trypsin-digest-protocol-to-analyze-retinal-vasculature-mouse

Trypsin Digest Protocol to Analyze the Retinal Vasculature of a Mouse Model

Trypsin digest is the gold standard method to analyze the retinal vasculature 1-5. It allows visualization of the entire network of complex ...

Many pathogens regulate or modify their immune-stimulating ligands to avoid detection by their infected hosts. Listeria monocytogenes, a facultative intracellular bacterial pathogen, interacts with multiple components of mammalian innate immunity during its infection cycle. During replication within the cytosol of infected cells, L. monocytogenes utilizes two multidrug efflux pumps, MdrM and MdrT, to secrete the small nucleic acid second messenger cyclic-di-AMP (c-di-AMP). Host recognition of c-di-AMP triggers the production of type I interferons, including beta interferon (IFN-Î²), which, surprisingly, promote L. monocytogenes virulence. In this study, we have examined the capacity of multiple laboratory and clinical isolates of L. monocytogenes to stimulate host production of IFN-Î². We have identified the L. monocytogenes strain LO28 as able to hyperinduce IFN-Î² production in infected cells âˆ¼30-fold more than the common laboratory clone L. monocytogenes strain 10403S. Genomic analyses determined that LO28 contains a naturally occurring loss-of-function allele of the transcriptional regulator BrtA and correspondingly derepresses expression of MdrT. Surprisingly, while derepression of MdrT resulted in hyperstimulation of IFN-Î², it results in significant attenuation in multiple mouse models of infection. While type I interferons may promote L. monocytogenes virulence, this study demonstrates that unregulated expression of the c-di-AMP-secreting efflux pump MdrT significantly restricts virulence in vivo by an unknown mechanism.

Stuart D. Rollins

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