Sugar-Phosphate Toxicities Attenuate Salmonella Fitness in the Gut.

Pathogens are becoming resistant to antimicrobials at an increasing rate, and novel therapeutic strategies are needed. Using Salmonella as a model, we have investigated the induction of sugar-phosphate toxicity as a potential therapeutic modality. The approach entails providing a nutrient while blocking the catabolism of that nutrient, resulting in the accumulation of a toxic intermediate. We hypothesize that this build-up will decrease the fitness of the organism during infection given nutrient availability. We tested this hypothesis using mutants lacking one of seven genes whose mutation is expected to cause the accumulation of a toxic metabolic intermediate. The araD, galE, rhaD, glpD, mtlD, manA, and galT mutants were then provided the appropriate sugars, either in vitro or during gastrointestinal infection of mice. All but the glpD mutant had nutrient-dependent growth defects in vitro, suggestive of sugar-phosphate toxicity. During gastrointestinal infection of mice, five mutants had decreased fitness. Providing the appropriate nutrient in the animal's drinking water was required to cause fitness defects with the rhaD and manA mutants and to enhance the fitness defect of the araD mutant. The galE and mtlD mutants were severely attenuated regardless of the nutrient being provided in the drinking water. Homologs of galE are widespread among bacteria and in humans, rendering the specific targeting of bacterial pathogens difficult. However, the araD, mtlD, and rhaD genes are not present in humans, appear to be rare in most phyla of bacteria, and are common in several genera of Enterobacteriaceae, making the encoded enzymes potential narrow-spectrum therapeutic targets. IMPORTANCE Bacterial pathogens are becoming increasingly resistant to antibiotics. There is an urgent need to identify novel drug targets and therapeutic strategies. In this work we have assembled and characterized a collection of mutations in our model pathogen, Salmonella enterica, that block a variety of sugar utilization pathways in such a way as to cause the accumulation of a toxic sugar-phosphate. Mutations in three genes, rhaD, araD, and mtlD, dramatically decrease the fitness of Salmonella in a mouse model of gastroenteritis, suggesting that RhaD, AraD, and MtlD may be good narrow-spectrum drug targets. The induction of sugar-phosphate toxicities may be a therapeutic strategy that is broadly relevant to other bacterial and fungal pathogens.

Rapid Aggregation of Staphylococcus aureus in Synovial Fluid Is Influenced by Synovial Fluid Concentration, Viscosity, and Fluid Dynamics, with Evidence of Polymer Bridging.

Early bacterial survival in the postsurgical joint is still a mystery. Recently, synovial fluid-induced aggregation was proposed as a potential mechanism of bacterial protection upon entry into the joint. As synovial fluid is secreted back into the joint cavity following surgery, rapid fluctuations in synovial fluid concentrations, composition, and viscosity occur. These changes, along with fluid movement resulting from postoperative joint motion, modify the environment and potentially affect the kinetics of aggregate formation. Through this work, we sought to evaluate the influence of exposure time, synovial fluid concentration, viscosity, and fluid dynamics on aggregation. Furthermore, we aimed to elucidate the primary mechanism of aggregate formation by assessing the interaction of bacterial adhesins with the synovial fluid polymer fibrinogen. Following incubation under each simulated postoperative joint condition, the aggregates were imaged using confocal microscopy. Our analysis revealed the formation of two distinct aggregate phenotypes, depending on whether the incubation was conducted under static or dynamic conditions. Using a surface adhesin mutant, we have narrowed down the genetic determinants for synovial fluid aggregate formation and identified essential host polymers. We report here that synovial fluid-induced aggregation is influenced by various changes specific to the postsurgical joint environment. While we now have evidence that select synovial fluid polymers facilitate bridging aggregation through essential bacterial adhesins, we suspect that their utility is limited by the increasing viscosity under static conditions. Furthermore, dynamic fluid movement recovers the ability of the bacteria with surface proteins present to aggregate under high-viscosity conditions, yielding large, globular aggregates. IMPORTANCE Infection is a major complication of knee and hip joint replacement surgery, which is used to treat arthritis or joint damage. We have shown that Staphylococcus aureus, a common bacterial pathogen, aggregates upon contact with synovial fluid. Within seconds, the bacterial cells interact with synovial fluid polymers in the joint fluid through their cell wall adhesins. The rapid formation of these aggregates likely aids in early bacterial survival in the joint, potentially contributing to the likelihood of developing an infection. By strengthening our basic understanding of the mechanics of synovial fluid aggregate formation under clinically relevant conditions, we hope to expand the knowledge of how to prevent or disrupt aggregation and reduce and more successfully treat these joint infections.