RESUMEN

Antimicrobial peptides (AMPs) play a pivotal role in the innate immune system as a frontline defense against microbial threats. AMPs can serve as biomarkers and alternative antibiotics, overcoming mortality related to multidrug-resistant pathogens in urinary tract infections (UTIs). While the relevance of AMPs in UTIs has been validated and AMP drugs approved by the US Food and Drug Administration are in clinical use, information about their modification status, regulation, and mechanism of action remains sparse. Only a small fraction of sequences with potential AMP activity, predicted on the basis of known AMP characteristics, have been validated. Elucidation of the global profile of AMPs in the bladder, kidney, and urine under UTI conditions would facilitate an in-depth, disease-specific understanding of the innate immune system and the development of tailored AMP biomarkers and antibiotics. This mini-review focuses on a comprehensive strategy for global profiling and validation of AMPs in UTIs that incorporates AMP data repositories, prediction algorithms, and proteomics for healthy individuals and UTI patients. PATIENT SUMMARY: Short protein molecules called peptides that have antimicrobial activity show promise for the treatment of urinary tract infections. More research and testing of naturally occurring and synthetic peptides with this activity are needed to fully understand how they can help in patient care.

RESUMEN

BACKGROUND AND OBJECTIVE: Urinary tract infections (UTIs) are common infections affecting the urinary system, predominantly caused by bacterial pathogens, with Escherichia coli being the most frequent pathogen. Infections of the kidney (eg, pyelonephritis) are severe and challenging to treat, due to the specific tissue microenvironment. In this study, the influence of different parameters mimicking the kidney environment on the effectiveness of antibiotics prescribed for pyelonephritis on the growth of uropathogenic strains was analyzed. METHODS: To investigate the influence of different factors mimicking the kidney environment, we tested the effect of different kidney-representative concentrations of sodium chloride and urea, and different pH values on the efficacy of ertapenem, levofloxacin, and ceftriaxone. The effectiveness was assessed by determining the minimal inhibitory concentrations (MICs) against various E. coli strains. KEY FINDINGS AND LIMITATIONS: The study revealed that pH significantly influences the MIC values of levofloxacin. Acidification of the pH led to an increase of the MIC values, while an alkaline pH had the opposite effect. The influence of sodium chloride and urea concentrations was strain and antibiotic specific. Since three different antibiotics were tested in this study, further research with additional antibiotics is warranted. CONCLUSIONS AND CLINICAL IMPLICATIONS: These results suggest that the physicochemical conditions within the kidney can substantially influence the success of antibiotic therapy for pyelonephritis. Therefore, it is crucial for clinicians to consider these factors when selecting and dosing antibiotics. Further research is needed to evaluate a broader range of antibiotics and additional environmental parameters, to develop a more comprehensive understanding of how the kidney environment affects antimicrobial activity. This knowledge will be vital in optimizing treatment strategies for pyelonephritis, ultimately improving patient outcomes. PATIENT SUMMARY: The physicochemical conditions within the kidney influence the success of antibiotic therapy for pyelonephritis. Our findings are vital in optimizing treatment strategies and will ultimately improve patient outcomes.

RESUMEN

Urinary tract infection (UTI) is a very common disease that is accompanied by various complications in the affected person. UTI triggers diverse inflammatory reactions locally in the infected urinary bladder and kidney, causing tissue destruction and organ failure. Moreover, systemic responses in the entire body carry the risk of urosepsis with far-reaching consequences. Understanding the cell-, organ-, and systemic mechanisms in UTI are crucial for prevention, early intervention, and current therapeutic approaches. This review summarizes the scientific advances over the last 10 years concerning pathogenesis, prevention, rapid diagnosis, and new treatment approaches. We also highlight the impact of the immune system and potential new therapies to reduce progressive and recurrent UTI.

