Background

Bacterial pathogens employ diverse strategies to manipulate host cellular function to establish infection [1]. Among the many approaches, modulation of host cells can occur at the level of immune signalling and immune cell function [1,2], cellular immune responses such as lysosomal and autophagy function [3–6], organelle function [7,8], or cytoskeletal dynamics [1,9,10]. The lysosomal pathway represents a fundamental signalling hub and cellular immune response pathway in eukaryotic cells [11,12]. Lysosomes are membrane-bound organelles responsible for the degradation of proteins, lipids, nucleic acids, and carbohydrates [11,12]. These vesicles exhibit a characteristically low pH that promotes the activation of over 60 different hydrolases, ultimately contributing to cellular homeostasis and clearance of intracellular pathogens [11,12]. Defects in lysosome function can lead to one of more than 50 different disorders, collectively defined as lysosome storage diseases, with implications in neurodegeneration and aging [12–15].

Given that lysosomes function as a primary organelle responsible for the clearance of intracellular bacterial pathogens, they represent an important target for pathogen manipulation. Intracellular bacteria have evolved diverse mechanisms to manipulate host function to evade lysosomal degradation [2,10]. Lysosomal evasion can occur through either direct or indirect means. An example of an indirect mechanism of evasion is the manipulation of host trafficking to acquire membrane from other organelles. For example, the pathogen Legionella pneumophila uses a cooperative approach involving a series of secreted virulence proteins to acquire membrane from the endoplasmic reticulum [16]. This acquisition of membrane disguises the pathogen-containing vacuole as the host compartment, ultimately evading detection by the lysosomal pathway [17]. Many pathogens have evolved more direct means of targeting lysosomal function [10,18]. For example, the foodborne pathogen Vibrio parahaemolyticus uses the secreted virulence protein VepA to target the lysosomal V-ATPase and induce lysosome rupture [5].

Effective characterization of pathogen-mediated suppression of host lysosome function involves identifying the mechanism of host evasion and ultimately establishing the mechanism of action of the virulence factor responsible. This requires the development of robust cell biological assays for the study of these phenotypes. Among the most commonly proposed direct mechanisms of evasion is the suppression of trafficking of host lysosomes [10, 18]. Here, we describe a host–pathogen infection assay that can be used to investigate this phenotype [19]. This fluorescence-based microscopy assay involves the use of a BODIPY TR-X conjugate of BSA (DQ-Red BSA). This highly labelled BSA derivative exists as a self-quenched, non-fluorescent form (Figure 1A) and, upon treatment of host cells, is internalised by endocytosis. Upon trafficking to functional lysosomes, the acquisition of lysosomal proteases results in BSA cleavage, resulting in unquenching and bright-red fluorescence [20]. DQ-Red BSA is a substrate for a broad array of lysosome hydrolases and, therefore, is capable of detecting global lysosome function [21].

Figure 1. DQ-Red BSA assay for bacterial pathogen–mediated suppression of host trafficking. A. DQ-Red BSA dye mechanism. The fluorophores within the DQ-Red BSA dye exist in a quenched form and therefore do not generate intrinsic fluorescence. When host cells are treated with the dye, the compound is internalised by general endocytic mechanisms, and is trafficked to intermediates of the endocytic pathway. When dye-containing vesicles are trafficked to lysosomes, active lysosomal proteases cleave the BSA backbone, resulting in unquenching of the fluorophores and the generation of bright-red fluorescence. B. Host–pathogen infection assay to assess suppression of host lysosomal trafficking. Host cells are seeded in a microscopy chamber slide and infected with the bacterial pathogen of interest expressing green fluorescent protein (GFP). At the timepoint of interest, cells are subjected to a pulse-chase with DQ-Red BSA. Infected host cells are imaged by fluorescence microscopy, and suppression of host lysosome trafficking is quantified by imaging software.

The following summarises 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 (Figure 1B). S. enterica is a Gram-negative pathogen that is among the leading causes of foodborne gastroenteritis worldwide [22]. As an intracellular vacuolar pathogen, S. Typhimurium evades targeting for death by lysosomes to establish a replicative niche within host cells [10]. S. Typhimurium was shown to suppress host trafficking to lysosomes (Figure 2) using this fluorescence microscopy–based imaging assay [19], which allows the visualization of trafficking to lysosomes in individual live-infected host cells alongside neighbouring non-infected host cell counterparts. This method was also used to identify the secreted virulence factor responsible for the phenotype, as well as domains within the bacterial protein that contribute to the phenotype [19]. The methods outlined highlight important considerations for adapting this method for the study of other host cell types and other bacterial pathogens, as well as for modifying the assay setup to tailor to the experimental question of interest.

Figure 2. Salmonella virulence factor–mediated suppression of host trafficking to lysosomes. Salmonella Typhimurium manipulates the host cytoskeleton to direct its uptake into host epithelial cells using early-secreted virulence proteins. Upon uptake, Salmonella-containing vacuoles (SCVs) begin to undergo interactions with early intermediates of the endocytic pathway. As a mechanism of evasion, Salmonella produces late-secreted virulence proteins to suppress trafficking to lysosomes and promote intracellular survival and replication.