RESUMEN
Mitochondrial gene expression in trypanosomes requires numerous multiprotein complexes that are unique to kinetoplastids. Among these, the most well characterized are RNA editing catalytic complexes (RECCs) that catalyze the guide RNA (gRNA)-specified insertion and deletion of uridines during mitochondrial mRNA maturation. This post-transcriptional resequencing of mitochondrial mRNAs can be extensive, involving dozens of different gRNAs and hundreds of editing sites with most of the mature mRNA sequences resulting from the editing process. Proper coordination of the editing with the cognate gRNAs is attributed to RNA editing substrate-binding complexes (RESCs), which are also required for RNA editing. Although the precise mechanism of RESC function is less well understood, their affinity for binding both editing substrates and products suggests that these complexes may provide a scaffold for RECC catalytic processing. KRGG1 has been shown to bind RNAs, and although affinity purification co-isolates RESC complexes, its role in RNA editing remains uncertain. We show here that KRGG1 is essential in BF parasites and required for normal editing. KRGG1 repression results in reduced amounts of edited A6 mRNA and increased amounts of edited ND8 mRNA. Sequence and structure analysis of KRGG1 identified a region of homology with RESC6, and both proteins have predicted tandem helical repeats that resemble ARM/HEAT motifs. The ARM/HEAT-like region is critical for function as exclusive expression of mutated KRGG1 results in growth inhibition and disruption of KRGG1 association with RESCs. These results indicate that KRGG1 is critical for RNA editing and its specific function is associated with RESC activity.
Asunto(s)
Edición de ARN , Trypanosoma brucei brucei , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Guía de Kinetoplastida/genética , ARN Protozoario/genética , ARN Protozoario/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
BACKGROUND: Group B Streptococcus (GBS) or Streptococcus agalactiae are ß-hemolytic gram-positive bacteria that colonize the lower genital tracts of women and are frequently associated with infections during pregnancy. Innate immune defenses are critical for controlling GBS dissemination and systemic infection. Mast cells are resident sentinel cells that come into contact with pathogens early during colonization and infection. OBJECTIVE: We aimed to investigate the contribution of chymase to systemic GBS infection and rates of preterm birth. METHODS: Pharmacologic and genetic approaches using mice deficient in mast cell protease (MCPT) 4, the mouse functional homologue of human chymase, were used. RESULTS: Our studies show that mast cells release a protease with chymotrypsin-like cleavage specificity in response to GBS. Additionally, increased GBS systemic infection and preterm births were observed in MCPT4-deficient mice versus MCPT4-sufficient mice. Furthermore, we observed that proteolytic cleavage of the host extracellular matrix protein fibronectin by peritoneal cell-derived mast cell lysates diminished GBS adherence. Consistent with this observation, the increase in GBS dissemination and preterm births observed in MCPT4-deficient mice was abolished when GBS was deficient in expression of the fibronectin-binding protein SfbA. CONCLUSIONS: Taken together, our results suggest that the protective effect of MCPT4 against GBS dissemination and preterm labor can be attributed in part to MCPT4-mediated proteolysis of fibronectin. Our studies reveal a novel role of mast cells in defense against bacterial infections.
Asunto(s)
Mastocitos/inmunología , Serina Endopeptidasas/inmunología , Infecciones Estreptocócicas/inmunología , Animales , Quimasas/inmunología , Femenino , Mastocitos/enzimología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Embarazo , Complicaciones Infecciosas del Embarazo/enzimología , Complicaciones Infecciosas del Embarazo/inmunología , Nacimiento Prematuro/inmunología , Nacimiento Prematuro/microbiologíaRESUMEN
Group B streptococci (GBS) are encapsulated, ß-hemolytic bacteria that are a common cause of infections in human newborns and certain adults. Two factors important for GBS virulence are the sialic acid capsular polysaccharide that promotes immune evasion and the hemolytic pigment that induces host cell cytotoxcity. These virulence factors are often oppositely regulated by the CovR/CovS two-component system. Clinical GBS strains exhibiting hyperhemolysis and low capsule due to pathoadaptive covR/S mutations have been isolated from patients. Given the importance of capsule to GBS virulence, we predicted that a decrease or loss of capsule would attenuate the virulence of covR/S mutants. Surprisingly, hyperhemolytic GBS with low or no capsule exhibit increased virulence, intracellular persistence, and blood-brain barrier penetration, which was independent of a Trojan horse mechanism of barrier penetration. Additionally, intracellular persistence enabled both hemolytic and hyperhemolytic GBS to evade antibiotics routinely used to treat these infections. The finding that diminished capsule expression promotes GBS virulence, intracellular persistence, and antibiotic evasion has important implications for sustained antibiotic therapy and efficacy of capsule-based vaccines.