Asunto(s)

Infecciones Urinarias , Humanos , Infecciones Urinarias/diagnóstico , Infecciones Urinarias/prevención & control , Antibacterianos/uso terapéutico

RESUMEN

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

Asunto(s)

Trastornos Congénitos de Glicosilación , Glicosiltransferasas , Humanos , Glicosilación , Glicosiltransferasas/genética , Trastornos Congénitos de Glicosilación/genética , Proteómica , Procesamiento Proteico-Postraduccional , Proteínas de la Membrana/genética , Manosiltransferasas

RESUMEN

Antimicrobial resistance (AMR) has emerged as a significant global healthcare problem. Antibiotic use has accelerated the physiologic process of AMR, particularly in Gram-negative pathogens. Urinary tract infections (UTIs) are predominantly of a Gram-negative nature. Uropathogens are evolutionarily highly adapted and selected strains with specific virulence factors, suggesting common mechanisms in how bacterial cells acquire virulence and AMR factors. The simultaneous increase in resistance and virulence is a complex and context-dependent phenomenon. Among known AMR mechanisms, the plenitude of different ß-lactamases is especially prominent. The risk for AMR in UTIs varies in different patient populations. A history of antibiotic consumption and the physiology of urinary flow are major factors that shape AMR prevalence. The urinary tract is in close crosstalk with the microbiome of other compartments, including the gut and genital tracts. In addition, pharmacokinetic properties and the physiochemical composition of urinary compartments can contribute to the emergence of AMR. Alternatives to antibiotic treatment and a broader approach to address bacterial infections are needed. Among the various alternatives studied, antimicrobial peptides and bacteriophage treatment appear to be highly promising approaches. We herein summarize the present knowledge of clinical and microbiological AMR in UTIs and discuss innovative approaches, namely new risk prediction tools and the use of non-antibiotic approaches to defend against uropathogenic microbes.

Asunto(s)

Infecciones Urinarias , Sistema Urinario , Humanos , Antibacterianos/uso terapéutico , Farmacorresistencia Bacteriana , Infecciones Urinarias/tratamiento farmacológico

RESUMEN

Introduction: Streptococcus pneumoniae is one of the main causes of community-acquired infections in the lung alveoli in children and the elderly. Alveolar macrophages (AM) patrol alveoli in homeostasis and under infectious conditions. However, the molecular adaptations of AM upon infections with Streptococcus pneumoniae are incompletely resolved. Methods: We used a comparative transcriptomic and proteomic approach to provide novel insights into the cellular mechanism that changes the molecular signature of AM during lung infections. Using a tandem mass spectrometry approach to murine cell-sorted AM, we revealed significant proteomic changes upon lung infection with Streptococcus pneumoniae. Results: AM showed a strong neutrophil-associated proteomic signature, such as expression of CD11b, MPO, neutrophil gelatinases, and elastases, which was associated with phagocytosis of recruited neutrophils. Transcriptomic analysis indicated intrinsic expression of CD11b by AM. Moreover, comparative transcriptomic and proteomic profiling identified CD11b as the central molecular hub in AM, which influenced neutrophil recruitment, activation, and migration. Discussion: In conclusion, our study provides novel insights into the intrinsic molecular adaptations of AM upon lung infection with Streptococcus pneumoniae and reveals profound alterations critical for effective antimicrobial immunity.

Asunto(s)

Antígeno CD11b , Neumonía Neumocócica , Animales , Ratones , Integrinas , Pulmón , Macrófagos Alveolares , Proteómica , Streptococcus pneumoniae , Transcriptoma

RESUMEN

Thyroid hormone (TH) transporter MCT8 deficiency causes severe locomotor disabilities likely due to insufficient TH transport across brain barriers and, consequently, compromised neural TH action. As an established animal model for this disease, Mct8/Oatp1c1 double knockout (DKO) mice exhibit strong central TH deprivation, locomotor impairments and similar histo-morphological features as seen in MCT8 patients. The pathways that cause these neuro-motor symptoms are poorly understood. In this paper, we performed proteome analysis of brain sections comprising cortical and striatal areas of 21-day-old WT and DKO mice. We detected over 2900 proteins by liquid chromatography mass spectrometry, 67 of which were significantly different between the genotypes. The comparison of the proteomic and published RNA-sequencing data showed a significant overlap between alterations in both datasets. In line with previous observations, DKO animals exhibited decreased myelin-associated protein expression and altered protein levels of well-established neuronal TH-regulated targets. As one intriguing new candidate, we unraveled and confirmed the reduced protein and mRNA expression of Pde10a, a striatal enzyme critically involved in dopamine receptor signaling, in DKO mice. As altered PDE10A activities are linked to dystonia, reduced basal ganglia PDE10A expression may represent a key pathogenic pathway underlying human MCT8 deficiency.

