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BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic may have impacted outpatient antibiotic prescribing in low- and middle-income countries such as Brazil. However, outpatient antibiotic prescribing in Brazil, particularly at the prescription level, is not well-described. METHODS: We used the IQVIA MIDAS database to characterize changes in prescribing rates of antibiotics commonly prescribed for respiratory infections (azithromycin, amoxicillin-clavulanate, levofloxacin/moxifloxacin, cephalexin, and ceftriaxone) among adults in Brazil overall and stratified by age and sex, comparing prepandemic (January 2019-March 2020) and pandemic periods (April 2020-December 2021) using uni- and multivariate Poisson regression models. The most common prescribing provider specialties for these antibiotics were also identified. RESULTS: In the pandemic period compared to the prepandemic period, outpatient azithromycin prescribing rates increased across all age-sex groups (incidence rate ratio [IRR] range, 1.474-3.619), with the greatest increase observed in males aged 65-74 years; meanwhile, prescribing rates for amoxicillin-clavulanate and respiratory fluoroquinolones mostly decreased, and changes in cephalosporin prescribing rates varied across age-sex groups (IRR range, 0.134-1.910). For all antibiotics, the interaction of age and sex with the pandemic in multivariable models was an independent predictor of prescribing changes comparing the pandemic versus prepandemic periods. General practitioners and gynecologists accounted for the majority of increases in azithromycin and ceftriaxone prescribing during the pandemic period. CONCLUSIONS: Substantial increases in outpatient prescribing rates for azithromycin and ceftriaxone were observed in Brazil during the pandemic with prescribing rates being disproportionally different by age and sex. General practitioners and gynecologists were the most common prescribers of azithromycin and ceftriaxone during the pandemic, identifying them as potential specialties for antimicrobial stewardship interventions.
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COVID-19 , Infecciones del Sistema Respiratorio , Adulto , Humanos , Masculino , Combinación Amoxicilina-Clavulanato de Potasio , Antibacterianos/uso terapéutico , Azitromicina , Brasil/epidemiología , Ceftriaxona , COVID-19/epidemiología , Pacientes Ambulatorios , Pandemias , Pautas de la Práctica en Medicina , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Infecciones del Sistema Respiratorio/epidemiología , Femenino , AncianoRESUMEN
BACKGROUND: High rates of antibiotic use (AU) among inpatients with coronavirus disease 2019 (COVID-19) despite low rates of bacterial coinfection and secondary infection have been reported. We evaluated the impact of the COVID-19 pandemic on AU in healthcare facilities (HCFs) in South America. METHODS: We conducted an ecologic evaluation of AU in inpatient adult acute care wards in 2 HCFs each in Argentina, Brazil, and Chile. The AU rates for intravenous antibiotics were calculated as the defined daily dose per 1000 patient-days, using pharmacy dispensing records and hospitalization data from March 2018-February 2020 (prepandemic) and March 2020-February 2021 (pandemic). Differences in median AU were compared between the prepandemic and pandemic periods, using the Wilcoxon rank sum test to determine significance. Interrupted time series analysis was used to analyze changes in AU during the COVID-19 pandemic. RESULTS: Compared with the prepandemic period, the median difference in AU rates for all antibiotics combined increased in 4 of 6 HCFs (percentage change, 6.7%-35.1%; P < .05). In the interrupted time series models, 5 of 6 HCFs had significant increases in use of all antibiotics combined immediately at the onset of the pandemic (immediate effect estimate range, 15.4-268), but only 1 of these 5 HCFs experienced a sustained increase over time (change in slope, +8.13; P < .01). The effect of the pandemic onset varied by antibiotic group and HCF. CONCLUSIONS: Substantial increases in AU were observed at the beginning of the COVID-19 pandemic, suggesting the need to maintain or strengthen antibiotic stewardship activities as part of pandemic or emergency HCF responses.
