Evaluación de la participación de la proteína YlbF en la formación de biofilm y hemólisis de Staphylococcus aureus y Klebsiella pneumoniae
ilustraciones, fotografías
- Autores:
-
Ávila Jiménez, Santiago
- Tipo de recurso:
- Fecha de publicación:
- 2022
- Institución:
- Universidad Nacional de Colombia
- Repositorio:
- Universidad Nacional de Colombia
- Idioma:
- spa
- OAI Identifier:
- oai:repositorio.unal.edu.co:unal/83350
- Palabra clave:
- 570 - Biología::572 - Bioquímica
Proteina
Bacteria
Protein
Staphylococcus aureus
Klebsiella pneumoniae
YlbF
Hemolisis
BIOFILM
- Rights
- openAccess
- License
- Reconocimiento 4.0 Internacional
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| dc.title.spa.fl_str_mv |
Evaluación de la participación de la proteína YlbF en la formación de biofilm y hemólisis de Staphylococcus aureus y Klebsiella pneumoniae |
| dc.title.translated.eng.fl_str_mv |
Evaluation of the participation of the YlbF protein in the formation of biofilm and hemolysis of Staphylococcus aureus and Klebsiella pneumoniae |
| title |
Evaluación de la participación de la proteína YlbF en la formación de biofilm y hemólisis de Staphylococcus aureus y Klebsiella pneumoniae |
| spellingShingle |
Evaluación de la participación de la proteína YlbF en la formación de biofilm y hemólisis de Staphylococcus aureus y Klebsiella pneumoniae 570 - Biología::572 - Bioquímica Proteina Bacteria Protein Staphylococcus aureus Klebsiella pneumoniae YlbF Hemolisis BIOFILM |
| title_short |
Evaluación de la participación de la proteína YlbF en la formación de biofilm y hemólisis de Staphylococcus aureus y Klebsiella pneumoniae |
| title_full |
Evaluación de la participación de la proteína YlbF en la formación de biofilm y hemólisis de Staphylococcus aureus y Klebsiella pneumoniae |
| title_fullStr |
Evaluación de la participación de la proteína YlbF en la formación de biofilm y hemólisis de Staphylococcus aureus y Klebsiella pneumoniae |
| title_full_unstemmed |
Evaluación de la participación de la proteína YlbF en la formación de biofilm y hemólisis de Staphylococcus aureus y Klebsiella pneumoniae |
| title_sort |
Evaluación de la participación de la proteína YlbF en la formación de biofilm y hemólisis de Staphylococcus aureus y Klebsiella pneumoniae |
| dc.creator.fl_str_mv |
Ávila Jiménez, Santiago |
| dc.contributor.advisor.none.fl_str_mv |
Castellanos Parra, Jaime Eduardo Corredor Rozo, Zayda Lorena |
| dc.contributor.author.none.fl_str_mv |
Ávila Jiménez, Santiago |
| dc.contributor.researchgroup.spa.fl_str_mv |
Laboratorio de Genética molecular bacteriana de la Universidad del Bosquer |
| dc.subject.ddc.spa.fl_str_mv |
570 - Biología::572 - Bioquímica |
| topic |
570 - Biología::572 - Bioquímica Proteina Bacteria Protein Staphylococcus aureus Klebsiella pneumoniae YlbF Hemolisis BIOFILM |
| dc.subject.lemb.spa.fl_str_mv |
Proteina Bacteria |
| dc.subject.lemb.eng.fl_str_mv |
Protein |
| dc.subject.proposal.spa.fl_str_mv |
Staphylococcus aureus Klebsiella pneumoniae YlbF Hemolisis |
| dc.subject.proposal.eng.fl_str_mv |
BIOFILM |
| description |
ilustraciones, fotografías |
| publishDate |
2022 |
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2022 |
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2023-02-07T15:41:03Z |
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2023-02-07T15:41:03Z |
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Trabajo de grado - Maestría |
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info:eu-repo/semantics/masterThesis |
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info:eu-repo/semantics/acceptedVersion |
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Text |
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http://purl.org/redcol/resource_type/TM |
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acceptedVersion |
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https://repositorio.unal.edu.co/handle/unal/83350 |
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Universidad Nacional de Colombia |
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Repositorio Institucional Universidad Nacional de Colombia |
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https://repositorio.unal.edu.co/ |
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https://repositorio.unal.edu.co/handle/unal/83350 https://repositorio.unal.edu.co/ |