Asunto(s)

Proteoma , Simportadores , Animales , Humanos , Ratones , Proteoma/metabolismo , Proteómica , Simportadores/genética , Transportadores de Ácidos Monocarboxílicos/genética , Transportadores de Ácidos Monocarboxílicos/metabolismo , Hormonas Tiroideas/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo

RESUMEN

SARS-CoV-2, responsible for the ongoing global pandemic, must overcome a conundrum faced by all viruses. To achieve its own replication and spread, it simultaneously depends on and subverts cellular mechanisms. At the early stage of infection, SARS-CoV-2 expresses the viral nonstructural protein 1 (NSP1), which inhibits host translation by blocking the mRNA entry tunnel on the ribosome; this interferes with the binding of cellular mRNAs to the ribosome. Viral mRNAs, on the other hand, overcome this blockade. We show that NSP1 enhances expression of mRNAs containing the SARS-CoV-2 leader. The first stem-loop (SL1) in the viral leader is both necessary and sufficient for this enhancement mechanism. Our analysis pinpoints specific residues within SL1 (three cytosine residues at the positions 15, 19, and 20) and another within NSP1 (R124), which are required for viral evasion, and thus might present promising drug targets. We target SL1 with the antisense oligo (ASO) to efficiently and specifically down-regulate SARS-CoV-2 mRNA. Additionally, we carried out analysis of a functional interactome of NSP1 using BioID and identified components of antiviral defense pathways. Our analysis therefore suggests a mechanism by which NSP1 inhibits the expression of host genes while enhancing that of viral RNA. This analysis helps reconcile conflicting reports in the literature regarding the mechanisms by which the virus avoids NSP1 silencing.

Asunto(s)

COVID-19 , SARS-CoV-2 , Proteínas no Estructurales Virales , COVID-19/virología , Humanos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosomas/metabolismo , SARS-CoV-2/metabolismo , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismo

RESUMEN

Platelets are components of the blood that are highly reactive, and they quickly respond to multiple physiological and pathophysiological processes. In the last decade, it became clear that platelets are the key components of circulation, linking hemostasis, innate, and acquired immunity. Protein composition, localization, and activity are crucial for platelet function and regulation. The current state of mass spectrometry-based proteomics has tremendous potential to identify and quantify thousands of proteins from a minimal amount of material, unravel multiple post-translational modifications, and monitor platelet activity during drug treatments. This review focuses on the role of proteomics in understanding the molecular basics of the classical and newly emerging functions of platelets. including the recently described role of platelets in immunology and the development of COVID-19.The state-of-the-art proteomic technologies and their application in studying platelet biogenesis, signaling, and storage are described, and the potential of newly appeared trapped ion mobility spectrometry (TIMS) is highlighted. Additionally, implementing proteomic methods in platelet transfusion medicine, and as a diagnostic and prognostic tool, is discussed.

Asunto(s)

Plaquetas/metabolismo , Espectrometría de Masas/métodos , Pruebas de Función Plaquetaria/métodos , Proteómica/métodos , Animales , Plaquetas/citología , Plaquetas/inmunología , COVID-19/inmunología , COVID-19/metabolismo , Humanos , Transfusión de Plaquetas , Procesamiento Proteico-Postraduccional , Transducción de Señal , Medicina Transfusional/métodos

RESUMEN

We introduce STAMPS, a pathway-centric web service for the development of targeted proteomics assays. STAMPS guides the user by providing several intuitive interfaces for a rapid and simplified method design. Applying our curated framework to signaling and metabolic pathways, we reduced the average assay development time by a factor of ∼150 and revealed that the insulin signaling is actively controlled by protein abundance changes in insulin-sensitive and -resistance states. Although at the current state STAMPS primarily contains mouse data, it was designed for easy extension with additional organisms.

