RESUMEN
BACKGROUND: Ventilator-associated pneumonia (VAP) is a leading hospital acquired infection in intensive care units despite improved patient care practices and advancements in endotracheal tube (ETT) designs. The ETT provides a conduit for bacterial access to the lower respiratory tract and a substratum for biofilm formation, both of which lead to VAP. A novel microscopic ordered surface topography, the Sharklet micro-pattern, has been shown to decrease surface attachment of numerous microorganisms, and may provide an alternative strategy for VAP prevention if included on the surface of an ETT. To evaluate the feasibility of this micro-pattern for this application, the microbial range of performance was investigated in addition to biofilm studies with and without a mucin-rich medium to simulate the tracheal environment in vitro. METHODS: The top five pathogens associated with ETT-related pneumonia, Methicillin-Resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa, Klebsiella pneumonia, Acinetobacter baumannii, and Escherichia coli, were evaluated for attachment to micro-patterned and un-patterned silicone surfaces in a short-term colonization assay. Two key pathogens, MRSA and Pseudomonas aeruginosa, were evaluated for biofilm formation in a nutrient rich broth for four days and minimal media for 24 hours, respectively, on each surface type. P. aeruginosa was further evaluated for biofilm formation on each surface type in a mucin-modified medium mimicking tracheal mucosal secretions. Results are reported as percent reductions and significance is based on t-tests and ANOVA models of log reductions. All experiments were replicated at least three times. RESULTS: Micro-patterned surfaces demonstrated reductions in microbial colonization for a broad range of species, with up to 99.9% (p < 0.05) reduction compared to un-patterned controls. Biofilm formation was also reduced, with 67% (p = 0.12) and 52% (p = 0.05) reductions in MRSA and P. aeruginosa biofilm formation, respectively. Further, a 58% (p < 0.01) reduction was demonstrated on micro-patterned surfaces for P. aeruginosa biofilms under clinically-simulated conditions when compared to un-patterned controls. CONCLUSIONS: This engineered micro-pattern reduces the colonization and biofilm formation of key VAP-associated pathogens in vitro. Future application of this micro-pattern on endotracheal tubes may prevent or prolong the onset of VAP without the need for antimicrobial agents.
RESUMEN
The Paf1 complex (Paf1, Ctr9, Cdc73, Rtf1, and Leo1) is normally associated with RNA polymerase II (Pol II) throughout the transcription cycle. However, the loss of either Rtf1 or Cdc73 results in the detachment of the Paf1 complex from Pol II and the chromatin form of actively transcribed genes. Using functionally tagged forms of the Paf1 complex factors, we have determined that, except for the more loosely associated Rtf1, the remaining components stay stably associated with one another in an RNase-resistant complex after dissociation from Pol II and chromatin. The loss of Paf1, Ctr9, or to a lesser extent Cdc73 or Rtf1 results in reduced levels of serine 2 phosphorylation of the Pol II C-terminal domain and in increased read through of the MAK21 polyadenylation site. We found that the cleavage and polyadenylation factor Cft1 requires the Pol II-associated form of the Paf1 complex for full levels of interaction with the serine 5-phosphorylated form of Pol II. When the Paf1 complex is dissociated from Pol II, a direct interaction between Cft1 and the Paf1 complex can be detected. These results are consistent with the Paf1 complex providing a point of contact for recruitment of 3'-end processing factors at an early point in the transcription cycle. The lack of this connection helps to explain the defects in 3'-end formation observed in the absence of Paf1.
Asunto(s)
Proteínas Nucleares/metabolismo , ARN Polimerasa II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas Nucleares/genética , Unión Proteica , ARN Polimerasa II/genética , Procesamiento Postranscripcional del ARN , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteína de Unión a TATA-Box/genética , Proteína de Unión a TATA-Box/metabolismo , Factores de Elongación Transcripcional , Factores de Escisión y Poliadenilación de ARNm/genéticaRESUMEN
The yeast Paf1 complex (Paf1C: Paf1, Cdc73, Ctr9, Rtf1, and Leo1) is associated with RNA Polymerase II (Pol II) at promoters and coding regions of transcriptionally active genes, but transcript abundance for only a small subset of genes is altered by loss of Paf1. By using conditional and null alleles of PAF1 and microarrays, we determined the identity of both primary and secondary targets of the Paf1C. Neither primary nor secondary Paf1C target promoters were responsive to loss of Paf1. Instead, Paf1 loss altered poly(A) site utilization of primary target genes SDA1 and MAK21, resulting in increased abundance of 3'-extended mRNAs. The 3'-extended MAK21 RNA is sensitive to nonsense-mediated decay (NMD), as revealed by its increased abundance in the absence of Upf1. Therefore, although the Paf1C is associated with Pol II at initiation and during elongation, these critical Paf1-dependent changes in transcript abundance are due to alterations in posttranscriptional processing.
Asunto(s)
Proteínas Nucleares/metabolismo , ARN Polimerasa II/metabolismo , Procesamiento Postranscripcional del ARN/fisiología , ARN Mensajero/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Sustancias Macromoleculares/metabolismo , Proteínas Nucleares/genética , ARN Mensajero/genética , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
The yeast Paf1 complex, minimally composed of Paf1, Ctr9, Cdc73, Rtf1, and Leo1, was originally isolated in association with RNA polymerase II (Pol II). Paf1 complex components are abundant and colocalize with Pol II on chromatin at promoters and in the coding regions of actively transcribed genes. Loss of Paf1 results in severe phenotypes and reduced amounts of other Paf1 factors, with little effect on abundance or chromatin distribution of Pol II, proteins important for transcriptional elongation (Spt5, Spt16), or RNA processing (Sub2). Loss of Paf1 factors causes a reduction of Pol II Ser2 phosphorylation and shortened poly(A) tails, suggesting that the complex facilitates linkage of transcriptional and posttranscriptional events. Surprisingly, loss of Rtf1 or Cdc73, with little phenotypic consequence, results in loss of Paf1 factors from chromatin and a significant reduction in Paf1/Pol II association. Therefore, the major functions of Paf1 can be independent of actively transcribing Pol II.