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Universidad Nacional de Colombia Repositorio Institucional Universidad Nacional de Colombia |
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spa |
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spa |
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Bryksin, A. V. & Matsumura, I. Overlap extension PCR cloning: A simple and reliable way to create recombinant plasmids. Biotechniques 48, 463–465 (2010). Peters, B. M., Jabra-Rizk, M. A., O’May, G. A., William Costerton, J. & Shirtliff, M. E. Polymicrobial interactions: Impact on pathogenesis and human disease. Clin. Microbiol. Rev. 25, 193–213 (2012). Miller, M. B. & Bassler, B. L. Quorum Sensing in Bacteria. Annu. Rev. Microbiol. 55, 165–199 (2001). Waters, C. M. & Bassler, B. L. QUORUM SENSING: Cell-to-Cell Communication in Bacteria. Annu. Rev. Cell Dev. Biol. 21, 319–346 (2005) Archer, N. K. et al. Staphylococcus aureus biofilms: Properties, regulation and roles in human disease. Virulence vol. 2 445–459 (2011). Balasubramanian, D., Harper, L., Shopsin, B. & Torres, V. J. Staphylococcus aureus pathogenesis in diverse host environments. Pathog. Dis. 75, (2017) Chambers, H. F. The changing epidemiology of staphylococcus aureus? in Emerging Infectious Diseases vol. 7 178–182 (Centers for Disease Control and Prevention (CDC), 2001) Iwase, T. et al. Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization. nature.com doi:10.1038/nature09074. Cervantes-García, E., García-González, R. & María Salazar-Schettino, P. Características generales del Staphylococcus aureus. Rev Latinoam Patol Clin Med Lab vol. 61 www.medigraphic.com/patologiaclinicawww.medigraphic.org.mx (2014). . Tumbarello, M. et al. Predictors of mortality in patients with bloodstream infections caused by extended-spectrum-beta-lactamase-producing Enterobacteriaceae: importance of inadequate initial antimicrobial treatment. Antimicrob. Agents Chemother. 51, 1987–1994 (2007). Paczosa, M. K. & Mecsas, J. Klebsiella pneumoniae: Going on the Offense with a Strong Defense. Microbiol. Mol. Biol. Rev. 80, 629–661 (2016). Botto, M. et al. Bacterial Biofilms: A Common Cause of Persistent Infections. Annu. Rev. Plant Physiol. Plant Mol. Biol vol. 64 www.sciencemag.org (1998). Otto, M. Staphylococcal Biofilms. in Gram-Positive Pathogens 699–711 (ASM Press, 2019). doi:10.1128/9781683670131.ch43. Piperaki, E. T., Syrogiannopoulos, G. A., Tzouvelekis, L. S. & Daikos, G. L. Klebsiella pneumoniae: Virulence, Biofilm and Antimicrobial Resistance. Pediatr. Infect. Dis. J. 36, 1002–1005 (2017). Soto, S. M. Importance of Biofilms in Urinary Tract Infections: New Therapeutic Approaches. (2014) doi:10.1155/2014/543974. Sowemimo-Coker, S. O. Red blood cell hemolysis during processing. Transfus. Med. Rev. 16, 46–60 (2002). Vandenesch, F., Lina, G. & Henry, T. Staphylococcus aureus hemolysins, bicomponent leukocidins, and cytolytic peptides: a redundant arsenal of membranedamaging virulence factors? Frontiers in cellular and infection microbiology vol. 2 12 (2012). Carabetta, V. J. et al. A complex of YlbF, YmcA and YaaT regulates sporulation, competence and biofilm formation by accelerating the phosphorylation of Spo0A. doi:10.1111/mmi.12186. Tortosa, P., Albano, M. & Dubnau, D. Characterization of ylbF, a new gene involved in competence development and sporulation in Bacillus subtilis. Mol. Microbiol. 35, 1110–1119 (2000). Adusei-Dans, F. et al. Structure-function studies of the Bacillus Subtilis RIC proteins identify the Fe-S cluster-ligating residues and their roles in development and RNA processing. MBio 10, (2019). Escobar Pérez, J. A. Identificación y caracterización de una proteína de unión al gen icaA y evaluación de su potencial participación en la formación de biofilm en Staphylococcus aureus. (2018) DeLoughery, A., Dengler, V., Chai, Y. & Losick, R. Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR. Mol. Microbiol. 99, 425–437 (2016) Ohniwa, R. L., Ushijima, Y., Saito, S. & Morikawa, K. Proteomic Analyses of Nucleoid-Associated Proteins in Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. PLoS One 6, e19172 (2011). Escobar-Perez, J. et al. Identification and “in silico” Structural Analysis of the Glutamine-rich Protein Qrp (YheA) in Staphylococcus Aureus. Open Bioinforma. J. 12, 18–29 (2019) DeLoughery, A., Lalanne, J. B., Losick, R. & Li, G. W. Maturation of polycistronic mRNAs by the endoribonuclease RNase Y and its associated Y-complex in Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 115, E5585–E5594 (2018). Tanner, A. W. et al. The RicAFT (YmcA-YlbF-YaaT) complex carries two [4Fe4S]2+ clusters and may respond to redox changes. Mol. Microbiol. 