High throughput methods to study protein-protein interactions during host-pathogen interactions.

The ability of a pathogen to survive and cause an infection is often determined by specific interactions between the host and pathogen proteins. Such interactions can be both intra- and extracellular and may define the outcome of an infection. There are a range of innovative biochemical, biophysical and bioinformatic techniques currently available to identify protein-protein interactions (PPI) between the host and the pathogen. However, the complexity and the diversity of host-pathogen PPIs has led to the development of several high throughput (HT) techniques that enable the study of multiple interactions at once and/or screen multiple samples at the same time, in an unbiased manner. We review here the major HT laboratory-based technologies employed for host-bacterial interaction studies.

A Strategic Target Rescues Trimethoprim Sensitivity in Escherichia coli.

Trimethoprim, a preferred treatment for urinary tract infections, is becoming obsolete owing to the rapid dissemination of resistant E. coli. Although direct resistance mechanisms such as overexpression of a mutant FolA and dfr enzymes are well characterized, associated alterations that drive or sustain resistance are unknown. We identify the repertoire of resistance-associated perturbations by constructing and interrogating a transcriptome-integrated functional interactome. From the cross talk between perturbations in stress-response and metabolic pathways, we identify the critical dependence on serine hydroxymethyltransferase (GlyA) as an emergent vulnerability. Through its deletion, we demonstrate that GlyA is necessary to sustain high levels of resistance in both laboratory-evolved resistant E. coli and a multidrug-resistant clinical isolate. Through comparative evolution, we show that the absence of GlyA activity decelerates the acquisition of resistance in E. coli. Put together, our results identify GlyA as a promising target, providing a basis for the rational design of drug combinations.

Rhizospheric life of Salmonella requires flagella-driven motility and EPS-mediated attachment to organic matter and enables cross-kingdom invasion.

Salmonella is an established pathogen of the members of the kingdom Animalia. Reports indicate that the association of Salmonella with fresh, edible plant products occurs at the pre-harvest state, i.e. in the field. In this study, we follow the interaction of Salmonella Typhimurium with the model plant Arabidopsis thaliana to understand the process of migration in soil. Plant factors like root exudates serve as chemo-attractants. Our ex situ experiments allowed us to track Salmonella from its free-living state to the endophytic state. We found that genes encoding two-component systems and proteins producing extracellular polymeric substances are essential for Salmonella to adhere to the soil and roots. To understand the trans-kingdom flow of Salmonella, we fed the contaminated plants to mice and observed that it invades and colonizes liver and spleen. To complete the disease cycle, we re-established the infection in plant by mixing the potting mixture with the fecal matter collected from the diseased animals. Our experiments revealed a cross-kingdom invasion by the pathogen via passage through a murine intermediate, a mechanism for its persistence in the soil and invasion in a non-canonical host. These results form a basis to break the life-cycle of Salmonella before it reaches its animal host and thus reduce Salmonella contamination of food products.

Rewiring of one carbon metabolism in Salmonella serves as an excellent live vaccine against systemic salmonellosis.

Live attenuated vaccines are superior to the killed or subunit vaccines. We designed a Salmonella Typhimurium strain by deleting folD gene (encoding methylenetetrahydrofolate dehydrogenase-cyclohydrolase) in the presence of a heterologous fhs gene (encoding formyltetrahydrofolate synthetase) and tested its vaccine potential under stringent conditions of lethal and sub-lethal challenges with virulent Salmonella in the murine model. The efficacy of the vaccine in conferring protection against Salmonella infection was determined in a wide range of host conditions of systemic infection, corresponding to human young adults, neonates, geriatric age and, importantly, to the immune compromised state of pregnancy. The standardized vaccination regime comprised a primary dose of 104 CFU/animal followed by a booster dose of 102 CFU/animal on day 7. Challenge with the virulent pathogen was done at day 7 post-administration of the booster. Subsequently, the mortality, morbidity, systemic colonization, antibody response and cytokine profiling were determined. The vaccinated cohort showed a strong protection against virulent pathogen in all models tested. The serum anti-Salmonella antibody titers and cytokine levels were significantly higher in the vaccinated cohort compared to the mock vaccinated cohort. Thus, we report the development and validation of a live attenuated vaccine candidate conferring excellent protection against Salmonellosis and typhoid fever.