respiratory tract of pigs and is the etiological agent of polyserositis. The metabolic enzymes of M. hyorhinis may play important roles in host-pathogen interactions. We immunized BALB/c mice with sodium deoxycholate-extracted antigens (DOC-Ags) and screened 10 hybridomas that secreted antibodies against various M. hyorhinis proteins. Pyruvate dehydrogenase E1 complex subunit alpha (PDHA) was identified as the protein that reacted with five of the 10 monoclonal antibodies (mAbs). Sequence analysis indicated that PDHA was highly conserved among M. hyorhinis strains, but not among other mycoplasmas. We predicted the three-dimensional structure of PDHA and identified three epitopes ((277)RTEEEEK(283), (299)KDKKYITDE(307), and (350)LKEQKQHAKDY(360)). The mAb 1H12 we generated was used to detect M. hyorhinis PDHA in vitro and in piglets infected with M. hyorhinis. We observed that PDHA was mainly located in the epithelial cells of the lungs. Our results indicate that the mAbs we generated could be used to further investigate the structure and function of M. hyorhinis PDHA. In addition, they could be used in the differential diagnosis of M. hyorhinis and other mycoplasmas.
Asunto(s)
Anticuerpos Monoclonales/análisis , Proteínas Bacterianas/inmunología , Infecciones por Mycoplasma/veterinaria , Mycoplasma hyorhinis/aislamiento & purificación , Piruvato Deshidrogenasa (Lipoamida)/inmunología , Enfermedades de los Porcinos/microbiología , Animales , Anticuerpos Monoclonales/inmunología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Mapeo Epitopo , Femenino , Ratones , Ratones Endogámicos BALB C , Infecciones por Mycoplasma/diagnóstico , Infecciones por Mycoplasma/inmunología , Infecciones por Mycoplasma/microbiología , Mycoplasma hyorhinis/enzimología , Mycoplasma hyorhinis/genética , Mycoplasma hyorhinis/inmunología , Piruvato Deshidrogenasa (Lipoamida)/química , Piruvato Deshidrogenasa (Lipoamida)/genética , Porcinos , Enfermedades de los Porcinos/diagnóstico , Enfermedades de los Porcinos/inmunologíaRESUMEN
Mycoplasmas are opportunistic parasites and some species are suggested to preferentially colonize tumor tissue in cancer patients. We could demonstrate that the annotated thymidine phosphorylase (TP) gene in the genome of Mycoplasma hyorhinis encodes a pyrimidine nucleoside phosphorylase (PyNPHyor) that not only efficiently catalyzes thymidine but also uridine phosphorolysis. The kinetic characteristics of PyNPHyor-catalyzed nucleoside and nucleoside analogue (NA) phosphorolysis were determined. We demonstrated that the expression of such an enzyme in mycoplasma-infected cell cultures dramatically alters the activity of various anticancer/antiviral NAs such as 5-halogenated pyrimidine nucleosides, including 5-trifluorothymidine (TFT). Due to their close association with human cancers, the presence of mycoplasmas may markedly influence the therapeutic efficiency of nucleoside-based drugs.
Asunto(s)
Antivirales/farmacología , Mycoplasma hyorhinis/enzimología , Pirimidina Fosforilasas/metabolismo , Trifluridina/farmacología , Línea Celular Tumoral , Humanos , Células MCF-7 , Mycoplasma hyorhinis/fisiología , Pirimidina Fosforilasas/genéticaRESUMEN
The intracellular metabolism and cytostatic activity of the anticancer drug gemcitabine (2',2'-difluoro-2'-deoxycytidine; dFdC) was severely compromised in Mycoplasma hyorhinis-infected tumor cell cultures. Pronounced deamination of dFdC to its less cytostatic metabolite 2',2'-difluoro-2'-deoxyuridine was observed, both in cell extracts and spent culture medium (i.e. tumor cell-free but mycoplasma-containing) of mycoplasma-infected tumor cells. This indicates that the decreased antiproliferative activity of dFdC in such cells is attributed to a mycoplasma cytidine deaminase causing rapid drug catabolism. Indeed, the cytostatic activity of gemcitabine could be restored by the co-administration of tetrahydrouridine (a potent cytidine deaminase inhibitor). Additionally, mycoplasma-derived pyrimidine nucleoside phosphorylase (PyNP) activity indirectly potentiated deamination of dFdC: the natural pyrimidine nucleosides uridine, 2'-deoxyuridine and thymidine inhibited mycoplasma-associated dFdC deamination but were efficiently catabolized (removed) by mycoplasma PyNP. The markedly lower anabolism and related cytostatic activity of dFdC in mycoplasma-infected tumor cells was therefore also (partially) restored by a specific TP/PyNP inhibitor (TPI), or by exogenous thymidine. Consequently, no effect on the cytostatic activity of dFdC was observed in tumor cell cultures infected with a PyNP-deficient Mycoplasma pneumoniae strain. Because it has been reported that some commensal mycoplasma species (including M. hyorhinis) preferentially colonize tumor tissue in cancer patients, our findings suggest that the presence of mycoplasmas in the tumor microenvironment could be a limiting factor for the anticancer efficiency of dFdC-based chemotherapy. Accordingly, a significantly decreased antitumor effect of dFdC was observed in mice bearing M. hyorhinis-infected murine mammary FM3A tumors compared with uninfected tumors.