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Antibacterianos , COVID-19 , Humanos , Adulto , Antibacterianos/uso terapéutico , COVID-19/epidemiología , Pacientes Internos , Pandemias , Chile/epidemiología , Argentina/epidemiología , BrasilRESUMEN
BACKGROUND: The impact of coronavirus disease 2019 (COVID-19) on antimicrobial use (AU) and resistance has not been well evaluated in South America. These data are critical to inform national policies and clinical care. METHODS: At a tertiary hospital in Santiago, Chile, between 2018 and 2022, subdivided into pre- (3/2018-2/2020) and post-COVID-19 onset (3/2020-2/2022), we evaluated intravenous AU and frequency of carbapenem-resistant Enterobacterales (CRE). We grouped monthly AU (defined daily doses [DDD]/1000 patient-days) into broad-spectrum ß-lactams, carbapenems, and colistin and used interrupted time-series analysis to compare AU during pre- and post-pandemic onset. We studied the frequency of carbapenemase-producing (CP) CRE and performed whole-genome sequencing analyses of all carbapenem-resistant (CR) Klebsiella pneumoniae (CRKpn) isolates collected during the study period. RESULTS: Compared with pre-pandemic, AU (DDD/1000 patient-days) significantly increased after the pandemic onset, from 78.1 to 142.5 (P < .001), 50.9 to 110.1 (P < .001), and 4.1 to 13.3 (P < .001) for broad-spectrum ß-lactams, carbapenems, and colistin, respectively. The frequency of CP-CRE increased from 12.8% pre-COVID-19 to 51.9% after pandemic onset (P < .001). The most frequent CRE species in both periods was CRKpn (79.5% and 76.5%, respectively). The expansion of CP-CRE harboring blaNDM was particularly noticeable, increasing from 40% (n = 4/10) before to 73.6% (n = 39/53) after pandemic onset (P < .001). Our phylogenomic analyses revealed the emergence of two distinct genomic lineages of CP-CRKpn: ST45, harboring blaNDM, and ST1161, which carried blaKPC. CONCLUSIONS: AU and the frequency of CP-CRE increased after COVID-19 onset. The increase in CP-CRKpn was driven by the emergence of novel genomic lineages. Our observations highlight the need to strengthen infection prevention and control and antimicrobial stewardship efforts.
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Antiinfecciosos , COVID-19 , Enterobacteriaceae Resistentes a los Carbapenémicos , Humanos , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Klebsiella pneumoniae/genética , Chile/epidemiología , Colistina , Pacientes Internos , Filogenia , Pandemias , COVID-19/epidemiología , Proteínas Bacterianas/genética , beta-Lactamasas/genética , Carbapenémicos/farmacología , Carbapenémicos/uso terapéutico , Hospitales , beta-Lactamas , Pruebas de Sensibilidad MicrobianaRESUMEN
Psittacosis is typically a mild febrile respiratory illness caused by infection with the bacterium Chlamydia psittaci and usually transmitted to humans by infected birds (1). On average, 11 psittacosis cases per year were reported in the United States during 2000-2017. During August-October 2018, the largest U.S. psittacosis outbreak in 30 years (82 cases identified*) occurred in two poultry slaughter plants, one each in Virginia and Georgia, that shared source farms (2). CDC used C. psittaci real-time polymerase chain reaction (PCR) to test 54 human specimens from this outbreak. This was the largest number of human specimens from a single outbreak ever tested for C. psittaci using real-time PCR, which is faster and more sensitive than commercially available serologic tests. This represented a rare opportunity to assess the utility of multiple specimen types for real-time PCR detection of C. psittaci. C. psittaci was detected more frequently in lower respiratory specimens (59% [10 of 17]) and stool (four of five) than in upper respiratory specimens (7% [two of 28]). Among six patients with sputum and nasopharyngeal swabs tested, C. psittaci was detected only in sputum in five patients. Cycle threshold (Ct) values suggested bacterial load was higher in lower respiratory specimens than in nasopharyngeal swabs. These findings support prioritizing lower respiratory specimens for real-time PCR detection of C. psittaci. Stool specimens might also have utility for diagnosis of psittacosis.
