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      Attenuated Salmonella Typhimurium Lacking the Pathogenicity Island-2 Type 3 Secretion System Grow to High Bacterial Numbers inside Phagocytes in Mice

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          Abstract

          Intracellular replication within specialized vacuoles and cell-to-cell spread in the tissue are essential for the virulence of Salmonella enterica. By observing infection dynamics at the single-cell level in vivo, we have discovered that the Salmonella pathogenicity island 2 (SPI-2) type 3 secretory system (T3SS) is dispensable for growth to high intracellular densities. This challenges the concept that intracellular replication absolutely requires proteins delivered by SPI-2 T3SS, which has been derived largely by inference from in vitro cell experiments and from unrefined measurement of net growth in mouse organs. Furthermore, we infer from our data that the SPI-2 T3SS mediates exit from infected cells, with consequent formation of new infection foci resulting in bacterial spread in the tissues. This suggests a new role for SPI-2 in vivo as a mediator of bacterial spread in the body. In addition, we demonstrate that very similar net growth rates of attenuated salmonellae in organs can be derived from very different underlying intracellular growth dynamics.

          Author Summary

          High quality science has been published concerning the dynamics of infectious disease spread through communities of people or animals, but less work has been done to understand infectious disease dynamics within the host. Many conclusions about how infectious agents work are based on experiments in isolated monocultures of cells or in somewhat crude experiments in whole animals. Understanding this complex process in whole animals is the next major challenge for infectious disease biologists, and is required if intervention strategies to prevent and cure infectious diseases are to be improved and targeted effectively. Bacteria of the species Salmonella enterica are a threat to public health, causing a wide range of life-threatening diseases in humans and animals world-wide. In vitro cell experiments and inference from measuring net growth kinetics in mouse organs suggest that intracellular replication of S. enterica requires proteins delivered by the Salmonella pathogenicity island 2 (SPI-2) type 3 secretion system (T3SS) and that mutants in SPI-2 cannot replicate efficiently intracellularly. However, by observing directly infection dynamics at the single-cell level in vivo, we show that SPI-2 T3SS mutants can replicate to high intracellular densities in phagocytes in the organs of infected animals, but appear unable to leave infected cells.

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          Most cited references 42

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          Aromatic-dependent Salmonella typhimurium are non-virulent and effective as live vaccines.

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            High efficiency transformation of E. coli by high voltage electroporation.

            E. coli can be transformed to extremely high efficiencies by subjecting a mixture of cells and DNA to brief but intense electrical fields of exponential decay waveform (electroporation). We have obtained 10(9) to 10(10) transformants/micrograms with strains LE392 and DH5 alpha, and plasmids pUC18 and pBR329. The process is highly dependent on two characteristics of the electrical pulse: the electric field strength and the pulse length (RC time constant). The frequency of transformation is a linear function of the DNA concentration over at least six orders of magnitude; and the efficiency of transformation is a function of the cell concentration. Most of the surviving cells are competent with up to 80% transformed at high DNA concentration. The mechanism does not appear to include binding of the DNA to the cells prior to entry. Possible mechanisms are discussed and a simple procedure for the practical use of this technique is presented.
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              Protein delivery into eukaryotic cells by type III secretion machines.

              Bacteria that have sustained long-standing close associations with eukaryotic hosts have evolved specific adaptations to survive and replicate in this environment. Perhaps one of the most remarkable of those adaptations is the type III secretion system (T3SS)--a bacterial organelle that has specifically evolved to deliver bacterial proteins into eukaryotic cells. Although originally identified in a handful of pathogenic bacteria, T3SSs are encoded by a large number of bacterial species that are symbiotic or pathogenic for humans, other animals including insects or nematodes, and plants. The study of these systems is leading to unique insights into not only organelle assembly and protein secretion but also mechanisms of symbiosis and pathogenesis.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                December 2012
                December 2012
                6 December 2012
                : 8
                : 12
                Affiliations
                Department of Veterinary Medicine, University of Cambridge, Cambridge, United Kingdom
                Stanford University School of Medicine, United States of America
                Author notes

                ¶ These authors are joint senior authors on this work.

                The authors have declared that no competing interests exist.

                Conceived and designed the experiments: AJG DJM PM. Performed the experiments: AJG FJEM GLF PM. Analyzed the data: AJG TJM DJM PM. Wrote the paper: AJG DJM PM. Obtained the funding: AJG DJM PM.

                Article
                PPATHOGENS-D-12-01815
                10.1371/journal.ppat.1003070
                3516571
                23236281

                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 credited.

                Page count
                Pages: 11
                Funding
                This work was supported by a Medical Research Council (MRC) grant G0801161 awarded to AJG, PM and DJM. TJM was supported by the Department for the Environment, Food and Rural Affairs/Higher Education Funding Council of England (grant number VT0105). GLF was supported by a Wellcome Trust PhD training studentship. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Genetics
                Genomics
                Immunology
                Microbiology
                Molecular Cell Biology
                Population Biology
                Mathematics
                Statistics
                Medicine
                Infectious Diseases

                Infectious disease & Microbiology

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