Asunto(s)

Redes y Vías Metabólicas , Proteómica/métodos , Transducción de Señal , Animales , Cromatografía Líquida de Alta Presión , Bases de Datos de Proteínas , Insulina/metabolismo , Redes y Vías Metabólicas/genética , Ratones , Péptidos/análisis , Transducción de Señal/genética , Espectrometría de Masas en Tándem

RESUMEN

Proteomic analysis of foodborne pathogen Listeria monocytogenes after treatment with three disinfectants based on ammonium salts of pyridoxal oxime (POD) reveal perturbation of cellular processes. These inhibitors caused disturbance in the synthesis of plasma membrane proteins and cell wall proteoglycans. Some of key proteins and proteoglycans from these two groups that are important for bacterial growth are down-regulated. Additionally, we demonstrated that the main bacterial toxin Listeriolysin O (LLO) is significantly down-regulated after treatment with each of three investigated inhibitors. These investigations confirm already postulated mechanism of action of POD-based inhibitors that results in disturbance of key cell surface proteins and proteoglycans in Gram-positive bacteria. Additionally, the use of some proteins such as LLO, as potential biomarker candidates of food poisoning with this bacterium is discussed.

Asunto(s)

Toxinas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Proteínas de Choque Térmico/metabolismo , Proteínas Hemolisinas/metabolismo , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/metabolismo , Piridoxal/análogos & derivados , Toxinas Bacterianas/genética , Cromatografía Liquida , Regulación hacia Abajo , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Proteómica , Piridoxal/farmacología , Espectrometría de Masas en Tándem/métodos

RESUMEN

Growth differentiation factor 11 (GDF11) is a TGF-ß superfamily circulating factor that regulates cardiomyocyte size in rodents, sharing 90% amino acid sequence identity in the active domains with myostatin (GDF8)-the major determinant of skeletal muscle mass. Conflicting data on age-related changes in circulating levels have been reported mainly due to the lack of specific detection methods. More recently, liquid chromatography tandem mass spectrometry (LC-MS/MS) based assay showed that the circulating levels of GDF11 do not change significantly throughout human lifespan, but GDF8 levels decrease with aging in men. Here a novel detection method is demonstrated based on parallel reaction monitoring LC-MS/MS assay combined with immunoprecipitation to reliably distinguish GDF11 and GDF8 as well as determine their endogenous levels in mouse serum. The data indicate that both GDF11 and GDF8 circulating levels significantly decline with aging in female mice.

Asunto(s)

Proteínas Morfogenéticas Óseas/sangre , Factores de Diferenciación de Crecimiento/sangre , Miostatina/sangre , Envejecimiento/fisiología , Animales , Cromatografía Liquida , Femenino , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Proteómica , Espectrometría de Masas en Tándem

RESUMEN

Extracellular vesicles (EVs) are membranous nanostructures that can indicate undergoing processes in organs and thus help in diagnostics and prognostics. They are secreted by all cells, contained in body fluids, and able to transfer proteins, lipids and nucleic acids to distant cells. Intracranial EVs were shown to change their composition after severe traumatic brain injury (TBI) and therefore to have biomarker potential to evaluate brain events. Properties of intracranial EVs early after TBI, however, have not been characterized. Here, we assessed cerebrospinal fluid (CSF) up to seven days after isolated severe TBI for physical properties of EVs and their proteins associated with neuroregeneration. These findings were compared with healthy controls and correlated to patient outcome. The study included 17 patients with TBI and 18 healthy controls. EVs in TBI-CSF were visualized by electron microscopy and confirmed by immunoblotting for membrane associated Flotillin-1 and Flotillin-2. Using nanoparticle tracking analysis, we detected the highest range in EV concentration at day 1 after injury and significantly increased EV size at days 4-7. CSF concentrations of neuroregeneration associated proteins Flotillin-1, ADP-ribosylation Factor 6 (Arf6), and Ras-related protein Rab7a (Rab7a) were monitored by enzyme-linked immunosorbent assays. Flotillin-1 was detected solely in TBI-CSF in about one third of tested patients. Unfavorable outcomes included decreasing Arf6 concentrations and a delayed Rab7a concentration increase in CSF. CSF concentrations of Arf6 and Rab7a were negatively correlated. Our data suggest that the brain response within several days after severe TBI includes shedding of EVs associated with neuroplasticity. Extended studies with a larger number of participants and CSF collected at shorter intervals are necessary to further evaluate neuroregeneration biomarker potential of Rab7a, Arf6, and Flotillin-1.