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Chlamydophila psittaci/aislamiento & purificación , Brotes de Enfermedades , Tamizaje Masivo/métodos , Psitacosis/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa , Adulto , Chlamydophila psittaci/genética , Heces/microbiología , Femenino , Georgia/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Psitacosis/epidemiología , Esputo/microbiología , Virginia/epidemiología , Adulto JovenRESUMEN
Host cytosolic proteins are endocytosed by Toxoplasma gondii and degraded in its lysosome-like compartment, the vacuolar compartment (VAC), but the dynamics and route of endocytic trafficking remain undefined. Conserved endocytic components and plant-like features suggest T. gondii endocytic trafficking involves transit through early and late endosome-like compartments (ELCs) and potentially the trans-Golgi network (TGN) as in plants. However, exocytic trafficking to regulated secretory organelles, micronemes and rhoptries, also proceeds through ELCs and requires classical endocytic components, including a dynamin-related protein, DrpB. Here, we show that host cytosolic proteins are endocytosed within 7 minutes post-invasion, trafficked through ELCs en route to the VAC, and degraded within 30 minutes. We could not definitively interpret if ingested protein is trafficked through the TGN. We also found that parasites ingest material from the host cytosol throughout the parasite cell cycle. Ingested host proteins colocalize with immature microneme proteins, proM2AP and proMIC5, in transit to the micronemes, but not with the immature rhoptry protein proRON4, indicating that endocytic trafficking of ingested protein intersects with exocytic trafficking of microneme proteins. Finally, we show that conditional expression of a DrpB dominant negative mutant increases T. gondii ingestion of host-derived proteins, suggesting that DrpB is not required for parasite endocytosis.
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Endocitosis , Exocitosis , Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Animales , Células CHO , Cricetinae , Cricetulus , Endosomas/metabolismo , Aparato de Golgi/metabolismoRESUMEN
In December 2019, a cluster of cases of pneumonia emerged in Wuhan City in central China's Hubei Province. Genetic sequencing of isolates obtained from patients with pneumonia identified a novel coronavirus (2019-nCoV) as the etiology (1). As of February 4, 2020, approximately 20,000 confirmed cases had been identified in China and an additional 159 confirmed cases in 23 other countries, including 11 in the United States (2,3). On January 17, CDC and the U.S. Department of Homeland Security's Customs and Border Protection began health screenings at U.S. airports to identify ill travelers returning from Wuhan City (4). CDC activated its Emergency Operations Center on January 21 and formalized a process for inquiries regarding persons suspected of having 2019-nCoV infection (2). As of January 31, 2020, CDC had responded to clinical inquiries from public health officials and health care providers to assist in evaluating approximately 650 persons thought to be at risk for 2019-nCoV infection. Guided by CDC criteria for the evaluation of persons under investigation (PUIs) (5), 210 symptomatic persons were tested for 2019-nCoV; among these persons, 148 (70%) had travel-related risk only, 42 (20%) had close contact with an ill laboratory-confirmed 2019-nCoV patient or PUI, and 18 (9%) had both travel- and contact-related risks. Eleven of these persons had laboratory-confirmed 2019-nCoV infection. Recognizing persons at risk for 2019-nCoV is critical to identifying cases and preventing further transmission. Health care providers should remain vigilant and adhere to recommended infection prevention and control practices when evaluating patients for possible 2019-nCoV infection (6). Providers should consult with their local and state health departments when assessing not only ill travelers from 2019-nCoV-affected countries but also ill persons who have been in close contact with patients with laboratory-confirmed 2019-nCoV infection in the United States.
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Betacoronavirus/aislamiento & purificación , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Brotes de Enfermedades/prevención & control , Tamizaje Masivo/estadística & datos numéricos , Neumonía Viral/virología , Adolescente , Adulto , Anciano , COVID-19 , Centers for Disease Control and Prevention, U.S. , Niño , Preescolar , Trazado de Contacto , Infecciones por Coronavirus/prevención & control , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pandemias , Medición de Riesgo , SARS-CoV-2 , Enfermedad Relacionada con los Viajes , Estados Unidos/epidemiología , Adulto JovenRESUMEN
During August-October, 2018, an outbreak of severe respiratory illness was reported among poultry slaughter plant workers in Virginia and Georgia, USA. A multiorganizational team investigated the cause and extent of illness, determined that the illness was psittacosis, and evaluated and recommended controls for health hazards in the workplace to prevent additional cases.