Asunto(s)
Antibacterianos/farmacología , Cápsulas Bacterianas/genética , Farmacorresistencia Bacteriana/genética , Streptococcus agalactiae/citología , Streptococcus agalactiae/patogenicidad , Animales , Barrera Hematoencefálica , Humanos , Macrófagos , Ratones , Ratones Endogámicos C57BL , Mutación , Streptococcus agalactiae/efectos de los fármacos , Streptococcus agalactiae/fisiología , VirulenciaRESUMEN
Group B streptococci (GBS) are Gram-positive bacteria that are a leading cause of neonatal infections. Most invasive isolates are ß-hemolytic, and hemolytic activity is critical for GBS virulence. Although nonhemolytic GBS strains are occasionally isolated, they are often thought to be virulence attenuated. In this study, we show that a nonhemolytic GBS strain (GB37) isolated from a septic neonate exhibits hypervirulence. Substitution of tryptophan to leucine (W297L) in the sensor histidine kinase CovS results in constitutive kinase signaling, leading to decreased hemolysis and increased activity of the GBS hyaluronidase, HylB. These results describe how nonpigmented and nonhemolytic GBS strains can exhibit hypervirulence.
Asunto(s)
Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/patogenicidad , Sustitución de Aminoácidos , Animales , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Histidina Quinasa/química , Histidina Quinasa/metabolismo , Humanos , Hialuronoglucosaminidasa/metabolismo , Recién Nacido , Leucina , Ratones , Infecciones Estreptocócicas/patología , Streptococcus agalactiae/genética , Triptófano , VirulenciaRESUMEN
Group B streptococci (GBS; Streptococcus agalactiae) are beta-hemolytic, Gram-positive bacteria that are common asymptomatic colonizers of healthy adults. However, these opportunistic bacteria also cause invasive infections in human newborns and in certain adult populations. To adapt to the various environments encountered during its disease cycle, GBS encodes a number of two-component signaling systems. Previous studies have indicated that the TCS comprising the sensor histidine kinase RgfC and the response regulator RgfA mediate GBS binding to extracellular matrix components, such as fibrinogen. However, in certain GBS clinical isolates, a point mutation in rgfA results in premature truncation of the response regulator. The truncated RgfA protein lacks the C-terminal DNA binding domain necessary for promoter binding and gene regulation. Here, we show that deletion of rgfC in GBS strains lacking a functional RgfA increased systemic infection. Furthermore, infection with the rgfC mutant increased induction of proinflammatory signaling pathways in vivo. Phosphoproteomic analysis revealed that 19 phosphopeptides corresponding to 12 proteins were differentially phosphorylated at aspartate, cysteine, serine, threonine, or tyrosine residues in the rgfC mutant. This included aspartate phosphorylation of a tyrosine kinase, CpsD, and a transcriptional regulator. Consistent with this observation, microarray analysis of the rgfC mutant indicated that >200 genes showed altered expression compared to the isogenic wild-type strain and included transcriptional regulators, transporters, and genes previously associated with GBS pathogenesis. Our observations suggest that in the absence of RgfA, nonspecific RgfC signaling affects the expression of virulence factors and GBS pathogenesis.