Asunto(s)

Biomarcadores/líquido cefalorraquídeo , Lesiones Traumáticas del Encéfalo/líquido cefalorraquídeo , Vesículas Extracelulares , Factor 6 de Ribosilación del ADP , Adulto , Anciano , Anciano de 80 o más Años , Lesiones Traumáticas del Encéfalo/fisiopatología , Femenino , Humanos , Masculino , Persona de Mediana Edad , Regeneración Nerviosa/fisiología , Recuperación de la Función/fisiología , Adulto Joven

RESUMEN

Autism spectrum disorders (ASDs) are associated with mutations affecting synaptic components, including GluN2B-NMDA receptors (NMDARs) and neurobeachin (NBEA). NBEA participates in biosynthetic pathways to regulate synapse receptor targeting, synaptic function, cognition, and social behavior. However, the role of NBEA-mediated transport in specific trafficking routes is unclear. Here, we highlight an additional function for NBEA in the local delivery and surface re-insertion of synaptic receptors in mouse neurons. NBEA dynamically interacts with Rab4-positive recycling endosomes, transiently enters spines in an activity-dependent manner, and regulates GluN2B-NMDAR recycling. Furthermore, we show that the microtubule growth inhibitor kinesin KIF21B constrains NBEA dynamics and is present in the NBEA-recycling endosome-NMDAR complex. Notably, Kif21b knockout decreases NMDAR surface expression and alters social behavior in mice, consistent with reported social deficits in Nbea mutants. The influence of NBEA-KIF21B interactions on GluN2B-NMDAR local recycling may be relevant to mechanisms underlying ASD etiology.

Asunto(s)

Conducta Animal , Proteínas Portadoras/metabolismo , Endocitosis , Cinesinas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Conducta Social , Animales , Células COS , Chlorocebus aethiops , Cognición , Espinas Dendríticas/efectos de los fármacos , Espinas Dendríticas/metabolismo , Dineínas/metabolismo , Endocitosis/efectos de los fármacos , Endosomas/metabolismo , Ácido Glutámico/farmacología , Aparato de Golgi/efectos de los fármacos , Aparato de Golgi/metabolismo , Proteínas de la Membrana , Ratones Noqueados , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Nocodazol/farmacología , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/metabolismo , Proteínas de Unión al GTP rab4/metabolismo

RESUMEN

Food borne pathogens, namely the Gram-positive bacterium Bacillus subtilis and the Gram-negative bacterium Escherichia coli, were grown under the inhibition with four different disinfectants based on chloride and bromide salts of pyridinium oxime. Bacterial samples were subjected to the sequential extraction of proteins and the in-solution tryptic digestion of obtained extracts was performed prior to the identification of proteins with LC-ESI-MS/MS. Proteomic analysis identified up- and down-regulated proteins in these bacteria after treatment with each compound. The tables with differently expressed proteins are presented with this article.

RESUMEN

A comprehensive proteomic analysis of food borne pathogens after treatment with disinfectants based on ammonium salts of pyridinium oxime was performed. Changes in proteomes of the Gram-positive bacterium Bacillus subtilis and the Gram-negative one, Escherichia coli, were evaluated. Up and down-regulated proteins in these bacteria after growth under the inhibition with four different disinfectants based on chloride and bromide salts of pyridinium oxime were identified and their cellular localizations and functions were determined by gene ontology searching. Proteome changes presented here demonstrate different mechanisms of action of these disinfectants. In the Gram-positive food pathogen Bacillus subtilis, the inhibitory substances seem to act mainly at the cell surface and cause significant alterations of membrane and cell surface proteins. On the other hand, intracellular proteins were more affected in the Gram-negative pathogen Escherichia coli. This research is a contribution to the investigation of the virulence and pathogenicity of food borne bacteria and their survival under stress conditions, and can also lead the way for further development of new inhibitors of microbial growth and studies of mechanism of their actions.