Asunto(s)
Antimetabolitos Antineoplásicos , Proteínas Bacterianas/metabolismo , Neoplasias de la Mama , Desoxicitidina/análogos & derivados , Neoplasias Mamarias Experimentales , Infecciones por Mycoplasma/enzimología , Mycoplasma hyorhinis/enzimología , Pirimidina Fosforilasas/metabolismo , Animales , Antimetabolitos Antineoplásicos/farmacocinética , Antimetabolitos Antineoplásicos/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/microbiología , Línea Celular Tumoral , Desoxicitidina/farmacocinética , Desoxicitidina/farmacología , Femenino , Humanos , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Neoplasias Mamarias Experimentales/metabolismo , Neoplasias Mamarias Experimentales/microbiología , Ratones , Tetrahidrouridina/farmacocinética , Tetrahidrouridina/farmacología , Timidina/metabolismo , Microambiente Tumoral/efectos de los fármacos , GemcitabinaRESUMEN
A mycoplasma-encoded purine nucleoside phosphorylase (designated PNPHyor) has been cloned and characterized for the first time. Efficient phosphorolysis of natural 6-oxopurine and 6-aminopurine nucleosides was observed, with adenosine the preferred natural substrate (Km = 61 µM). Several cytostatic purine nucleoside analogs proved to be susceptible to PNPHyor-mediated phosphorolysis, and a markedly decreased or increased cytostatic activity was observed in Mycoplasma hyorhinis-infected human breast carcinoma MCF-7 cell cultures (MCF-7.Hyor), depending on the properties of the released purine base. We demonstrated an â¼10-fold loss of cytostatic activity of cladribine in MCF-7.Hyor cells and observed a rapid and complete phosphorolysis of this drug when it was exposed to the supernatant of mycoplasma-infected cells. This conversion (inactivation) could be prevented by a specific PNP inhibitor. These findings correlated well with the high efficiency of PNPHyor-catalyzed phosphorolysis of cladribine to its less toxic base 2-chloroadenine (Km = 80 µM). In contrast, the cytostatic activity of nucleoside analogs carrying a highly toxic purine base and being a substrate for PNPHyor, but not human PNP, was substantially increased in MCF-7.Hyor cells (â¼130-fold for fludarabine and â¼45-fold for 6-methylpurine-2'-deoxyriboside). Elimination of the mycoplasma from the tumor cell cultures or selective inhibition of PNPHyor by a PNP inhibitor restored the cytostatic activity of the purine-based nucleoside drugs. Since several studies suggest a high and preferential colonization or association of tumor tissue in cancer patients with different prokaryotes (including mycoplasmas), the data presented here may be of relevance for the optimization of purine nucleoside-based anticancer drug treatment.