Asunto(s)
Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Quinasas/genética , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/genética , Streptococcus agalactiae/patogenicidad , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/metabolismo , Encéfalo/metabolismo , Encéfalo/microbiología , Encéfalo/patología , Línea Celular , Células Endoteliales/metabolismo , Células Endoteliales/microbiología , Células Endoteliales/patología , Perfilación de la Expresión Génica , Histidina Quinasa , Humanos , Masculino , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , Mutación , Fosforilación , Regiones Promotoras Genéticas , Proteínas Quinasas/metabolismo , Alineación de Secuencia , Transducción de Señal , Infecciones Estreptocócicas/patología , Streptococcus agalactiae/metabolismo , Transcripción Genética , VirulenciaRESUMEN
The type III secretion system (T3SS) is a complex macromolecular machinery employed by a number of Gram-negative pathogens to inject effectors directly into the cytoplasm of eukaryotic cells. ExoU from the opportunistic pathogen Pseudomonas aeruginosa is one of the most aggressive toxins injected by a T3SS, leading to rapid cell necrosis. Here we report the crystal structure of ExoU in complex with its chaperone, SpcU. ExoU folds into membrane-binding, bridging, and phospholipase domains. SpcU maintains the N-terminus of ExoU in an unfolded state, required for secretion. The phospholipase domain carries an embedded catalytic site whose position within ExoU does not permit direct interaction with the bilayer, which suggests that ExoU must undergo a conformational rearrangement in order to access lipids within the target membrane. The bridging domain connects catalytic domain and membrane-binding domains, the latter of which displays specificity to PI(4,5)P2. Both transfection experiments and infection of eukaryotic cells with ExoU-secreting bacteria show that ExoU ubiquitination results in its co-localization with endosomal markers. This could reflect an attempt of the infected cell to target ExoU for degradation in order to protect itself from its aggressive cytotoxic action.
Asunto(s)
Proteínas Bacterianas , Sistemas de Secreción Bacterianos , Toxinas Bacterianas , Pliegue de Proteína , Infecciones por Pseudomonas/metabolismo , Pseudomonas aeruginosa , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Células HeLa , Humanos , Chaperonas Moleculares/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/química , Pseudomonas aeruginosa/metabolismo , Relación Estructura-Actividad , UbiquitinaciónRESUMEN
Invasive bacterial infections remain a major cause of human morbidity. Group B streptococcus (GBS) are Gram-positive bacteria that cause invasive infections in humans. Here, we show that factor XIIIA-deficient (FXIIIA-deficient) female mice exhibited significantly increased susceptibility to GBS infections. Additionally, female WT mice had increased levels of FXIIIA and were more resistant to GBS infection compared with isogenic male mice. We observed that administration of exogenous FXIIIA to male mice increased host resistance to GBS infection. Conversely, administration of a FXIIIA transglutaminase inhibitor to female mice decreased host resistance to GBS infection. Interestingly, male gonadectomized mice exhibited decreased sensitivity to GBS infection, suggesting a role for gonadal androgens in host susceptibility. FXIIIA promoted GBS entrapment within fibrin clots by crosslinking fibronectin with ScpB, a fibronectin-binding GBS surface protein. Thus, ScpB-deficient GBS exhibited decreased entrapment within fibrin clots in vitro and increased dissemination during systemic infections. Finally, using mice in which FXIIIA expression was depleted in mast cells, we observed that mast cell-derived FXIIIA contributes to host defense against GBS infection. Our studies provide insights into the effects of sexual dimorphism and mast cells on FXIIIA expression and its interactions with GBS adhesins that mediate bacterial dissemination and pathogenesis.
Asunto(s)
Factor XIIIa , Infecciones Estreptocócicas , Andrógenos/metabolismo , Animales , Factor XIIIa/metabolismo , Femenino , Fibrina/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Masculino , Mastocitos/metabolismo , Ratones , Infecciones Estreptocócicas/genética , Streptococcus agalactiae/metabolismo , Transglutaminasas/metabolismoRESUMEN
Invasive bacterial infections during pregnancy are a major risk factor for preterm birth, stillbirth, and fetal injury. Group B streptococci (GBS) are Gram-positive bacteria that asymptomatically colonize the lower genital tract but infect the amniotic fluid and induce preterm birth or stillbirth. Experimental models that closely emulate human pregnancy are pivotal for the development of successful strategies to prevent these adverse pregnancy outcomes. Using a unique nonhuman primate model that mimics human pregnancy and informs temporal events surrounding amniotic cavity invasion and preterm labor, we show that the animals inoculated with hyaluronidase (HylB)-expressing GBS consistently exhibited microbial invasion into the amniotic cavity, fetal bacteremia, and preterm labor. Although delayed cytokine responses were observed at the maternal-fetal interface, increased prostaglandin and matrix metalloproteinase levels in these animals likely mediated preterm labor. HylB-proficient GBS dampened reactive oxygen species production and exhibited increased resistance to neutrophils compared to an isogenic mutant. Together, these findings demonstrate how a bacterial enzyme promotes GBS amniotic cavity invasion and preterm labor in a model that closely resembles human pregnancy.IMPORTANCE Group B streptococci (GBS) are bacteria that commonly reside in the female lower genital tract as asymptomatic members of the microbiota. However, during pregnancy, GBS can infect tissues at the maternal-fetal interface, leading to preterm birth, stillbirth, or fetal injury. Understanding how GBS evade host defenses during pregnancy is key to developing improved preventive therapies for these adverse outcomes. In this study, we used a unique nonhuman primate model to show that an enzyme secreted by GBS, hyaluronidase (HylB) promotes bacterial invasion into the amniotic cavity and fetus. Although delayed immune responses were seen at the maternal-fetal interface, animals infected with hyaluronidase-expressing GBS exhibited premature cervical ripening and preterm labor. These observations reveal that HylB is a crucial GBS virulence factor that promotes bacterial invasion and preterm labor in a pregnancy model that closely emulates human pregnancy. Therefore, hyaluronidase inhibitors may be useful in therapeutic strategies against ascending GBS infection.
