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Development and Characterization of an Unmarked hemO Cluster Knockout in Hypervirulent Acinetobacter baumannii LAC-4
Başlık:
Development and Characterization of an Unmarked hemO Cluster Knockout in Hypervirulent Acinetobacter baumannii LAC-4
Yazar:
Ewing, Peter James, author.
ISBN:
9780355981827
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (80 pages)
Genel Not:
Source: Masters Abstracts International, Volume: 57-06M(E).
Advisors: Howard H. Xu Committee members: Nancy McQueen; Hyunsook Park.
Özet:
Acinetobacter baumannii is an emerging Gram negative pathogen that has been a major contributor to the significant increases in multi-drug resistant (MDR) nosocomial infections worldwide. This bacterium has been known to rapidly acquire mobile genetic elements, facilitating the emergence of MDR and pan-resistant strains. With the emergence of strains greatly increasing in resistance, treatment with conventional antibiotics has become limited. Furthermore, the development of new antibiotics has not caught up with the spread of antibiotic resistance, and the pharmaceutical antibiotic drug development pipeline is scarce. While antibiotic resistance poses great risk to human health, it also contributes to a significant barrier and burden in studying MDR organisms. In MDR A. baumannii for instance, the mechanisms of resistance are well understood, but the determinants of virulence are poorly understood. Yet scientists are limited in the molecular tools they can use to study A. baumannii due to its resistance to common antibiotic selective markers. One factor of virulence that has become a growing field of study in the past two decades is the role of iron metabolism in virulence. Iron is an important nutrient for all forms of life, and a necessity for growth and replication. While mammalian hosts evolved an array of iron limiting mechanisms to sequester the iron from pathogens, the pathogens themselves have evolved strategies to acquire iron for both colonization and growth. And while the general understanding of bacterial iron metabolism is progressing, investigations into iron utilization in A. baumannii have been limited. Previously, we and our collaborators showed that a strain of A. baumannii, LAC-4, isolated from a Los Angeles County hospital outbreak, exhibited hypervirulence in mice and high serum resistance. The strain was found to express a highly efficient heme utilization system, and after treatment with a heme utilization inhibitor, the hypervirulence was attenuated. Bioinformatics analyses indicated that all A. baumannii strains contained a ubiquitous heme utilization gene cluster, but only some strains, including LAC-4, possess a second heme utilization gene cluster containing a heme oxygenase ( hemO) gene. In this study we have generated an unmarked knockout (KO) mutant of LAC-4 in which the hemO gene cluster has been deleted. Construction of this mutant utilized an optimized gene replacement system using homologous recombination of a knockout cassette containing upstream and downstream flanking regions to the hemO gene cluster cloned into the suicide vector pMo130. MDR LAC-4 had previously been determined susceptible to apramycin, enabling the use of an apramycin resistance gene linked to the end of the KO cassette to provide selection for the single-crossover of the suicide plasmid. The resistance gene was later removed from the chromosome along with the plasmid by a second-crossover event identified by the efficient use of both sacB counter selection and the xylE reporter gene. The resulting mutant was evaluated against an established mouse model of bacterial pneumonia and was found to be significantly less virulent than the LAC-4 wildtype strain. Overall, our study shows that an unmarked knockout construct design used in conjunction with sacB and xylE is an effective method for generating easily identifiable unmarked mutants. Furthermore, and of greater significance in elucidation of heme metabolism, it was shown that the hemO cluster appears to contribute to LAC-4's hypervirulence.
Notlar:
School code: 0962
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Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(687568.1) | 687568-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
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