Asunto(s)

Bacillus subtilis/efectos de los fármacos , Desinfectantes/farmacología , Proteínas de Escherichia coli/metabolismo , Escherichia coli/efectos de los fármacos , Microbiología de Alimentos/métodos , Enfermedades Transmitidas por los Alimentos/prevención & control , Proteómica/métodos , Piridoxal/análogos & derivados , Bacillus subtilis/crecimiento & desarrollo , Bacillus subtilis/metabolismo , Bacillus subtilis/patogenicidad , Biomarcadores/metabolismo , Cromatografía Liquida , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Escherichia coli/patogenicidad , Enfermedades Transmitidas por los Alimentos/microbiología , Viabilidad Microbiana/efectos de los fármacos , Piridoxal/farmacología , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en Tándem , Virulencia/efectos de los fármacos

RESUMEN

Francisella is a gram-negative bacterial pathogen, which causes tularemia in humans and animals. A crucial step of Francisella infection is its invasion of macrophage cells. Biogenesis of the Francisella-containing phagosome (FCP) is arrested for ~15 min at the endosomal stage, followed by gradual bacterial escape into the cytosol, where the microbe proliferates. The crucial step in pathogenesis of tularemia is short and transient presence of the bacterium within phagosome. Isolation of FCPs for further studies has been challenging due to the short period of time of bacterial residence in it and the characteristics of the FCP. Here, we will for the first time present the method for isolation of the FCPs from infected human monocytes-derived macrophages (hMDMs). For elimination of lysosomal compartment these organelles were pre-loaded with dextran coated colloidal iron particles prior infection and eliminated by magnetic separation of the post-nuclear supernatant (PNS). We encountered the challenge that mitochondria has similar density to the FCP. To separate the FCP in the PNS from mitochondria, we utilized iodophenylnitrophenyltetrazolium, which is converted by the mitochondrial succinate dehydrogenase into formazan, leading to increased density of the mitochondria and allowing separation by the discontinuous sucrose density gradient ultracentrifugation. The purity of the FCP preparation and its acquisition of early endosomal markers was confirmed by Western blots, confocal and transmission electron microscopy. Our strategy to isolate highly pure FCPs from macrophages should facilitate studies on the FCP and its biogenesis.

Asunto(s)

Francisella/fisiología , Monocitos/microbiología , Fagosomas/microbiología , Tularemia/microbiología , Células Cultivadas , Francisella/aislamiento & purificación , Humanos , Macrófagos/química , Macrófagos/microbiología , Monocitos/química , Fagosomas/química

RESUMEN

Legionnaires' disease is an acute fibrinopurulent pneumonia. During infection Legionella pneumophila adheres to the alveolar lining and replicates intracellularly within recruited macrophages. Here we provide a sequence and domain composition analysis of the L. pneumophila PilY1 protein, which has a high homology to PilY1 of Pseudomonas aeruginosa. PilY1 proteins of both pathogens contain a von Willebrand factor A (vWFa) and a C-terminal PilY domain. Using cellular fractionation, we assigned the L. pneumophila PilY1 as an outer membrane protein that is only expressed during the transmissive stationary growth phase. PilY1 contributes to infection of human lung tissue explants (HLTEs). A detailed analysis using THP-1 macrophages and A549 lung epithelial cells revealed that this contribution is due to multiple effects depending on host cell type. Deletion of PilY1 resulted in a lower replication rate in THP-1 macrophages but not in A549 cells. Further on, adhesion to THP-1 macrophages and A549 epithelial cells was decreased. Additionally, the invasion into non-phagocytic A549 epithelial cells was drastically reduced when PilY1 was absent. Complementation variants of a PilY1-negative mutant revealed that the C-terminal PilY domain is essential for restoring the wild type phenotype in adhesion, while the putatively mechanosensitive vWFa domain facilitates invasion into non-phagocytic cells. Since PilY1 also promotes twitching motility of L. pneumophila, we discuss the putative contribution of this newly described virulence factor for bacterial dissemination within infected lung tissue.

