Bacterial Pathogen-Mediated Suppression of Host Trafficking to Lysosomes: Fluorescence Microscopy-Based DQ-Red BSA Analysis.

Intracellular bacterial pathogens have evolved to be adept at manipulating host cellular function for the benefit of the pathogen, often by means of secreted virulence factors that target host pathways for modulation. The lysosomal pathway is an essential cellular response pathway to intracellular pathogens and, as such, represents a common target for bacterial-mediated evasion. Here, we describe a method to quantitatively assess bacterial pathogen-mediated suppression of host cell trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of epithelial cells as a model. This live-cell imaging assay involves the use of a BODIPY TR-X conjugate of BSA (DQ-Red BSA) that traffics to and fluoresces in functional lysosomes. This method can be adapted to study infection with a broad array of pathogens in diverse host cell types. It is capable of being applied to identify secreted virulence factors responsible for a phenotype of interest as well as domains within the bacterial protein that are important for mediating the phenotype. Collectively, these tools can provide invaluable insight into the mechanisms of pathogenesis of a diverse array of pathogenic bacteria, with the potential to uncover virulence factors that may be suitable targets for therapeutic intervention. Key features • Infection-based analysis of bacterial-mediated suppression of host trafficking to lysosomes, using Salmonella enterica serovar Typhimurium infection of human epithelial cells as a model. • Live microscopy-based analysis allows for the visualization of individually infected host cells and is amenable to phenotype quantification. • Assay can be adapted to a broad array of pathogens and diverse host cell types. • Assay can identify virulence factors mediating a phenotype and protein domains that mediate a phenotype.

The progress of type II persisters of Escherichia coli O157:H7 to a non-culturable state during prolonged exposure to antibiotic stress with revival being aided through acid-shock treatment and provision of methyl pyruvate.

Persisters are a form of dormancy in bacteria that provide temporary resistance to antibiotics. The following reports on the formation of Escherichia coli O157:H7 E318 type II persisters from a protracted (8 days) challenge with ampicillin. Escherichia coli O157:H7 followed a multiphasic die-off pattern with an initial rapid decline (Phase I) of susceptible cells that transitioned to a slower rate representing tolerant cells (Phase II). After 24 h post-antibiotic challenge, the E. coli O157:H7 levels remained relatively constant at 2 log CFU/mL (Phase III), but became non-culturable within 8-days (Phase IV). The revival of persisters in Phase III could be achieved by the removal of antibiotic stress, although those in Phase IV required an extended incubation period or application of acid-shock. The carbon utilization profile of persister cells was less diverse compared with non-persisters, with only methyl pyruvate being utilized from the range tested. Inclusion of methyl pyruvate in tryptic soy agar revived non-cultural persisters, presumably by stimulating metabolism. The results suggest that persisters could be subdivided into culturable or non-culturable cells, with the former representing a transition state to the latter. The study provided insights into how to revive cells from dormancy to aid enumeration and control.