As molecular studies of disease-causing microbes advanced, Stanley Falkow modified Koch’s postulates in order to apply them to the molecular genetics of pathogenicity. When the era of molecular genetics began nearly a century later, the need arose to redefine Koch’s postulates. Finally, the etiological agent must be able to be reisolated from the inoculated host, and be identified as identical to the original causative agent. Furthermore, the etiological agents of the disease must be able to be isolated from an infected individual, and the isolated microbe should subsequently cause disease when introduced into a healthy individual. Koch’s postulates stated that the causative agent of a disease must be found in abundance in all organisms suffering from the condition, but should not be found in association with healthy individuals. In 1884, Robert Koch proposed a set of four criteria designed to establish a causative relationship between a microbe and a disease that defined infectious disease research in the 19 th and 20 th centuries. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Ĭompeting interests: The authors have declared that no competing interests exist. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedĭata Availability: All relevant data are within the paper and its supporting information files.įunding: This research was supported by the National Health and Medical Research Council,, APP1049716 to JAF. Received: JanuAccepted: FebruPublished: April 9, 2015Ĭopyright: © 2015 Arras et al. PLoS ONE 10(4):Īcademic Editor: Kirsten Nielsen, University of Minnesota, UNITED STATES Combined, these data confirm that we have successfully created a much-needed molecular resource for the Cryptococcus community, enabling the reliable fulfillment of the molecular Koch’s postulates.Ĭitation: Arras SDM, Chitty JL, Blake KL, Schulz BL, Fraser JA (2015) A Genomic Safe Haven for Mutant Complementation in Cryptococcus neoformans. qRT-PCR of the flanking genes on either side of the safe haven site following integration of the targeting vector revealed no changes in their expression, and no secondary phenotypes were observed in a range of phenotypic assays including an intranasal murine infection model. The plasmid vector integrates with high frequency, effectively complementing a mutant strain without disrupting adjacent genes. neoformans genome dubbed the “safe haven”, and constructed a plasmid vector that targets DNA constructs to this preselected site. We have identified and characterized a small gene-free region in the C. To counter the drawbacks of the current approaches to complementation we have created a new tool to assist in this key step in the study of a gene’s role in virulence. ![]() Depending on which approach is utilized, the mutant may be complemented but other genes are potentially disrupted in the process. Currently, one of three approaches will be taken: the gene is reintroduced at the original locus, the gene is reintroduced into a random site in the genome, or the mutant is not complemented at all. ![]() However, the complementation of these mutants is less straightforward. These approaches are widely employed in Cryptococcus neoformans, where gene deletion via biolistic transformation is a well-established technique. Likewise, its reintroduction should restore virulence to the mutant. Fundamentally, these molecular postulates state that if a gene is involved in virulence, its removal will compromise virulence. Just as Koch’s postulates formed the foundation of early infectious disease study, Stanley Falkow’s molecular Koch’s postulates define best practice in determining whether a specific gene contributes to virulence of a pathogen.
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