104, 837–850 (2017) Gheorghe, I., Popa, M. & Gabriela Măruţescu, L. Molecular Features of Virulence and Resistance Mechanisms in Nosocomial and Community-Acquired Staphylococcus aureus. in Staphylococcus Aureus (IntechOpen, 2019). doi:10.5772/intechopen.75191. Kluytmans, J., Van Belkum, A. & Verbrugh, H. Nasal Carriage of Staphylococcus aureus: Epidemiology, Underlying Mechanisms, and Associated Risks. Am Soc Microbiol vol. 10 http://cmr.asm.org/ (1997). Chambers, H. F. & DeLeo, F. R. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nature Reviews Microbiology vol. 7 629–641 (2009). Kuroda, M. et al. Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Elsevier. Valle, J. et al. SarA and not σB is essential for biofilm development by Staphylococcus aureus. Mol. Microbiol. 48, 1075–1087 (2003) Martí, M. et al. Extracellular proteases inhibit protein-dependent biofilm formation in Staphylococcus aureus. Microbes Infect. 12, 55–64 (2010). O’Rourke, J. P. et al. Development of a Mimotope Vaccine Targeting the Staphylococcus aureus Quorum Sensing Pathway. PLoS One 9, e111198 (2014). Otto, M. Staphylococcal biofilms. Current Topics in Microbiology and Immunology vol. 322 207–228 (2008). O’Neill, E. et al. A novel Staphylococcus aureus biofilm phenotype mediated by the fibronectin-binding proteins, FnBPA and FnBPB. J. Bacteriol. 190, 3835–3850 (2008). Rodriguez Tamayo, E. A. & Quinceno, Jimenez, J. N. Factores relacionados con la colonización por Staphylococcus aureus. 66–77 (2015). Yamashita, K. et al. Crystal structure of the octameric pore of staphylococcal γhemolysin reveals the β-barrel pore formation mechanism by two components. Proc. Natl. Acad. Sci. U. S. A. 108, 17314–17319 (2011). Camussone, C. M. & Calvinho, L. F. Virulence factors of Staphylococcus aureus associated with intramammary infections in cows: Relevance and role as immunogens. Rev. Argent. Microbiol. 45, 119–130 (2013) Berube, B. J. & Bubeck Wardenburg, J. Staphylococcus aureus α-Toxin: Nearly a Century of Intrigue. Toxins (Basel). 5, 1140–1166 (2013). Otto, M. & Gov, M. N. Staphylococcus aureus toxins. doi:10.1016/j.mib.2013.11.004. Verdon, J., Girardin, N., Lacombe, C., Berjeaud, J. M. & Héchard, Y. δ-hemolysin, an update on a membrane-interacting peptide. Peptides vol. 30 817–823 (2009). Dubnau, E. J. et al. A protein complex supports the production of Spo0A-P and plays additional roles for biofilms and the K-state in Bacillus subtilis. Mol. Microbiol. 101, 606–624 (2016). Kearns, D. B., Chu, F., Branda, S. S., Kolter, R. & Losick, R. A master regulator for biofilm formation by Bacillus subtilis. Mol. Microbiol. 55, 739–749 (2005). Parashar, V., Konkol, M. A., Kearns, D. B. & Neiditch, M. B. A plasmid-encoded phosphatase regulates bacillus subtilis biofilm architecture, sporulation, and genetic competence. J. Bacteriol. 195, 2437–2448 (2013). Branda, S. S. et al. Genes Involved in Formation of Structured Multicellular Communities by Bacillus subtilis. J. Bacteriol. 186, 3970–3979 (2004). . Khemici, V., Prados, J., Linder, P. & Redder, P. Decay-Initiating Endoribonucleolytic Cleavage by RNase Y Is Kept under Tight Control via Sequence Preference and Sub-cellular Localisation. PLOS Genet. 11, e1005577 (2015). Bonnin, R. A. & Bouloc, P. RNA Degradation in Staphylococcus aureus: Diversity of Ribonucleases and Their Impact. (2015) doi:10.1155/2015/395753. Marincola, G. et al. RNase Y of Staphylococcus aureus and its role in the activation of virulence genes. Mol. Microbiol. 85, 817–832 (2012). Marincola, G. & Wolz, C. Downstream element determines RNase Y cleavage of the saePQRS operon in Staphylococcus aureus. Nucleic Acids Res. 45, 5980–5994 (2017). Ashurst, J. V. & Dawson, A. Klebsiella Pneumonia. StatPearls (2018). Lopez Vargas, J. A. & Echeverri Toro, L. M. K. pneumoniae: ¿la nueva “superbacteria”? Patogenicidad, epidemiología y mecanismos de resistencia | Iatreia. https://revistas.udea.edu.co/index.php/iatreia/article/view/11129 (2010). Tzouvelekis, L. S., Markogiannakis, A., Psichogiou, M., Tassios, P. T. & Daikos, G. L. Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: An evolving crisis of global dimensions. Clin. Microbiol. Rev. 25, 682–707 (2012). Van Duijn, P. J., Dautzenberg, M. J. D. & Oostdijk, E. A. N. Recent trends in antibiotic resistance in European ICUs. Curr. Opin. Crit. Care 17, 658–665 (2011) Andrade, G. et al. Comité Editorial: MENSAJE BIOQUÍMICO. (2020). Podschun, R. & Ullmann, U. Klebsiella spp. as nosocomial pathogens: Epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin. Microbiol. Rev. 11, 589–603 (1998). Clements, A. et al. The Major Surface-Associated Saccharides of Klebsiella pneumoniae Contribute to Host Cell Association. PLoS One 3, e3817 (2008). Hornick, D. B., Allen, B. L., Horn, M. A. & Clegg, S. Adherence to respiratory epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae type 3 fimbrial gene products. Infect. Immun. 60, 1577 (1992). Tarkkanen, A. M., Virkola, R., Clegg, S. & Korhonen, T. K. Binding of the type 3 fimbriae of Klebsiella pneumoniae to human endothelial and urinary bladder cells. Infect. Immun. 65, 1546 (1997). Jagnow, J. & Clegg, S. Klebsiella pneumoniae MrkD-mediated biofilm formation on extracellular matrix- and collagen-coated surfaces. Microbiology 149, 2397–2405 (2003). Langstraat, J., Bohse, M. & Clegg, S. Type 3 fimbrial shaft (MrkA) of Klebsiella pneumoniae, but not the fimbrial adhesin (MrkD), facilitates biofilm formation. Infect. Immun. 69, 5805–5812 (2001). Hornick, D. B., Thommandru, J., Smits, W. & Clegg, S. Adherence properties of an mrkD-negative mutant of Klebsiella pneumoniae. Infect. Immun. 63, 2026–2032 (1995). Arnaud, M., Chastanet, A. & Débarbouillé, M. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. Appl. Environ. Microbiol. 70, 6887–6891 (2004). Yansura, D. G. & Henner, D. J. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 81, 439–443 (1984). Tran, D. T. M. et al. Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis. Microb. Cell Fact. 16, 1–10 (2017). Deloughery, A., Dengler, V., Chai, Y. & Losick, R. Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR. Mol. Microbiol. 99, 425–437 (2016). Vergara-Irigaray, M. et al. Relevant Role of Fibronectin-Binding Proteins in Staphylococcus aureus Biofilm-Associated Foreign-Body Infections †. Infect. Immun. 77, 3978–3991 (2009). George, E. A. & Muir, T. W. Molecular mechanisms of agr quorum sensing in virulent staphylococci. ChemBioChem vol. 8 847–855 (2007). Pilar Trotonda, M., Manna, A. C., Cheung, A. L., Lasa, I. & Penadés, J. R. SarA Positively Controls Bap-Dependent Biofilm Formation in Staphylococcus aureus. J. Bacteriol. 187, 5790–5798 (2005) Liu, Q., Yeo, W. S. & Bae, T. The SaeRS two-component system of Staphylococcus aureus. Genes vol. 7 (2016). Charpentier, E. et al. Novel Cassette-Based Shuttle Vector System for GramPositive Bacteria. Appl. Environ. Microbiol. 70, 6076–6085 (2004). Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583 (2021). Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004). Lehnik-Habrink, M., Lewis, R. J., Mäder, U. & Stülke, J. RNA degradation in Bacillus subtilis: An interplay of essential endo- and exoribonucleasesmmi. Mol. Microbiol. 84, 1005–1017 (2012). |
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Universidad Nacional de Colombia |
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Bogotá - Ciencias - Maestría en Ciencias - Bioquímica |
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Facultad de Ciencias |
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Bogotá, Colombia |
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Universidad Nacional de Colombia - Sede Bogotá |
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Reconocimiento 4.0 Internacionalhttp://creativecommons.org/licenses/by/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Castellanos Parra, Jaime Eduardod005422b5147ff1af3f046ffaf4328e6Corredor Rozo, Zayda Lorena159a74b683c58884227442ac5a501f42Ávila Jiménez, Santiago25490c13ff66f56a3d0162c3bbe33627Laboratorio de Genética molecular bacteriana de la Universidad del Bosquer2023-02-07T15:41:03Z2023-02-07T15:41:03Z2022https://repositorio.unal.edu.co/handle/unal/83350Universidad Nacional de ColombiaRepositorio Institucional Universidad Nacional de Colombiahttps://repositorio.unal.edu.co/ilustraciones, fotografíasStaphylococcus aureus y Klebsiella pneumoniae son bacterias reconocidas como patógenas de infecciones intrahospitalarias y comunitarias a nivel mundial. Estas se caracterizan por presentar genes que codifican diferentes factores de adaptación al medio y factores de virulencia como la formación de