Asunto(s)
Antineoplásicos/farmacología , Mycoplasma hyorhinis/enzimología , Purina-Nucleósido Fosforilasa/metabolismo , Purinas/farmacología , Antineoplásicos/metabolismo , Línea Celular Tumoral , Cladribina/metabolismo , Cladribina/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Cinética , Mycoplasma hyorhinis/genética , Nucleósidos de Purina/metabolismo , Nucleósidos de Purina/farmacología , Purina-Nucleósido Fosforilasa/antagonistas & inhibidores , Purina-Nucleósido Fosforilasa/genética , Purinas/metabolismo , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Mycoplasma hyorhinis, the major contaminant of tissue cultures, has been implicated in a variety of diseases in swine. Most human and animal mycoplasmas remain attached to the surface of epithelial cells. Nonetheless, we have recently shown that M. hyorhinis is able to invade and survive within nonphagocytic melanoma cells. The invasion process may require the damaging of the host cell membrane by either chemical, physical or enzymatic means. In this study, we show that M. hyorhinis membranes possess a nonspecific phospholipase A (PLA) activity capable of hydrolyzing both position 1 and position 2 of 1-acyl-2-(12-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] aminododecanoyl) phosphatidylcholine. In silico analysis of the M. hyorhinis genome shows that the PLA of M. hyorhinis shares no homology to described phospholipases. The PLA activity of M. hyorhinis was neither stimulated by Ca (2+) nor inhibited by EGTA and had a broad pH spectrum. Mycoplasma hyorhinis also possess a potent glycerophosphodiesterase (GPD), which apparently cleaves the glycerophosphodiester formed by PLA to yield glycerol-3-phosphate. Possible roles of PLA and GPD in invading host eukaryotic cells and in forming mediators upon the interaction of M. hyorhinis with eukaryotic cells are suggested.
Asunto(s)
Proteínas Bacterianas/metabolismo , Mycoplasma hyorhinis/enzimología , Fosfolipasas A/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , 4-Cloro-7-nitrobenzofurazano/análogos & derivados , 4-Cloro-7-nitrobenzofurazano/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Membrana Celular/enzimología , Colorantes Fluorescentes , Hidrólisis , Datos de Secuencia Molecular , Mycoplasma hyorhinis/citología , Fosfatidilcolinas/metabolismo , Fosfolipasas A/química , Hidrolasas Diéster Fosfóricas/química , Alineación de SecuenciaRESUMEN
In the present paper we demonstrate that the cytostatic and antiviral activity of pyrimidine nucleoside analogues is markedly decreased by a Mycoplasma hyorhinis infection and show that the phosphorolytic activity of the mycoplasmas is responsible for this. Since mycoplasmas are (i) an important cause of secondary infections in immunocompromised (e.g. HIV infected) patients and (ii) known to preferentially colonize tumour tissue in cancer patients, catabolic mycoplasma enzymes may compromise efficient chemotherapy of virus infections and cancer. In the genome of M. hyorhinis, a TP (thymidine phosphorylase) gene has been annotated. This gene was cloned, expressed in Escherichia coli and kinetically characterized. Whereas the mycoplasma TP efficiently catalyses the phosphorolysis of thymidine (Km=473 µM) and deoxyuridine (Km=578 µM), it prefers uridine (Km=92 µM) as a substrate. Our kinetic data and sequence analysis revealed that the annotated M. hyorhinis TP belongs to the NP (nucleoside phosphorylase)-II class PyNPs (pyrimidine NPs), and is distinct from the NP-II class TP and NP-I class UPs (uridine phosphorylases). M. hyorhinis PyNP also markedly differs from TP and UP in its substrate specificity towards therapeutic nucleoside analogues and susceptibility to clinically relevant drugs. Several kinetic properties of mycoplasma PyNP were explained by in silico analyses.
Asunto(s)
Neoplasias de la Mama/virología , Infecciones por Mycoplasma , Mycoplasma hyorhinis/enzimología , Nucleósidos de Pirimidina/metabolismo , Timidina Fosforilasa/metabolismo , Uridina Fosforilasa/metabolismo , Secuencia de Aminoácidos , Antivirales/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/enzimología , Bromodesoxiuridina/análogos & derivados , Bromodesoxiuridina/farmacología , Biología Computacional , Femenino , Humanos , Idoxuridina/farmacología , Cinética , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Células Tumorales Cultivadas , Virus/efectos de los fármacosRESUMEN
The fluorinated pyrimidine family of nucleosides continues to represent major current chemotherapeutic agents for treating solid tumors. We herein report their phosphate prodrugs, ProTides, as promising new derivatives, which partially bypass the dependence of the current drugs on active transport and nucleoside kinase-mediated activation. They are also resistant to metabolic deactivation by phosphorolytic enzymes. We report 39 ProTides of the fluorinated pyrimidine FUDR with variation in the aryl, ester, and amino acid regions. Notably, only certain ProTide motifs are successful in delivering the nucleoside monophosphate into intact cells. We also find that the ProTides retain activity in mycoplasma infected cells, unlike FUDR. Data suggest these compounds to be worthy of further progression.