Asunto(s)

Adhesión Bacteriana/genética , Proteínas Fimbrias/genética , Legionella pneumophila/fisiología , Enfermedad de los Legionarios/microbiología , Pulmón/microbiología , Proteínas Fimbrias/química , Proteínas Fimbrias/metabolismo , Regulación Bacteriana de la Expresión Génica , Interacciones Huésped-Patógeno , Humanos , Enfermedad de los Legionarios/patología , Pulmón/patología , Mutación , Dominios Proteicos , Transporte de Proteínas , Factores de Virulencia/química , Factores de Virulencia/genética , Factores de Virulencia/metabolismo

RESUMEN

The use of implantable cardioverter defibrillators (ICD) has favorably impacted the prevention and treatment of sudden cardiac death (SCD) associated with ventricular arrhythmias. However, there are situations where an ICD cannot be immediately implanted, even though the patient is at high risk for SCD. The wearable cardioverter defibrillator (WCD) is a unique technology that can bridge this gap for patients. The WCD has been demonstrated to terminate ventricular tachycardia/fibrillation if worn and used correctly. With proper training, it is relatively easy to put on, maintain, and use. Most patients are compliant and are able to consistently wear the device. The WCD negates the infection risk or procedural complications associated with insertion and possible extraction of leads, as with an ICD. In terms of primary prevention of ventricular tachycardia/fibrillation in patients with a left ventricular ejection fraction ≤35%, prospective, randomized studies evaluating the survival of patients utilizing the WCD will need to be performed before evidenced-based criteria for its use can be established. On the basis of current data, WCD use for those awaiting heart transplant, for those with ICD indications status post-ICD explant, and for high-risk SCD patients with possible reversible cardiomyopathy appears to be a reasonable approach on the basis of current data.

Asunto(s)

Muerte Súbita Cardíaca/prevención & control , Desfibriladores , Humanos , Cooperación del Paciente , Selección de Paciente

RESUMEN

L. pneumophila-containing vacuoles (LCVs) exclude endocytic and lysosomal markers in human macrophages and protozoa. We screened a L. pneumophila mini-Tn10 transposon library for mutants, which fail to inhibit the fusion of LCVs with lysosomes by loading of the lysosomal compartment with colloidal iron dextran, mechanical lysis of infected host cells, and magnetic isolation of LCVs that have fused with lysosomes. In silico analysis of the mutated genes, D. discoideum plaque assays and infection assays in protozoa and U937 macrophage-like cells identified well established as well as novel putative L. pneumophila virulence factors. Promising candidates were further analyzed for their co-localization with lysosomes in host cells using fluorescence microscopy. This approach corroborated that the O-methyltransferase, PilY1, TPR-containing protein and polyketide synthase (PKS) of L. pneumophila interfere with lysosomal degradation. Competitive infections in protozoa and macrophages revealed that the identified PKS contributes to the biological fitness of pneumophila strains and may explain their prevalence in the epidemiology of Legionnaires' disease.

Asunto(s)

Interacciones Huésped-Patógeno , Legionella pneumophila/fisiología , Enfermedad de los Legionarios/microbiología , Lisosomas/metabolismo , Sintasas Poliquetidas/metabolismo , Vacuolas/microbiología , Factores de Virulencia/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Línea Celular , Elementos Transponibles de ADN , Dictyostelium/microbiología , Humanos , Legionella pneumophila/genética , Legionella pneumophila/crecimiento & desarrollo , Monocitos/microbiología , Mutagénesis Insercional , Sintasas Poliquetidas/genética , Vacuolas/metabolismo , Factores de Virulencia/genética