biofilm y la lisis de eritrocitos, los cuales son controlados por una red compleja de reguladores transcripcionales y ribonucleasas. En Bacillus subtilis se ha demostrado que las proteínas del complejo Y (YlbF, YmcA y YaaT), que se caracterizan por presentar un dominio Com_ylbF, están relacionadas con la regulación de la formación de biofilm, competencia y esporulación. Recientemente, en S. aureus se identificó una proteína con dominio Com_ylbF denominada Qrp (YheA), la cual al ser delecionada del genoma, afecta la formación de biofilm y la hemólisis. S. aureus expresa las proteínas YmcA, YaaT y YlbF, pero a la fecha no se tiene información sobre su participación en la regulación de estos factores de virulencia. Por otra parte, en K. pneumoniae no se han reportado los genes que codifican para estas proteínas, ni la relación que pueden tener con los factores de virulencia, pero se ha encontrado la proteína YlbF en una cepa de esta bacteria. El objetivo de este trabajo fue evaluar la participación de la proteína YlbF en los procesos de hemólisis y formación de biofilm en S. aureus y K. pneumoniae, para ello se realizaron ensayos de deleción (S. aureus) y complementación (K. pneumoniae) de las bacterias con el gen ylbF y se evaluó mediante ensayos de formación de biofilm y ensayos de hemolisis si existía alguna variación en estos procesos al realizar la deleción o complementación de este gen. Se logró realizar la deleción del gen ylbF del genoma de S. aureus y la complementación del gen ylbF en K. pneumoniae y se encontró que las cepas estudiadas presentan una variación en la formación de biofilm y en la hemolisis de eritrocitos cuando se varia la presencia de esta proteína. Esto no indicaría que esta proteína YlbF, perteneciente al complejo Y puede ser de gran importancia para los procesos básicos de colonización y secreción de factores de virulencia por parte de estas bacterias. (Texto tomado de la fuente)Staphylococcus aureus and Klebsiella pneumoniae are bacteria recognized as pathogens of nosocomial and community infections worldwide. These are characterized by presenting genes that encode different adaptation factors to the environment and virulence factors such as biofilm formation and erythrocyte lysis, which are controlled by a complex network of transcriptional regulators and ribonucleases. In Bacillus subtilis it has been shown that the proteins of the Y complex (YlbF, YmcA and YaaT), which are characterized by having a Com_ylbF domain, are related to the regulation of biofilm formation, competition, and sporulation. Recently, a protein with a Com_ylbF domain called Qrp (YheA) was identified in S. aureus, which, when deleted from the genome, affects biofilm formation and hemolysis. S. aureus expresses YmcA, YaaT and YlbF proteins, but to date there is no information on their participation in the regulation of these virulence factors. On the other hand, the genes that code for these proteins have not been reported in K. pneumoniae, nor the relationship they may have with virulence factors, but the YlbF protein has been found in a strain of this bacterium. The objective of this work was to evaluate the participation of the YlbF protein in the processes of hemolysis and biofilm formation in S. aureus and K. pneumoniae, for which deletion (S. aureus) and complementation (K. pneumoniae) assays were performed. of the bacteria with the ylbF gene and it was evaluated by means of biofilm formation assays and hemolysis assays if there was any variation in these processes when performing the deletion or complementation of this gene. It was possible to carry out the deletion of the ylbF gene from the S. aureus genome and the complementation of the ylbF gene in K. pneumoniae and it was found that the studied strains present a variation in the formation of biofilm and in the hemolysis of erythrocytes when the presence is varied. of this protein. This would not indicate that this YlbF protein, belonging to the Y complex, may be of great importance for the basic processes of colonization and secretion of virulence factors by these bacteriaCOLCIENCIASMaestríaGENETICA MOLECULAR BACTERIANABIOQUIMICAPROTEOMICAEstudio de factores de virulencia bacterianosxx, 86 páginasapplication/pdfspaUniversidad Nacional de ColombiaBogotá - Ciencias - Maestría en Ciencias - BioquímicaFacultad de CienciasBogotá, ColombiaUniversidad Nacional de Colombia - Sede Bogotá570 - Biología::572 - BioquímicaProteinaBacteriaProteinStaphylococcus aureusKlebsiella pneumoniaeYlbFHemolisisBIOFILMEvaluación de la participación de la proteína YlbF en la formación de biofilm y hemólisis de Staphylococcus aureus y Klebsiella pneumoniaeEvaluation of the participation of the YlbF protein in the formation of biofilm and hemolysis of Staphylococcus aureus and Klebsiella pneumoniaeTrabajo de grado - Maestríainfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/acceptedVersionTexthttp://purl.org/redcol/resource_type/TMBryksin, A. V. & Matsumura, I. Overlap extension PCR cloning: A simple and reliable way to create recombinant plasmids. Biotechniques 48, 463–465 (2010).Peters, B. M., Jabra-Rizk, M. A., O’May, G. A., William Costerton, J. & Shirtliff, M. E. Polymicrobial interactions: Impact on pathogenesis and human disease. Clin. Microbiol. Rev. 25, 193–213 (2012).Miller, M. B. & Bassler, B. L. Quorum Sensing in Bacteria. Annu. Rev. Microbiol. 55, 165–199 (2001).Waters, C. M. & Bassler, B. L. QUORUM SENSING: Cell-to-Cell Communication in Bacteria. Annu. Rev. Cell Dev. Biol. 21, 319–346 (2005)Archer, N. K. et al. Staphylococcus aureus biofilms: Properties, regulation and roles in human disease. Virulence vol. 2 445–459 (2011).Balasubramanian, D., Harper, L., Shopsin, B. & Torres, V. J. Staphylococcus aureus pathogenesis in diverse host environments. Pathog. Dis. 75, (2017)Chambers, H. F. The changing epidemiology of staphylococcus aureus? in Emerging Infectious Diseases vol. 7 178–182 (Centers for Disease Control and Prevention (CDC), 2001)Iwase, T. et al. Staphylococcus epidermidis Esp inhibits Staphylococcus aureus biofilm formation and nasal colonization. nature.com doi:10.1038/nature09074.Cervantes-García, E., García-González, R. & María Salazar-Schettino, P. Características generales del Staphylococcus aureus. Rev Latinoam Patol Clin Med Lab vol. 61 www.medigraphic.com/patologiaclinicawww.medigraphic.org.mx (2014).. Tumbarello, M. et al. Predictors of mortality in patients with bloodstream infections caused by extended-spectrum-beta-lactamase-producing Enterobacteriaceae: importance of inadequate initial antimicrobial treatment. Antimicrob. Agents Chemother. 51, 1987–1994 (2007).Paczosa, M. K. & Mecsas, J. Klebsiella pneumoniae: Going on the Offense with a Strong Defense. Microbiol. Mol. Biol. Rev. 80, 629–661 (2016).Botto, M. et al. Bacterial Biofilms: A Common Cause of Persistent Infections. Annu. Rev. Plant Physiol. Plant Mol. Biol vol. 64 www.sciencemag.org (1998).Otto, M. Staphylococcal Biofilms. in Gram-Positive Pathogens 699–711 (ASM Press, 2019). doi:10.1128/9781683670131.ch43.Piperaki, E. T., Syrogiannopoulos, G. A., Tzouvelekis, L. S. & Daikos, G. L. Klebsiella pneumoniae: Virulence, Biofilm and Antimicrobial Resistance. Pediatr. Infect. Dis. J. 36, 1002–1005 (2017).Soto, S. M. Importance of Biofilms in Urinary Tract Infections: New Therapeutic Approaches. (2014) doi:10.1155/2014/543974.Sowemimo-Coker, S. O. Red blood cell hemolysis during processing. Transfus. Med. Rev. 16, 46–60 (2002).Vandenesch, F., Lina, G. & Henry, T. Staphylococcus aureus hemolysins, bicomponent leukocidins, and cytolytic peptides: a redundant arsenal of membranedamaging virulence factors? Frontiers in cellular and infection microbiology vol. 2 12 (2012).Carabetta, V. J. et al. A complex of YlbF, YmcA and YaaT regulates sporulation, competence and biofilm formation by accelerating the phosphorylation of Spo0A. doi:10.1111/mmi.12186.Tortosa, P., Albano, M. & Dubnau, D. Characterization of ylbF, a new gene involved in competence development and sporulation in Bacillus subtilis. Mol. Microbiol. 35, 1110–1119 (2000).Adusei-Dans, F. et al. Structure-function studies of the Bacillus Subtilis RIC proteins identify the Fe-S cluster-ligating residues and their roles in development and RNA processing. MBio 10, (2019).Escobar Pérez, J. A. Identificación y caracterización de una proteína de unión al gen icaA y evaluación de su potencial participación en la formación de biofilm en Staphylococcus aureus. (2018)DeLoughery, A., Dengler, V., Chai, Y. & Losick, R. Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR. Mol. Microbiol. 