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Mataderos , Psitacosis/epidemiología , Adulto , Georgia/epidemiología , Historia del Siglo XXI , Humanos , Persona de Mediana Edad , Psitacosis/historia , Psitacosis/microbiología , Vigilancia en Salud Pública , Virginia/epidemiología , Adulto JovenRESUMEN
During 2014-2017, CDC Emerging Infections Program surveillance data reported that the occurrence of invasive methicillin-resistant Staphylococcus aureus (MRSA) infections associated with injection drug use doubled among persons aged 18-49 years residing in Monroe County in western New York.* Unpublished surveillance data also indicate that an increasing proportion of all Candida spp. bloodstream infections in Monroe County and invasive group A Streptococcus (GAS) infections in 15 New York counties are also occurring among persons who inject drugs. In addition, across six surveillance sites nationwide, the proportion of invasive MRSA infections that occurred in persons who inject drugs increased from 4.1% of invasive MRSA cases in 2011 to 9.2% in 2016 (1). To better understand the types and frequency of these infections and identify prevention opportunities, CDC and public health partners conducted a rapid assessment of bacterial and fungal infections among persons who inject drugs in western New York. The goals were to assess which bacterial and fungal pathogens most often cause infections in persons who inject drugs, what proportion of persons who inject use opioids, and of these, how many were offered medication-assisted treatment for opioid use disorder. Medication-assisted treatment, which includes use of medications such as buprenorphine, methadone, and naltrexone, reduces cravings and has been reported to lower the risk for overdose death and all-cause mortality in persons who use opioids (2,3). In this assessment, nearly all persons with infections who injected drugs used opioids (97%), but half of inpatients (22 of 44) and 12 of 13 patients seen only in the emergency department (ED) were not offered medication-assisted treatment. The most commonly identified pathogen was S. aureus (80%), which is frequently found on skin. Health care visits for bacterial and fungal infections associated with injection opioid use are an opportunity to treat the underlying opioid use disorder with medication-assisted treatment. Routine care for patients who continue to inject should include advice on hand hygiene and not injecting into skin that has not been cleaned or to use any equipment contaminated by reuse, saliva, soil, or water (4,5).
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Infecciones Bacterianas/epidemiología , Micosis/epidemiología , Vigilancia de la Población , Abuso de Sustancias por Vía Intravenosa/complicaciones , Abuso de Sustancias por Vía Intravenosa/epidemiología , Adolescente , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , New York/epidemiología , Adulto JovenRESUMEN
Eukaryotes critically rely on endocytosis of autologous and heterologous material to maintain homeostasis and to proliferate. Although mechanisms of endocytosis have been extensively identified in mammalian and plant systems along with model systems including budding yeast, relatively little is known about endocytosis in protozoan parasites including those belonging to the phylum Apicomplexa. Whereas it has been long established that the apicomplexan agents of malaria (Plasmodium spp.) internalize and degrade hemoglobin from infected red blood cells to acquire amino acids for growth, that the related and pervasive parasite Toxoplasma gondii has a functional and active endocytic system was only recently discovered. Here we discuss emerging and hypothesized mechanisms of endocytosis in Toxoplasma gondii with reference to model systems and malaria parasites. Establishing a framework for potential mechanisms of endocytosis in Toxoplasma gondii will help guide future research aimed at defining the molecular basis and biological relevance of endocytosis in this tractable and versatile parasite.
RESUMEN
BACKGROUND: The Coronavirus Disease 2019 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), evolved rapidly in the United States. This report describes the demographic, clinical, and epidemiologic characteristics of 544 U.S. persons under investigation (PUI) for COVID-19 with complete SARS-CoV-2 testing in the beginning stages of the pandemic from January 17 through February 29, 2020. METHODS: In this surveillance cohort, the U.S. Centers for Disease Control and Prevention (CDC) provided consultation to public health and healthcare professionals to identify PUI for SARS-CoV-2 testing by quantitative real-time reverse-transcription PCR. Demographic, clinical, and epidemiologic characteristics of PUI were reported by public health and healthcare professionals during consultation with on-call CDC clinicians and subsequent submission of a CDC PUI Report Form. Characteristics of laboratory-negative and laboratory-positive persons were summarized as proportions for the period of January 17-February 29, and characteristics of all PUI were compared before and after February 12 using prevalence ratios. RESULTS: A total of 36 PUI tested positive for SARS-CoV-2 and were classified as confirmed cases. Confirmed cases and PUI testing negative for SARS-CoV-2 had similar demographic, clinical, and epidemiologic characteristics. Consistent with changes in PUI evaluation criteria, 88% (13/15) of confirmed cases detected before February 12, 2020, reported travel from China. After February 12, 57% (12/21) of confirmed cases reported no known travel- or contact-related exposures. CONCLUSIONS: These findings can inform preparedness for future pandemics, including capacity for rapid expansion of novel diagnostic tests to accommodate broad surveillance strategies to assess community transmission, including potential contributions from asymptomatic and presymptomatic infections.