Asunto(s)
Hialuronoglucosaminidasa/metabolismo , Neutrófilos/inmunología , Trabajo de Parto Prematuro/inmunología , Infecciones Estreptocócicas/inmunología , Streptococcus agalactiae/metabolismo , Líquido Amniótico/microbiología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Humanos , Hialuronoglucosaminidasa/genética , Inflamación , Pulmón/microbiología , Pulmón/patología , Macaca nemestrina , Neutrófilos/microbiología , Embarazo , Nacimiento Prematuro , Primates , Infecciones Estreptocócicas/metabolismo , Infecciones Estreptocócicas/microbiología , Streptococcus agalactiae/enzimología , Streptococcus agalactiae/genética , Streptococcus agalactiae/inmunologíaRESUMEN
Most Apicomplexa reside and multiply in the cytoplasm of their host cell, within a parasitophorous vacuole (PV) originating from both parasite and host cell components. Trafficking of parasite-encoded proteins destined to membrane compartments beyond the confine of the parasite plasma membrane is a process that offers a rich territory to explore novel mechanisms of protein-membrane interactions. Here, we focus on the PVs formed by the asexual stages of two pathogens of medical importance, Plasmodium and Toxoplasma. We compare the PVs of both parasites, with a particular emphasis on their evolutionary divergent compartmentalization within the host cell. We also discuss the existence of peculiar export mechanisms and/or sorting determinants that are potentially involved in the post-secretory targeting of parasite proteins to the PV subcompartments.
Asunto(s)
Apicomplexa/metabolismo , Apicomplexa/patogenicidad , Vacuolas/metabolismo , Animales , Apicomplexa/citología , Membrana Celular/metabolismo , Interacciones Huésped-Parásitos , Plasmodium falciparum/metabolismo , Plasmodium falciparum/patogenicidad , Toxoplasma/metabolismo , Toxoplasma/patogenicidadRESUMEN
A critical step in infection by the apicomplexan parasite Toxoplasma gondii is the formation of a membrane-bound compartment within which the parasite proliferates. This process relies on a set of secretory organelles that discharge their contents into the host cell upon invasion. Among these organelles, the dense granules are specialized in the export of transmembrane (TM) GRA proteins, which are major components of the mature parasitophorous vacuole (PV) membrane. How eukaryotic pathogens export and sort membrane-bound proteins destined for the host cell is still poorly understood at the mechanistic level. In this study, we show that soluble trafficking of the PV-targeted GRA5 TM protein is parasite specific: when expressed in mammalian cells, GRA5 is targeted to the plasma membrane and behaves as an integral membrane protein with a type I toplogy. We also demonstrate the dual role of the GRA5 N-terminal ectodomain, which is sufficient to prevent membrane integration within the parasite and is essential for both sorting and post-secretory membrane insertion into the vacuolar membrane. These results contrast with the general rule that states that information contained within the cytoplasmic tail and/or the TM domain of integral membrane proteins dictates their cellular localization. They also highlight the diversity of sorting mechanisms that leads to the specialization of secretory processes uniquely adapted to intracellular parasitism.