99, 425–437 (2016)Ohniwa, R. L., Ushijima, Y., Saito, S. & Morikawa, K. Proteomic Analyses of Nucleoid-Associated Proteins in Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus aureus. PLoS One 6, e19172 (2011).Escobar-Perez, J. et al. Identification and “in silico” Structural Analysis of the Glutamine-rich Protein Qrp (YheA) in Staphylococcus Aureus. Open Bioinforma. J. 12, 18–29 (2019)DeLoughery, A., Lalanne, J. B., Losick, R. & Li, G. W. Maturation of polycistronic mRNAs by the endoribonuclease RNase Y and its associated Y-complex in Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 115, E5585–E5594 (2018).Tanner, A. W. et al. The RicAFT (YmcA-YlbF-YaaT) complex carries two [4Fe4S]2+ clusters and may respond to redox changes. Mol. Microbiol. 104, 837–850 (2017)Gheorghe, I., Popa, M. & Gabriela Măruţescu, L. Molecular Features of Virulence and Resistance Mechanisms in Nosocomial and Community-Acquired Staphylococcus aureus. in Staphylococcus Aureus (IntechOpen, 2019). doi:10.5772/intechopen.75191.Kluytmans, J., Van Belkum, A. & Verbrugh, H. Nasal Carriage of Staphylococcus aureus: Epidemiology, Underlying Mechanisms, and Associated Risks. Am Soc Microbiol vol. 10 http://cmr.asm.org/ (1997).Chambers, H. F. & DeLeo, F. R. Waves of resistance: Staphylococcus aureus in the antibiotic era. Nature Reviews Microbiology vol. 7 629–641 (2009).Kuroda, M. et al. Whole genome sequencing of meticillin-resistant Staphylococcus aureus. Elsevier.Valle, J. et al. SarA and not σB is essential for biofilm development by Staphylococcus aureus. Mol. Microbiol. 48, 1075–1087 (2003)Martí, M. et al. Extracellular proteases inhibit protein-dependent biofilm formation in Staphylococcus aureus. Microbes Infect. 12, 55–64 (2010).O’Rourke, J. P. et al. Development of a Mimotope Vaccine Targeting the Staphylococcus aureus Quorum Sensing Pathway. PLoS One 9, e111198 (2014).Otto, M. Staphylococcal biofilms. Current Topics in Microbiology and Immunology vol. 322 207–228 (2008).O’Neill, E. et al. A novel Staphylococcus aureus biofilm phenotype mediated by the fibronectin-binding proteins, FnBPA and FnBPB. J. Bacteriol. 190, 3835–3850 (2008).Rodriguez Tamayo, E. A. & Quinceno, Jimenez, J. N. Factores relacionados con la colonización por Staphylococcus aureus. 66–77 (2015).Yamashita, K. et al. Crystal structure of the octameric pore of staphylococcal γhemolysin reveals the β-barrel pore formation mechanism by two components. Proc. Natl. Acad. Sci. U. S. A. 108, 17314–17319 (2011).Camussone, C. M. & Calvinho, L. F. Virulence factors of Staphylococcus aureus associated with intramammary infections in cows: Relevance and role as immunogens. Rev. Argent. Microbiol. 45, 119–130 (2013)Berube, B. J. & Bubeck Wardenburg, J. Staphylococcus aureus α-Toxin: Nearly a Century of Intrigue. Toxins (Basel). 5, 1140–1166 (2013).Otto, M. & Gov, M. N. Staphylococcus aureus toxins. doi:10.1016/j.mib.2013.11.004.Verdon, J., Girardin, N., Lacombe, C., Berjeaud, J. M. & Héchard, Y. δ-hemolysin, an update on a membrane-interacting peptide. Peptides vol. 30 817–823 (2009).Dubnau, E. J. et al. A protein complex supports the production of Spo0A-P and plays additional roles for biofilms and the K-state in Bacillus subtilis. Mol. Microbiol. 101, 606–624 (2016).Kearns, D. B., Chu, F., Branda, S. S., Kolter, R. & Losick, R. A master regulator for biofilm formation by Bacillus subtilis. Mol. Microbiol. 55, 739–749 (2005).Parashar, V., Konkol, M. A., Kearns, D. B. & Neiditch, M. B. A plasmid-encoded phosphatase regulates bacillus subtilis biofilm architecture, sporulation, and genetic competence. J. Bacteriol. 195, 2437–2448 (2013).Branda, S. S. et al. Genes Involved in Formation of Structured Multicellular Communities by Bacillus subtilis. J. Bacteriol. 186, 3970–3979 (2004).. Khemici, V., Prados, J., Linder, P. & Redder, P. Decay-Initiating Endoribonucleolytic Cleavage by RNase Y Is Kept under Tight Control via Sequence Preference and Sub-cellular Localisation. PLOS Genet. 11, e1005577 (2015).Bonnin, R. A. & Bouloc, P. RNA Degradation in Staphylococcus aureus: Diversity of Ribonucleases and Their Impact. (2015) doi:10.1155/2015/395753.Marincola, G. et al. RNase Y of Staphylococcus aureus and its role in the activation of virulence genes. Mol. Microbiol. 