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COVID-19/diagnóstico , COVID-19/epidemiología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Prueba de Ácido Nucleico para COVID-19 , Centers for Disease Control and Prevention, U.S. , Niño , Preescolar , Estudios de Cohortes , Monitoreo Epidemiológico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Salud Pública , SARS-CoV-2/aislamiento & purificación , Viaje , Enfermedad Relacionada con los Viajes , Estados Unidos/epidemiología , Adulto JovenRESUMEN
Calcium sensing receptor (CaSR) mutations implicated in familial hypocalciuric hypercalcemia, pancreatitis and idiopathic epilepsy syndrome map to an extended arginine-rich region in the proximal carboxyl terminus. Arginine-rich motifs mediate endoplasmic reticulum retention and/or retrieval of multisubunit proteins so we asked whether these mutations, R886P, R896H or R898Q, altered CaSR targeting to the plasma membrane. Targeting was enhanced by all three mutations, and Ca(2+)-stimulated ERK1/2 phosphorylation was increased for R896H and R898Q. To define the role of the extended arginine-rich region in CaSR trafficking, we independently determined the contributions of R890/R891 and/or R896/K897/R898 motifs by mutation to alanine. Disruption of the motif(s) significantly increased surface expression and function relative to wt CaSR. The arginine-rich region is flanked by phosphorylation sites at S892 (protein kinase C) and S899 (protein kinase A). The phosphorylation state of S899 regulated recognition of the arginine-rich region; S899D showed increased surface localization. CaSR assembles in the endoplasmic reticulum as a covalent disulfide-linked dimer and we determined whether retention requires the presence of arginine-rich regions in both subunits. A single arginine-rich region within the dimer was sufficient to confer intracellular retention comparable to wt CaSR. We have identified an extended arginine-rich region in the proximal carboxyl terminus of CaSR (residues R890 - R898) which fosters intracellular retention of CaSR and is regulated by phosphorylation. Mutation(s) identified in chronic pancreatitis and idiopathic epilepsy syndrome therefore increase plasma membrane targeting of CaSR, likely contributing to the altered Ca(2+) signaling characteristic of these diseases.
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Epilepsia/metabolismo , Pancreatitis/metabolismo , Receptores Sensibles al Calcio/metabolismo , Proteínas 14-3-3/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Señalización del Calcio , Línea Celular , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Epilepsia/genética , Humanos , Mutagénesis Sitio-Dirigida , Pancreatitis/genética , Fosforilación , Receptores Sensibles al Calcio/genéticaRESUMEN
The intracellular parasite Toxoplasma gondii resides within a membrane-bound parasitophorous vacuole (PV) and secretes an array of proteins to establish this replicative niche. It has been shown previously that Toxoplasma secretes kinases and that numerous proteins are phosphorylated after secretion. Here, we assess the role of the phosphorylation of strand-forming protein 1 (SFP1) and the related protein GRA29, two secreted proteins with unknown function. We show that both proteins form stranded structures in the PV that are independent of the previously described intravacuolar network or actin. SFP1 and GRA29 can each form these structures independently of other Toxoplasma secreted proteins, although GRA29 appears to regulate SFP1 strands. We show that an unstructured region at the C termini of SFP1 and GRA29 is required for the formation of strands and that mimicking the phosphorylation of this domain of SFP1 negatively regulates strand development. When tachyzoites convert to chronic-stage bradyzoites, both proteins show a dispersed localization throughout the cyst matrix. Many secreted proteins are reported to dynamically redistribute as the cyst forms, and secreted kinases are known to play a role in cyst formation. Using quantitative phosphoproteome and proteome analyses comparing tachyzoite and early bradyzoite stages, we reveal widespread differential phosphorylation of secreted proteins. While we found no direct evidence for phosphorylation playing a dominant role for SFP1/GRA29 redistribution in the cyst, these data support a model in which secreted kinases and phosphatases contribute to the regulation of secreted proteins during stage conversion.IMPORTANCEToxoplasma gondii is a common parasite that infects up to one-third of the human population. Initially, the parasite grows rapidly, infecting and destroying cells of the host, but subsequently switches to a slow-growing form and establishes chronic infection. In both stages, the parasite lives within a membrane-bound vacuole within the host cell, but in the chronic stage, a durable cyst wall is synthesized, which provides protection to the parasite during transmission to a new host. Toxoplasma secretes proteins into the vacuole to build its replicative niche, and previous studies identified many of these proteins as phosphorylated. We investigate two secreted proteins and show that a phosphorylated region plays an important role in their regulation in acute stages. We also observed widespread phosphorylation of secreted proteins when parasites convert from acute to chronic stages, providing new insight into how the cyst wall may be dynamically regulated.