Asunto(s)
Antígenos de Protozoos/metabolismo , Interacciones Huésped-Parásitos/fisiología , Proteínas Protozoarias/metabolismo , Toxoplasma , Vacuolas/parasitología , Animales , Antígenos de Protozoos/genética , Antígenos de Protozoos/fisiología , Línea Celular , Gránulos Citoplasmáticos/fisiología , Gránulos Citoplasmáticos/ultraestructura , Fibroblastos/metabolismo , Fibroblastos/parasitología , Fibroblastos/fisiología , Fibroblastos/ultraestructura , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestructura , Microscopía Fluorescente , Transporte de Proteínas/fisiología , Proteínas Protozoarias/genética , Proteínas Protozoarias/fisiología , Vías Secretoras , Toxoplasma/metabolismo , Toxoplasma/patogenicidad , Toxoplasma/fisiología , Transfección , Vacuolas/metabolismo , Vacuolas/fisiología , Vacuolas/ultraestructuraRESUMEN
Thirteen percent of pregnancies result in preterm birth or stillbirth, accounting for fifteen million preterm births and three and a half million deaths annually. A significant cause of these adverse pregnancy outcomes is in utero infection by vaginal microorganisms. To establish an in utero infection, vaginal microbes enter the uterus by ascending infection; however, the mechanisms by which this occurs are unknown. Using both in vitro and murine models of vaginal colonization and ascending infection, we demonstrate how a vaginal microbe, group B streptococcus (GBS), which is frequently associated with adverse pregnancy outcomes, uses vaginal exfoliation for ascending infection. GBS induces vaginal epithelial exfoliation by activation of integrin and ß-catenin signaling. However, exfoliation did not diminish GBS vaginal colonization as reported for other vaginal microbes. Rather, vaginal exfoliation increased bacterial dissemination and ascending GBS infection, and abrogation of exfoliation reduced ascending infection and improved pregnancy outcomes. Thus, for some vaginal bacteria, exfoliation promotes ascending infection rather than preventing colonization. Our study provides insight into mechanisms of ascending infection by vaginal microbes.
Asunto(s)
Células Epiteliales/inmunología , Infecciones Estreptocócicas/inmunología , Streptococcus agalactiae/inmunología , Vagina/inmunología , Vaginosis Bacteriana/inmunología , Animales , Células Epiteliales/microbiología , Células Epiteliales/patología , Femenino , Ratones , Ratones Noqueados , Infecciones Estreptocócicas/patología , Vagina/microbiología , Vagina/patología , Vaginosis Bacteriana/microbiología , Vaginosis Bacteriana/patologíaRESUMEN
Preterm birth is a leading cause of neonatal morbidity and mortality. Although microbial invasion of the amniotic cavity (MIAC) is associated with the majority of early preterm births, the temporal events that occur during MIAC and preterm labor are not known. Group B Streptococci (GBS) are ß-hemolytic, gram-positive bacteria, which commonly colonize the vagina but have been recovered from the amniotic fluid in preterm birth cases. To understand temporal events that occur during MIAC, we utilized a unique chronically catheterized nonhuman primate model that closely emulates human pregnancy. This model allows monitoring of uterine contractions, timing of MIAC and immune responses during pregnancy-associated infections. Here, we show that adverse outcomes such as preterm labor, MIAC, and fetal sepsis were observed more frequently during infection with hemolytic GBS when compared to nonhemolytic GBS. Although MIAC was associated with systematic progression in chorioamnionitis beginning with chorionic vasculitis and progressing to neutrophilic infiltration, the ability of the GBS hemolytic pigment toxin to induce neutrophil cell death and subvert killing by neutrophil extracellular traps (NETs) in placental membranes in vivo facilitated MIAC and fetal injury. Furthermore, compared to maternal neutrophils, fetal neutrophils exhibit decreased neutrophil elastase activity and impaired phagocytic functions to GBS. Collectively, our studies demonstrate how a unique bacterial hemolytic lipid toxin enables GBS to circumvent neutrophils and NETs in placental membranes to induce fetal injury and preterm labor.
