Salmonella cause over 40,000 reported infections and 500 deaths annually at a cost of $50M. The most dangerous one escape the early neutrophil (PMN) reaction in the lamina propria and disseminate. Are some Salmonella typhimurium more resistant to oxygen-independent killing by PMN cationic antimicrobial proteins (CAP) and therefore more likely to disseminate? Recently, genetically determined changes in lipid A have been implicated in such resistance.
Aim 1 : To study the resistance of Salmonella to CAP with standard molecular genetics, to genetically and physically characterize the gene(s) that control it, and to identify the mechanisms;
Aim 2 : To study binding of CAP by the mutant Salmonella, the permeabilizing action of CAP, and the disorganizing action of CAP on LPS and the outer membrane;
Aim 3 : To examine LPS structure of mutant alleles;
and Aim 4 : to compare side by side in the same PMN monolayers the resistance of Salmonella mutants and the parent strain O2-independent antimicrobial action. Spontaneous and Tn10 insertional mutants resistant to both polymyxin B and CAP or resistant only to CAP will be used. Techniques: insertional mutagenesis, generalized transduction enzymes, agarose gel electrophoresis, and probing or Southern transfer with a Tn10 probe. Gene(s) will be cloned with established techniques or radioiodinated, biologically active cationic proteins will be studied. Studies on permeabilization of strains and LPS release from strains will be done with established methods. The structure of lipid A will be studied with fast-atom bombardment electron mass spectrometry, laser desorption, and nuclear magnetic resonance spectroscopy. (The mutations (in the wild-type background), together with wild-type Salmonella, will be compared side by side in the same monolayer of human PMN to test resistance to killing in anaerobic conditions.) We use drug resistance or metabolic markers to separate and count mutant and parent strains surviving side by side in PMN. Bacterial survival rates are calculated from inputs estimated from metabolically incorporated 3H-uracil and 35S- methionine.