Asunto(s)
Antineoplásicos/síntesis química , Floxuridina/análogos & derivados , Floxuridina/síntesis química , Compuestos Organofosforados/síntesis química , Profármacos/síntesis química , Antineoplásicos/farmacología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Resistencia a Antineoplásicos , Ensayos de Selección de Medicamentos Antitumorales , Tranportador Equilibrativo 1 de Nucleósido/genética , Floxuridina/farmacología , Humanos , Mycoplasma hyorhinis/enzimología , Compuestos Organofosforados/farmacología , Profármacos/farmacología , Pirimidina Fosforilasas/metabolismo , Relación Estructura-ActividadRESUMEN
Nucleoside analogues are widely used as chemotherapeutic agents in the treatment of cancer. Several cancers are reported to be associated with mycoplasmas (i.e. Mycoplasma hyorhinis), which contain a number of nucleoside-metabolizing enzymes. Pyrimidine nucleoside analogues, such as 5-fluoro-2'-deoxyuridine (FdUrd), 5-trifluorothymidine (TFT) and 5-halogenated 2'-deoxyuridines can be degraded by thymidine phosphorylase (TP) to their inactive bases. We found in M. hyorhinis-infected MCF-7 breast carcinoma cells (MCF-7/HYOR) a mycoplasma-encoded TP that dramatically (20-150-fold) reduces the cytostatic activity of these compounds. The reduction in cytostatic activity could be fully restored in the presence of TPI (5-chloro-6-[1-(2-iminopyrrolidinyl)methyl]uracil hydrochloride), a known inhibitor of human TP. This observation is in agreement with the markedly decreased formation of active metabolite (i.e. FdUMP for FdUrd) or diminished drug incorporation into nucleic acids (i.e. for TFT and 5-bromo-2'-deoxyuridine) in MCF-7/HYOR cells compared with uninfected MCF-7 cells. Antimetabolite formation is fully restored in the presence of TPI. In contrast, 5-fluoro-5'-deoxyuridine (5'DFUR), an intermediate metabolite of capecitabine, was markedly more cytostatic in MCF-7/HYOR cells than in uninfected cells, due to the activation of this prodrug by the mycoplasma-encoded TP. Thus, our data reveal that M. hyorhinis expresses a TP that activates 5'DFUR but inactivates FdUrd, TFT and 5-halogenated 2'-deoxyuridines, and that is highly sensitive to the inhibitory effect of the TP inhibitor TPI. Given the association of M. hyorhinis with several human cancers, our findings suggest that pyrimidine nucleoside-based but not 5FU-based anti-cancer therapy might be more effective when combined with a mycoplasmal TP inhibitor.
Asunto(s)
Antineoplásicos/farmacología , Infecciones por Mycoplasma , Mycoplasma hyorhinis/enzimología , Nucleósidos de Pirimidina/farmacología , Timidina Fosforilasa/antagonistas & inhibidores , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Técnicas de Cocultivo , Humanos , Timidina Fosforilasa/metabolismoRESUMEN
BACKGROUND: Mycoplasma contaminations are a recurrent problem in the use of cultured cells, including human cells, especially as it has been shown to impede cell cycle, triggering cell death under various conditions. More specific consequences on cell metabolism are poorly known. RESULTS: Here we report the lack of significant consequence of a heavy contamination by the frequently encountered mycoplasma strain, M. hyorhinis, on the determination of respiratory chain activities, but the potential interference when assaying citrate synthase. Contamination by M. hyorhinis was detected by fluorescent imaging and further quantified by the determination of the mycoplasma-specific phosphate acetyltransferase activity. Noticeably, this latter activity was not found equally distributed in various mycoplasma types, being exceptionally high in M. hyorhinis. CONCLUSION: While we observed a trend for respiration reduction in heavily contaminated cells, no significant and specific targeting of any respiratory chain components could be identified. This suggested a potential interference with cell metabolism rather than direct interaction with respiratory chain components.