RESUMEN
Group B Streptococci (GBS) are ß-hemolytic, gram-positive bacteria that are typically associated with infections in human newborns or immunocompromised adults. However, mutation in the two-component regulator CovR/S relieves repression of hemolysin, potentially increasing virulence of GBS. We report the isolation of hyperhemolytic/hyperpigmented GBS strain from an adolescent patient who presented to the University of Washington clinic with symptoms of sore throat. While the patient also tested positive for mononucleosis, a GBS strain with increased hemolysis was isolated from the throat swab obtained from the patient. As hyperhemolytic/hyperpigmented GBS strains are typically associated with mutations in the regulator CovR/CovS, we sequenced the covR/S loci in the clinical isolate. An adenine to cytosine mutation resulting in a change in amino acid coding sequence from glutamine at position 120 to proline in CovR (Q120P) was identified. Introduction of the Q120P amino acid substitution in a CovR complementation plasmid abolished complementation of a ΔcovR mutant derived from the wild type GBS serotype Ia strain A909; these results confirm that the hyperhemolysis observed in the clinical isolate is due to the Q120P substitution in CovR. Antibiotic was prescribed and the patient's symptoms resolved without reported complications. This study represents the first report of the isolation of a hyperhemolytic/hyperpigmented GBS strain due to a covR/S mutation from an adolescent patient with persistent sore throat who was also diagnosed with mononucleosis. The isolation of GBS CovR/S mutants indicates their presence in settings of co-infections and includes adolescents.
RESUMEN
Ascending infection of microbes from the lower genital tract into the amniotic cavity increases the risk of preterm birth, stillbirth, and newborn infections. Host defenses that are critical for preventing ascending microbial infection are not completely understood. Group B Streptococcus (GBS) are Gram-positive bacteria that frequently colonize the lower genital tract of healthy women but cause severe infections during pregnancy, leading to preterm birth, stillbirth, or early-onset newborn infections. We recently described that the GBS pigment is hemolytic, and increased pigment expression promotes GBS penetration of human placenta. Here, we show that the GBS hemolytic pigment/lipid toxin and hyperpigmented GBS strains induce mast cell degranulation, leading to the release of preformed and proinflammatory mediators. Mast cell-deficient mice exhibit enhanced bacterial burden, decreased neutrophil mobilization, and decreased immune responses during systemic GBS infection. In a vaginal colonization model, hyperpigmented GBS strains showed increased persistence in mast cell-deficient mice compared to mast cell-proficient mice. Consistent with these observations, fewer rectovaginal GBS isolates from women in their third trimester of pregnancy were hyperpigmented/hyperhemolytic. Our work represents the first example of a bacterial hemolytic lipid that induces mast cell degranulation and emphasizes the role of mast cells in limiting genital colonization by hyperpigmented GBS.
RESUMEN
Group B streptococci (GBS) are Gram-positive bacteria that cause infections in utero and in newborns. We recently showed that the GBS pigment is hemolytic and increased pigment production promotes bacterial penetration of human placenta. However, mechanisms utilized by the hemolytic pigment to induce host cell lysis and the consequence on fetal injury are not known. Here, we show that the GBS pigment induces membrane permeability in artificial lipid bilayers and host cells. Membrane defects induced by the GBS pigment trigger K(+) efflux leading to osmotic lysis of red blood cells or pyroptosis in human macrophages. Macrophages lacking the NLRP3 inflammasome recovered from pigment-induced cell damage. In a murine model of in utero infection, hyperpigmented GBS strains induced fetal injury in both an NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent manner. These results demonstrate that the dual mechanism of action of the bacterial pigment/lipid toxin leading to hemolysis or pyroptosis exacerbates fetal injury and suggest that preventing both activities of the hemolytic lipid is likely critical to reduce GBS fetal injury and preterm birth.