85, 817–832 (2012).Marincola, G. & Wolz, C. Downstream element determines RNase Y cleavage of the saePQRS operon in Staphylococcus aureus. Nucleic Acids Res. 45, 5980–5994 (2017).Ashurst, J. V. & Dawson, A. Klebsiella Pneumonia. StatPearls (2018).Lopez Vargas, J. A. & Echeverri Toro, L. M. K. pneumoniae: ¿la nueva “superbacteria”? Patogenicidad, epidemiología y mecanismos de resistencia | Iatreia. https://revistas.udea.edu.co/index.php/iatreia/article/view/11129 (2010).Tzouvelekis, L. S., Markogiannakis, A., Psichogiou, M., Tassios, P. T. & Daikos, G. L. Carbapenemases in Klebsiella pneumoniae and other Enterobacteriaceae: An evolving crisis of global dimensions. Clin. Microbiol. Rev. 25, 682–707 (2012).Van Duijn, P. J., Dautzenberg, M. J. D. & Oostdijk, E. A. N. Recent trends in antibiotic resistance in European ICUs. Curr. Opin. Crit. Care 17, 658–665 (2011)Andrade, G. et al. Comité Editorial: MENSAJE BIOQUÍMICO. (2020).Podschun, R. & Ullmann, U. Klebsiella spp. as nosocomial pathogens: Epidemiology, taxonomy, typing methods, and pathogenicity factors. Clin. Microbiol. Rev. 11, 589–603 (1998).Clements, A. et al. The Major Surface-Associated Saccharides of Klebsiella pneumoniae Contribute to Host Cell Association. PLoS One 3, e3817 (2008).Hornick, D. B., Allen, B. L., Horn, M. A. & Clegg, S. Adherence to respiratory epithelia by recombinant Escherichia coli expressing Klebsiella pneumoniae type 3 fimbrial gene products. Infect. Immun. 60, 1577 (1992).Tarkkanen, A. M., Virkola, R., Clegg, S. & Korhonen, T. K. Binding of the type 3 fimbriae of Klebsiella pneumoniae to human endothelial and urinary bladder cells. Infect. Immun. 65, 1546 (1997).Jagnow, J. & Clegg, S. Klebsiella pneumoniae MrkD-mediated biofilm formation on extracellular matrix- and collagen-coated surfaces. Microbiology 149, 2397–2405 (2003).Langstraat, J., Bohse, M. & Clegg, S. Type 3 fimbrial shaft (MrkA) of Klebsiella pneumoniae, but not the fimbrial adhesin (MrkD), facilitates biofilm formation. Infect. Immun. 69, 5805–5812 (2001).Hornick, D. B., Thommandru, J., Smits, W. & Clegg, S. Adherence properties of an mrkD-negative mutant of Klebsiella pneumoniae. Infect. Immun. 63, 2026–2032 (1995).Arnaud, M., Chastanet, A. & Débarbouillé, M. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, gram-positive bacteria. Appl. Environ. Microbiol. 70, 6887–6891 (2004).Yansura, D. G. & Henner, D. J. Use of the Escherichia coli lac repressor and operator to control gene expression in Bacillus subtilis. Proc. Natl. Acad. Sci. U. S. A. 81, 439–443 (1984).Tran, D. T. M. et al. Development of inducer-free expression plasmids based on IPTG-inducible promoters for Bacillus subtilis. Microb. Cell Fact. 16, 1–10 (2017).Deloughery, A., Dengler, V., Chai, Y. & Losick, R. Biofilm formation by Bacillus subtilis requires an endoribonuclease-containing multisubunit complex that controls mRNA levels for the matrix gene repressor SinR. Mol. Microbiol. 99, 425–437 (2016).Vergara-Irigaray, M. et al. Relevant Role of Fibronectin-Binding Proteins in Staphylococcus aureus Biofilm-Associated Foreign-Body Infections †. Infect. Immun. 77, 3978–3991 (2009).George, E. A. & Muir, T. W. Molecular mechanisms of agr quorum sensing in virulent staphylococci. ChemBioChem vol. 8 847–855 (2007).Pilar Trotonda, M., Manna, A. C., Cheung, A. L., Lasa, I. & Penadés, J. R. SarA Positively Controls Bap-Dependent Biofilm Formation in Staphylococcus aureus. J. Bacteriol. 187, 5790–5798 (2005)Liu, Q., Yeo, W. S. & Bae, T. The SaeRS two-component system of Staphylococcus aureus. Genes vol. 7 (2016).Charpentier, E. et al. Novel Cassette-Based Shuttle Vector System for GramPositive Bacteria. Appl. Environ. Microbiol. 70, 6076–6085 (2004).Jumper, J. et al. Highly accurate protein structure prediction with AlphaFold. Nature 596, 583 (2021).Pettersen, E. F. et al. UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004).Lehnik-Habrink, M., Lewis, R. J., Mäder, U. & Stülke, J. RNA degradation in Bacillus subtilis: An interplay of essential endo- and exoribonucleasesmmi. Mol. Microbiol. 84, 1005–1017 (2012).Público generalLICENSElicense.txtlicense.txttext/plain; charset=utf-85879https://repositorio.unal.edu.co/bitstream/unal/83350/1/license.txteb34b1cf90b7e1103fc9dfd26be24b4aMD51ORIGINAL1014249157.2022.pdf1014249157.2022.pdfTesis de Maestría en Ciencias - Bioquímicaapplication/pdf1679705https://repositorio.unal.edu.co/bitstream/unal/83350/2/1014249157.2022.pdf6167e94651820dc3250ae12e1d4d008dMD52THUMBNAIL1014249157.2022.pdf.jpg1014249157.2022.pdf.jpgGenerated Thumbnailimage/jpeg5182https://repositorio.unal.edu.co/bitstream/unal/83350/3/1014249157.2022.pdf.jpg5ecab0a8a9ead6c9692d85ed5b4d4e6bMD53unal/83350oai:repositorio.unal.edu.co:unal/833502023-08-15 23:04:18.562Repositorio Institucional Universidad Nacional de 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