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

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.