Salmonella promotes virulence by repressing cellulose production

Cellulose is the most abundant organic polymer on Earth. In bacteria, cellulose confers protection against environmental insults and is a constituent of biofilms typically formed on abiotic surfaces. We report that, surprisingly, Salmonella enterica serovar Typhimurium makes cellulose when inside macrophages. We determine that preventing cellulose synthesis increases virulence, whereas stimulation of cellulose synthesis inside macrophages decreases virulence. An attenuated mutant lacking the mgtC gene exhibited increased cellulose levels due to increased expression of the cellulose synthase gene bcsA and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of bcsA restored wild-type virulence to the Salmonella mgtC mutant, but not to other attenuated mutants displaying a wild-type phenotype regarding cellulose. Our findings indicate that a virulence determinant can promote pathogenicity by repressing a pathogen''s antivirulence trait. Moreover, they suggest that controlling antivirulence traits increases long-term pathogen fitness by mediating a trade-off between acute virulence and transmission.Bacterial pathogens encode genes that promote virulence. Virulence genes increase the fitness of pathogens by fostering replication at the expense of their hosts (1). Typically, virulence genes function by providing protection from host antimicrobial products, enabling the synthesis of nutrients that are limiting in host tissues and by manipulating host pathways in ways that favor pathogen survival at preferred sites. Notably, pathogens also may encode antivirulence genes, that is, genes that hamper pathogens'' virulence (2–5). Here we provide a singular example of a virulence protein that promotes pathogenicity by interfering with the production of an antivirulence factor.Cellulose is a polysaccharide composed of β(1→4)-linked d-glucose units. As a major structural component of the cell walls of plants and many eukaryotic microorganisms, cellulose accounts for ∼1.5 × 10¹² tons of the annual biomass on Earth, making it the most abundant organic polymer on the planet (6). In bacteria, cellulose is an exopolysaccharide normally synthesized in the context of organized bacterial communities known as biofilms. Cellulose inhibits bacterial motility by hindering flagellar rotation (7), and provides cohesion and structural integrity to mature biofilms (8–10).The facultative intracellular pathogen Salmonella enterica serovar Typhimurium causes gastroenteriditis in humans and a systemic infection in mice that resembles typhoid fever (11). During systemic infection, Salmonella survives and replicates in specialized membrane-bound mildly acidic vacuoles within host phagocytic cells (12, 13). Growth within these specialized compartments requires the coordinated expression of an array of virulence determinants (14), including the MgtC protein (15). MgtC is a unique virulence factor because it interacts with and inhibits the activity of Salmonella’s F₁F_o ATP synthase (16), a protein complex that is responsible for synthesis of the majority of the ATP in the bacterium (17) and is also required for virulence (18). MgtC’s action prevents a nonphysiological increase in cytosolic ATP and decrease in cytosolic pH taking place during growth in mildly acidic environments, such as that experienced by Salmonella inside a macrophage phagosome (16).In addition to its role in promoting intramacrophage survival, MgtC enables Salmonella (15, 19) and a number of phylogenetically distant intracellular bacterial pathogens (20–24) to grow normally in low-Mg²⁺ laboratory media. In Salmonella, growth in low-Mg²⁺ media also promotes mgtC expression, even when Salmonella experiences a neutral pH (15, 19). Notably, the mgtC mutant harbors higher ATP levels than the wild-type (WT) strain when grown in low-Mg²⁺ media, similar to what it exhibits on mild acidification of its surroundings (16). These findings suggest that a rise in ATP levels leads to physiological alterations that hinder growth in low-Mg²⁺ media and attenuated virulence.We now report that, surprisingly, Salmonella produces cellulose when inside macrophages. We establish that the MgtC protein promotes Salmonella virulence by limiting cellulose production during infection. We determine that MgtC controls both expression of the cellulose synthase complex and the intracellular levels of cyclic diguanylate (c-di-GMP), the cellulose synthase’s allosteric activator. Virulence can be restored to the mgtC mutant simply by preventing cellulose biosynthesis, which does not affect ATP levels. Our findings illustrate how Salmonella uses a virulence protein to repress the expression of an antivirulence trait during infection of a mammalian host, and they define cellulose as an antivirulence determinant. Moreover, they suggest that pathogens use antivirulence traits to balance acute virulence and transmission.