RHS-elements function as type II toxin-antitoxin modules that regulate intra-macrophage replication of Salmonella Typhimurium

RHS elements are components of conserved toxin-delivery systems, wide-spread within the bacterial kingdom and some of the most positively selected genes known. However, very little is known about how Rhs toxins affect bacterial biology. Salmonella Typhimurium contains a full-length rhs gene and an adjacent orphan rhs gene, which lacks the conserved delivery part of the Rhs protein. Here we show that, in addition to the conventional delivery, Rhs toxin-antitoxin pairs encode for functional type-II toxin-antitoxin (TA) loci that regulate S. Typhimurium proliferation within macrophages. Mutant S. Typhimurium cells lacking both Rhs toxins proliferate 2-times better within macrophages, mainly because of an increased growth rate. Thus, in addition to providing strong positive selection for the rhs loci under conditions when there is little or no toxin delivery, internal expression of the toxin-antitoxin system regulates growth in the stressful environment found inside macrophages. Author summary Bacteria that reside and multiply inside of phagocytic cells are hard to treat with common antibiotics, partly because subpopulations of bacteria are non-growing. Very little is known about how bacteria regulate their growth in the phagocytic vesicle. We show that RHS elements, previously known to function as mobilizable toxins that inhibit growth of neighboring bacteria, also function as internally expressed toxin-antitoxin systems that regulate Salmonella Typhimurium growth in macrophages. RHS elements were discovered more than 30 years ago, but their role in biology has long remained unclear even though they are some of the most positively selected genes known. Our results suggest an explanation to why rhs genes are under such strong positive selection in addition to suggesting a novel function for these toxins in regulating bacterial growth.