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Proteínas Protozoarias/metabolismo , Toxoplasma/patogenicidad , Vacuolas/metabolismo , Animales , Transporte Biológico , Fibroblastos/parasitología , Prepucio/citología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Fosforilación , Proteoma , Proteínas Protozoarias/genética , Organismos Libres de Patógenos Específicos , Toxoplasma/genética , Vacuolas/parasitologíaRESUMEN
Globally, nearly 2 billion people are infected with the intracellular protozoan Toxoplasma gondii1. This persistent infection can cause severe disease in immunocompromised people and is epidemiologically linked to major mental illnesses2 and cognitive impairment3. There are currently no options for curing this infection. The lack of effective therapeutics is due partly to a poor understanding of the essential pathways that maintain long-term infection. Although it is known that Toxoplasma replicates slowly within intracellular cysts demarcated with a cyst wall, precisely how it sustains itself and remodels organelles in this niche is unknown. Here, we identify a key role for proteolysis within the parasite lysosomal organelle (the vacuolar compartment or VAC) in turnover of autophagosomes and persistence during neural infection. We found that disrupting a VAC-localized cysteine protease compromised VAC digestive function and markedly reduced chronic infection. Death of parasites lacking the VAC protease was preceded by accumulation of undigested autophagosomes in the parasite cytoplasm. These findings suggest an unanticipated function for parasite lysosomal degradation in chronic infection, and identify an intrinsic role for autophagy in the T. gondii parasite and its close relatives. This work also identifies a key element of Toxoplasma persistence and suggests that VAC proteolysis is a prospective target for pharmacological development.
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Autofagosomas/metabolismo , Interacciones Huésped-Patógeno , Lisosomas/metabolismo , Toxoplasma/fisiología , Animales , Supervivencia Celular , Células Cultivadas , Proteasas de Cisteína/genética , Proteasas de Cisteína/metabolismo , Fibroblastos/parasitología , Técnicas de Inactivación de Genes , Humanos , Ratones Endogámicos C57BL , Neuronas/parasitología , Proteolisis , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Toxoplasma/enzimología , Toxoplasma/metabolismoRESUMEN
How the protozoan pathogen Toxoplasma gondii and related parasites shuttle proteins through their intricate system of endomembranous compartments remains unclear. Sangaré et al. show that the Toxoplasma retromer complex is essential for parasite viability through its role in protein targeting to multiple locales and its interactions with newly identified partners.
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Proteínas Protozoarias/metabolismo , Toxoplasma/metabolismo , Animales , Interacciones Huésped-Parásitos , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Orgánulos/metabolismo , Transporte de Proteínas/genética , Proteínas Protozoarias/genética , Proteínas de Unión al GTP rab/metabolismoRESUMEN
The passage of genetic information during meiosis requires exceptionally high fidelity to prevent birth defects and infertility. Accurate chromosome segregation during the first meiotic division relies on the formation of crossovers between homologous chromosomes and a series of precisely controlled steps to exchange genetic information. Many studies have hinted at a role for p53 in meiosis, but how it functions in this process is poorly understood. Here, we have identified a cooperative role for the p53-like protein CEP-1 and the meiotic protein HIM-5 in maintaining genome stability in the C. elegans germline. Loss of cep-1 and him-5 results in synthetic lethality that is dependent on the upstream DNA damage checkpoint but independent of the downstream core apoptotic pathway. We show that this synthetic lethality is the result of defective crossover formation due to reduced SPO-11-dependent double-strand breaks. Using cep-1 separation-of-function alleles, we show that cep-1 and him-5 also suppress inappropriate activation of the nonhomologous end joining (NHEJ) pathway. This work reveals an ancestral function for the p53 family in ensuring the fidelity of meiosis and establishes CEP-1 as a critical determinant of repair pathway choice.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Regulación de la Expresión Génica/fisiología , Meiosis/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/genética , Epigénesis Genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Crossover (CO) recombination creates a physical connection between homologs that promotes their proper segregation at meiosis I (MI). Failure to realize an obligate CO causes homologs to attach independently to the MI spindle and separate randomly, leading to nondisjunction. However, mechanisms that determine whether homolog pairs have received crossovers remain mysterious. Here we describe a surveillance system in C. elegans that monitors recombination intermediates and couples their formation to meiotic progression. Recombination intermediates are required to activate the system, which then delays further processing if crossover precursors are lacking on even one chromosome. The synaptonemal complex, a specialized, proteinaceous structure connecting homologous chromosomes, is stabilized in cis on chromosomes that receive a crossover and is destabilized on those lacking crossovers, a process that is dependent on the function of the polo-like kinase PLK-2. These results reveal a new layer of communication between crossover-committed intermediates and the synaptonemal complex that functions as a cis-acting, obligate, crossover-counting mechanism.