Asunto(s)
Toxinas Bacterianas , Permeabilidad de la Membrana Celular , Enfermedades Fetales , Lípidos de la Membrana , Piroptosis/inmunología , Infecciones Estreptocócicas , Streptococcus agalactiae , Animales , Toxinas Bacterianas/inmunología , Toxinas Bacterianas/toxicidad , Línea Celular Tumoral , Femenino , Enfermedades Fetales/inmunología , Enfermedades Fetales/microbiología , Enfermedades Fetales/patología , Humanos , Masculino , Lípidos de la Membrana/inmunología , Lípidos de la Membrana/toxicidad , Ratones , Ratones Noqueados , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/patología , Streptococcus agalactiae/inmunología , Streptococcus agalactiae/patogenicidadRESUMEN
Two key biological features distinguish Trypanosoma evansi from the T. brucei group: independence from the tsetse fly as obligatory vector, and independence from the need for functional mitochondrial DNA (kinetoplast or kDNA). In an effort to better understand the molecular causes and consequences of these differences, we sequenced the genome of an akinetoplastic T. evansi strain from China and compared it to the T. b. brucei reference strain. The annotated T. evansi genome shows extensive similarity to the reference, with 94.9% of the predicted T. b. brucei coding sequences (CDS) having an ortholog in T. evansi, and 94.6% of the non-repetitive orthologs having a nucleotide identity of 95% or greater. Interestingly, several procyclin-associated genes (PAGs) were disrupted or not found in this T. evansi strain, suggesting a selective loss of function in the absence of the insect life-cycle stage. Surprisingly, orthologous sequences were found in T. evansi for all 978 nuclear CDS predicted to represent the mitochondrial proteome in T. brucei, although a small number of these may have lost functionality. Consistent with previous results, the F1FO-ATP synthase γ subunit was found to have an A281 deletion, which is involved in generation of a mitochondrial membrane potential in the absence of kDNA. Candidates for CDS that are absent from the reference genome were identified in supplementary de novo assemblies of T. evansi reads. Phylogenetic analyses show that the sequenced strain belongs to a dominant group of clonal T. evansi strains with worldwide distribution that also includes isolates classified as T. equiperdum. At least three other types of T. evansi or T. equiperdum have emerged independently. Overall, the elucidation of the T. evansi genome sequence reveals extensive similarity of T. brucei and supports the contention that T. evansi should be classified as a subspecies of T. brucei.
Asunto(s)
Genoma de Protozoos , Filogenia , Proteínas Protozoarias/metabolismo , Trypanosoma/clasificación , Trypanosoma/genética , Regulación de la Expresión Génica , Repeticiones de Microsatélite , Polimorfismo de Nucleótido Simple , Análisis de Componente Principal , Proteínas Protozoarias/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/genética , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismoRESUMEN
How eukaryotic pathogens export and sort membrane-bound proteins destined for host-cell compartments is still poorly understood. The dense granules of the intracellular protozoan Toxoplasma gondii constitute an unusual secretory pathway that allows soluble export of the GRA proteins which become membrane-associated within the parasite replicative vacuole. This process relies on both the segregation of the proteins routed to the dense granules from those destined to the parasite plasma membrane and on the sorting of the secreted GRA proteins to their proper final membranous system. Here, we provide evidence that the soluble trafficking of GRA6 to the dense granules relies on the N-terminal domain of the protein, which is sufficient to prevent GRA6 targeting to the parasite plasma membrane. We also show that the GRA6 N-terminal domain, possibly by interacting with negatively charged lipids, is fundamental for proper GRA6 association with the vacuolar membranous network of nanotubes. These results support our emerging model: sorting of transmembrane GRA proteins to the host cell vacuole is mainly driven by the dual role of their N-terminal hydrophilic domain and is compartmentally regulated.
Asunto(s)
Antígenos de Protozoos/química , Antígenos de Protozoos/metabolismo , Membrana Celular/metabolismo , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Toxoplasmosis/metabolismo , Vacuolas/metabolismo , Antígenos de Protozoos/genética , Membrana Celular/química , Membrana Celular/genética , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/parasitología , Humanos , Estructura Terciaria de Proteína , Transporte de Proteínas , Proteínas Protozoarias/genética , Toxoplasma/química , Toxoplasma/genética , Toxoplasmosis/parasitologíaRESUMEN
Yersinia pestis, the causative agent of bubonic plague, employs its type III secretion system to inject toxins into target cells, a crucial step in infection establishment. LcrV is an essential component of the T3SS of Yersinia spp, and is able to associate at the tip of the secretion needle and take part in the translocation of anti-host effector proteins into the eukaryotic cell cytoplasm. Upon cell contact, LcrV is also released into the surrounding medium where it has been shown to block the normal inflammatory response, although details of this mechanism have remained elusive. In this work, we reveal a key aspect of the immunomodulatory function of LcrV by showing that it interacts directly and with nanomolar affinity with the inflammatory cytokine IFNγ. In addition, we generate specific IFNγ mutants that show decreased interaction capabilities towards LcrV, enabling us to map the interaction region to two basic C-terminal clusters of IFNγ. Lastly, we show that the LcrV-IFNγ interaction can be disrupted by a number of inhibitors, some of which display nanomolar affinity. This study thus not only identifies novel potential inhibitors that could be developed for the control of Yersinia-induced infection, but also highlights the diversity of the strategies used by Y. pestis to evade the immune system, with the hijacking of pleiotropic cytokines being a long-range mechanism that potentially plays a key role in the severity of plague.