Abstract
Within deep tissue sites, extracellular bacterial pathogens often replicate in clusters that are surrounded by immune cells. Disease is modulated by interbacterial interactions as well as bacterial-host cell interactions resulting in microbial growth, phagocytic attack and secretion of host antimicrobial factors. To overcome the limited ability to manipulate these infection sites, we established a system for Yersinia pseudotuberculosis (Yptb) growth in microfluidics-driven microdroplets that regenerates microbial social behavior in tissues. Chemical generation of nitric oxide (NO) in the absence of immune cells was sufficient to reconstruct microbial social behavior, as witnessed by expression of the NO-inactivating protein Hmp on the extreme periphery of microcolonies, mimicking spatial regulation in tissues. Similarly, activated macrophages that expressed inducible NO synthase (iNOS) drove peripheral expression of Hmp, allowing regeneration of social behavior observed in tissues. These results argue that topologically correct microbial tissue growth and associated social behavior can be reconstructed in culture.
Highlights
A variety of bacterial pathogens colonize and replicate within tissues despite the presence of the host immune system (Carter & Collins, 1974; Cheng et al, 2009; Simonet et al, 1990).Growth in tissue sites involves the formation of distinct foci of replication, which can develop into either abscesses, granulomas, or poorly defined clusters of bacteria (Cheng et al, 2011; Pagan & Ramakrishnan, 2018)
For pathogens as diverse as Mycobacterium tuberculosis and Staphylococcus aureus, growth in tissues is followed by recruitment of neutrophils, macrophages and inflammatory monocytes, which can lead to the formation of abscesses or granulomas when the infection is not eradicated (Cheng et al, 2011; Pagan & Ramakrishnan, 2018)
These pathogen-immune cell aggregates are intricate structures in which the pathogen interfaces with host cells, and result in significant self and social interactions, making it difficult to reproduce in culture the entire set of events present in tissue
Summary
A variety of bacterial pathogens colonize and replicate within tissues despite the presence of the host immune system (Carter & Collins, 1974; Cheng et al, 2009; Simonet et al, 1990).Growth in tissue sites involves the formation of distinct foci of replication, which can develop into either abscesses, granulomas, or poorly defined clusters of bacteria (Cheng et al, 2011; Pagan & Ramakrishnan, 2018). Extracellular bacterial pathogens, in particular, can establish a tissue niche and replicate to high numbers These clusters of bacteria in tissues are often clonal, result in distinct microcolonies, and are surrounded by host innate immune cells. Yptb establishes extracellular foci of replication, resulting in the formation of microcolonies that develop into lesions that are densely populated by immune cells (Simonet et al, 1990). The resulting neutrophil-bacterium interface appears to result in a stable relationship, in which frustrated phagocytes bind, but do not internalize the nearby bacteria Surrounding this sphere of neutrophils are macrophages, monocytes, and other immune cells, which appear to be recruited as a response to failure to clear the focus of infection
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