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Caenorhabditis elegans/genética , Intercambio Genético/genética , Meiosis , Complejo Sinaptonémico/genética , AnimalesRESUMEN
The protozoan parasite Toxoplasma gondii resides within a nonfusogenic vacuole during intracellular replication. Although the limiting membrane of this vacuole provides a protective barrier to acidification and degradation by lysosomal hydrolases, it also physically segregates the parasite from the host cytosol. Accordingly, it has been suggested that T. gondii acquires material from the host via membrane channels or transporters. The ability of the parasite to internalize macromolecules via endocytosis during intracellular replication has not been tested. Here, we show that Toxoplasma ingests host cytosolic proteins and digests them using cathepsin L and other proteases within its endolysosomal system. Ingestion was reduced in mutant parasites lacking an intravacuolar network of tubular membranes, implicating this apparatus as a possible conduit for trafficking to the parasite. Genetic ablation of proteins involved in the pathway is associated with diminished parasite replication and virulence attenuation. We show that both virulent type I and avirulent type II strain parasites ingest and digest host-derived protein, indicating that the pathway is not restricted to highly virulent strains. The findings provide the first definitive evidence that T. gondii internalizes proteins from the host during intracellular residence and suggest that protein digestion within the endolysosomal system of the parasite contributes to toxoplasmosis. Importance: Toxoplasma gondii causes significant disease in individuals with weak immune systems. Treatment options for this infection have drawbacks, creating a need to understand how this parasite survives within the cells it infects as a prelude to interrupting its survival strategies. This study reveals that T. gondii internalizes proteins from the cytoplasm of the cells it infects and degrades such proteins within a digestive compartment within the parasite. Disruption of proteins involved in the pathway reduced parasite replication and lessened disease severity. The identification of a novel parasite ingestion pathway opens opportunities to interfere with this process and improve the outcome of infection.
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Citosol/metabolismo , Proteínas/metabolismo , Toxoplasma/metabolismo , Toxoplasma/patogenicidad , Toxoplasmosis/metabolismo , Animales , Citosol/parasitología , Femenino , RatonesRESUMEN
Mutations in the him-5 gene in Caenorhabditis elegans strongly reduce the frequency of crossovers on the X chromosome, with lesser effects on the autosomes. him-5 mutants also show a change in crossover distribution on both the X and autosomes. These phenotypes are accompanied by a delayed entry into pachytene and premature desynapsis of the X chromosome. The nondisjunction, progression defects and desynapsis can be rescued by an exogenous source of double strand breaks (DSBs), indicating that the role of HIM-5 is to promote the formation of meiotic DSBs. Molecular cloning of the gene shows that the inferred HIM-5 product is a highly basic protein of 252 amino acids with no clear orthologs in other species, including other Caenorhabditis species. Although him-5 mutants are defective in segregation of the X chromosome, HIM-5 protein localizes preferentially to the autosomes. The mutant phenotypes and localization of him-5 are similar but not identical to the results seen with xnd-1, although unlike xnd-1, him-5 has no apparent effect on the acetylation of histone H2A on lysine 5 (H2AacK5). The localization of HIM-5 to the autosomes depends on the activities of both xnd-1 and him-17 allowing us to begin to establish pathways for the control of crossover distribution and frequency.