RESUMO
Bacteriophages (phages) are the most abundant biological entity in the human body, but until recently the role that phages play in human health was not well characterized. Although phages do not cause infections in human cells, phages can alter the severity of bacterial infections by the dissemination of virulence factors amongst bacterial hosts. Recent studies, made possible with advances in genome engineering and microscopy, have uncovered a novel role for phages in the human body - the ability to modulate the physiology of the mammalian cells that can harbor intracellular bacteria. In this review, we synthesize key results on how phages traverse through mammalian cells - including uptake, distribution, and interaction with intracellular receptors - highlighting how these steps in turn influence host cell killing of bacteria. We discuss the implications of the growing field of phage-mammalian cell interactions for phage therapy.
Assuntos
Bacteriófagos/metabolismo , Células/metabolismo , Células/virologia , Interações Hospedeiro-Patógeno , Mamíferos , Animais , Bacteriófagos/genética , Células/citologia , Citosol/microbiologia , Citosol/virologia , DNA Viral , Humanos , Camundongos , Fagossomos/microbiologia , Fagossomos/virologia , Prófagos/genética , Prófagos/metabolismo , Internalização do VírusRESUMO
The gut microbiome is dominated by lysogens, bacteria that carry bacterial viruses (phages). Uncovering the function of phages in the microbiome and observing interactions between phages, bacteria, and mammalian cells in real time in specific cell types are limited by the difficulty of engineering fluorescent markers into large, lysogenic phage genomes. Here, we present a method to multiplex the engineering of life-cycle reporters into lysogenic phages and how to infect macrophages with engineered lysogens to study these interactions at the single-cell level. For complete details on the use and execution of this protocol, please refer to Bodner et al. (2020).
Assuntos
Bacteriófagos/genética , Macrófagos/virologia , Análise de Célula Única/métodos , Bactérias/virologia , Bioengenharia/métodos , Microbioma Gastrointestinal/genética , Microbioma Gastrointestinal/fisiologia , Engenharia Genética/métodos , Lisogenia , Prófagos/genética , Ativação Viral/fisiologiaRESUMO
Half of the bacteria in the human gut microbiome are lysogens containing integrated prophages, which may activate in stressful immune environments. Although lysogens are likely to be phagocytosed by macrophages, whether prophage activation occurs or influences the outcome of bacterial infection remains unexplored. To study the dynamics of bacteria-phage interactions in living cells-in particular, the macrophage-triggered induction and lysis of dormant prophages in the phagosome-we adopted a tripartite system where murine macrophages engulf E. coli, which are lysogenic with an engineered bacteriophage λ, containing a fluorescent lysis reporter. Pre-induced prophages are capable of lysing the host bacterium and propagating infection to neighboring bacteria in the same phagosome. A non-canonical pathway, mediated by PhoP, is involved with the native λ phage induction inside phagocytosed E. coli. These findings suggest two possible mechanisms by which induced prophages may function to aid the bactericidal activity of macrophages.
Assuntos
Lisogenia/fisiologia , Imagem Molecular/métodos , Ativação Viral/fisiologia , Animais , Bactérias , Bacteriófago lambda/fisiologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Microbioma Gastrointestinal , Engenharia Genética/métodos , Células HEK293 , Humanos , Macrófagos/metabolismo , Camundongos , Prófagos/metabolismo , Prófagos/fisiologia , Células RAW 264.7RESUMO
Aptamers are chemically synthesized oligonucleotides or peptides with molecular recognition capabilities. We investigated recognition of substrate-tethered small-molecule targets, using neurotransmitters as examples, and fluorescently labeled DNA aptamers. Substrate regions patterned via microfluidic channels with dopamine or â¯â¯l-tryptophan were selectively recognized by previously identified dopamine or l-tryptophan aptamers, respectively. The on-substrate dissociation constant determined for the dopamine aptamer was comparable to, though, slightly greater than the previously determined solution dissociation constant. Using prefunctionalized neurotransmitter-conjugated oligo(ethylene glycol) alkanethiols and microfluidics patterning, we produced multiplexed substrates to capture and to sort aptamers. Substrates patterned with l-3,4-dihydroxyphenylalanine, l- threo-dihydroxyphenylserine, and l-5-hydroxytryptophan enabled comparison of the selectivity of the dopamine aptamer for different targets via simultaneous determination of in situ binding constants. Thus, beyond our previous demonstrations of recognition by protein binding partners (i.e., antibodies and G-protein-coupled receptors), strategically optimized small-molecule-functionalized substrates show selective recognition of nucleic acid binding partners. These substrates are useful for side-by-side target comparisons and future identification and characterization of novel aptamers targeting neurotransmitters or other important small molecules.