Pseudomonas aeruginosa Microcolony Aggregates Elicit Neutrophil Swarming - PROJECT SUMMARY/ABSTRACT Pseudomonas aeruginosa is a ubiquitous, opportunistic bacterial pathogen adept at developing antimicrobial resistance and capable of colonizing a variety of host tissue sites to manifest disease. The immunocompromised lung, damaged skin from burn or injury, or scratched cornea represent vulnerable sites where P. aeruginosa infection establishes to cause significant pathology. P. aeruginosa exhibit both planktonic and biofilm lifestyles in the course of infection. Host cells are infiltrated by individual bacteria damaging affected tissue while communities of bacteria emerge in extracellular space as biofilms. These colonization tactics create formidable targets that challenge the defensive capabilities of individual neutrophils recruited as part of the innate immune response. In turn, neutrophils have evolved a strategy of working together as a population to synergize their collective arsenals against large target pathogenic threats that lie beyond the capacity of individual neutrophils to phagocytose and clear. This strategy reflects an emergent property of the collective called neutrophil swarming whereby individual neutrophils coordinate to surround a target (an area of infection or damage) so that the swarm can isolate and neutralize the threat. A greater understanding of the cellular and molecular mechanisms of neutrophil swarming has tremendous potential to reveal novel anti-infective therapeutic strategies with improved efficiency geared toward harnessing the power of the swarm to eradicate intractable infections. This proposal is timely as multidrug resistant strains of P. aeruginosa have been on the rise in recent years and pose an increasingly alarming burden to healthcare providers. We unveil herein a novel ex vivo model of neutrophil swarming against a laboratory cultivated large bacterial target. The bacterial target consists of P. aeruginosa combined with agar to create beads of imbedded bacteria growing as microcolonies on the bead surface. We demonstrate that neutrophils rapidly swarm in response to these large bacterial targets and the swarm can temporarily restrict bacterial growth. We seek to leverage this new model to identify P. aeruginosa and neutrophil factors that drive this multifaceted innate immune response. Our data suggests that elements of the bacterial type three secretion system (T3SS) play a central role from the pathogen perspective. Additionally, we will explore contributions of the neutrophil swarm amplifying agent, leukotriene B4, and mechanisms by which it is biosynthesized by a neutrophil community in the context of swarm development. Overall, we contend that our approach contributes a new tool for investigators to use when seeking to understand cellular and molecular mechanisms of neutrophil swarming. In leveraging this tool, we aim to add to this knowledge base by executing of our proposed mechanistic studies focused on both host and pathogenic factors. Results of our investigations can serve as foundational toward development of novel anti-infective strategies that bolster host innate immune responses rather than targeting the infecting organism, which may reduce development of drug resistance and provide efficacy to a variety of infectious organisms with limited treatment options that cause disease in humans.
