RESUMO
Brucella abortus is a facultative, intracellular, zoonotic pathogen that resides inside macrophages during infection. This is a specialized niche where B. abortus encounters various stresses as it navigates through the macrophage. In order to survive this harsh environment, B. abortus utilizes post-transcriptional regulation of gene expression through the use of small regulatory RNAs (sRNAs). Here, we characterize a Brucella sRNAs called MavR (for MurF- and virulence-regulating sRNA), and we demonstrate that MavR is required for the full virulence of B. abortus in macrophages and in a mouse model of chronic infection. Transcriptomic and proteomic studies revealed that a major regulatory target of MavR is MurF. MurF is an essential protein that catalyzes the final cytoplasmic step in peptidoglycan (PG) synthesis; however, we did not detect any differences in the amount or chemical composition of PG in the ΔmavR mutant. A 6-nucleotide regulatory seed region within MavR was identified, and mutation of this seed region resulted in dysregulation of MurF production, as well as significant attenuation of infection in a mouse model. Overall, the present study underscores the importance of sRNA regulation in the physiology and virulence of Brucella.
Assuntos
Brucelose , Pequeno RNA não Traduzido , Animais , Camundongos , Brucella abortus/metabolismo , Regulação da Expressão Gênica , Macrófagos , Camundongos Endogâmicos BALB C , Proteômica , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismoRESUMO
Interest in evaluating individual cellular populations in the central nervous system has prompted the development of several techniques enabling the enrichment of single-cell populations. Herein we detail a relatively inexpensive method to specifically isolate neurons, astrocytes, and microglia from a mixed homogenate utilizing magnetic beads conjugated to cell-type specific antibodies. We have used this technique to isolate astrocytes across development and into late adulthood. Finally, we detail the utilization of this technique in novel astrocyte and astrocyte/neuron co-culture paradigms. © 2019 by John Wiley & Sons, Inc.