Abstract
Edwardsiella piscicida (E. piscicida) is an intracellular pathogen within a broad spectrum of hosts. Essential to E. piscicida virulence is its ability to survive and replicate inside host cells, yet the underlying mechanisms and the nature of the replicative compartment remain unclear. Here, we characterized its intracellular lifestyle in non-phagocytic cells and showed that intracellular replication of E. piscicida in non-phagocytic cells is dependent on its type III secretion system. Following internalization, E. piscicida is contained in vacuoles that transiently mature into early endosomes, but subsequently bypasses the classical endosome pathway and fusion with lysosomes which depends on its T3SS. Following a rapid escape from the degradative pathway, E. piscicida was found to create a specialized replication-permissive niche characterized by endoplasmic reticulum (ER) markers. We also found that a T3SS effector EseJ is responsible for intracellular replication of E. piscicida by preventing endosome/lysosome fusion. Furthermore, in vivo experiments confirmed that EseJ is necessary for bacterial colonization of E. piscicida in both mice and zebrafish. Thus, this work elucidates the strategies used by E. piscicida to survive and proliferate within host non-phagocytic cells.
Author summary E. piscicida is a facultative intracellular bacterium associated with septicemia and fatal infections in many animals, including fish and humans. However, little is known about its intracellular life, which is important for successful invasion of the host. The present study is the first comprehensive characterization of E. piscicida’s intracellular life-style in host cells. Upon internalization, E. piscicida is transiently contained in Rab5-positive vacuoles, but the pathogen prevents further endosome maturation and fusion with lysosomes by utilizing an T3SS effector EseJ. In addition, the bacterium creates an specialized replication niche for rapid growth via an interaction with the ER. Our study provides new insights into the strategies used by E. piscicida to successfully establishes an intracellular lifestyle that contributes to its survival and dissemination during infection.
Introduction
Intracellular pathogens often invade host cells as a means of escaping extracellular immune defenses and creating a safe niche for replication. However, internalized pathogens are not entirely protected, as they are normally routed to lysosomes for degradation. Invasive pathogens must devise strategies to avoid this. Typically, intracellular pathogens either (i) reside within a customized, membrane-bound compartment, which limits trafficking along the endosomal pathway, as observed for Legionella [1], Brucella subspp [2] and Salmonella [3], or (ii) rupture and escape their vacuole to reside and replicate in the host cytosol, as in the case for Shigella, Listeria, and Rickettsia subspp [4].
Many pathogenic bacteria are found to proliferate in a membrane-bound compartment. These bacteria adopt different strategies to survive after phagocytosis. Some bacteria, such as Salmonella [5] and Coxiella burnetiid [6], survive and proliferate in an acidic compartment. Other pathogens avoid lysosomal fusion by blocking phagosome maturation, such as Mycobacterium tuberculosis [7], or by hijacking the eukaryotic secretory pathway, such as Legionella pneumophila [1]. E. piscicida was reported to reside within membrane-bound vacuoles (ECVs) after infection of both phagocytic and non-phagocytic cells [8,9]. However, the mechanism by which the bacterium evade lysosomal degradation remains unclear.
Host cell manipulation by pathogenic bacteria is largely mediated through the delivery of an arsenal of virulence proteins called effectors to the host cell cytosol [10,11]. Legionella pneumophila produce multiple effector proteins which specifically target host proteins such as Arf1, Rab1 and Sec22b to ultimately create a replicative organelle [12]. Salmonella typhi serovar Typhimurium is known to regulate Salmonella-containing vacuole (SCV) trafficking via the action of SPI-2 T3SS-delivered effectors [3]. For example, SifA targets the host GTPase Rab9 to inhibit the process of Rab9-dependent M6PR recycling [13] and SopD2 targets the host GTPase Rab7 to perturb endocytic trafficking [14]. Previous studies have shown that T3SS and T6SS mechanisms are essential for the virulence of E. piscicida [15]. An increasing number of T3SS and T6SS effectors have been identified, including EseG [16], EseJ [17], EseH [18], EseK [19] and EvpP [20]. EseG was reported to localize to the ECV membrane, but its function remains undefined [21]. EseJ was reported to be involved in the adhesion stage during infection [17]. However, the virulence factors involved in the regulation of replication of E. piscicida in host cells remain unknown.
Given previous findings supporting the ability of E. piscicida to invade, survive, and replicate within non-phagocytic cells [8], the goal of the present work was to uncover the strategies and molecular mechanisms used by this pathogen to circumvent lysosomal routing and establish a replicative niche within the host. We have identified an effector EseJ that is required for intracellular replication in a specialized vacuole that is important for E. piscicida replication inside host cells. We found that EseJ acts by inhibiting lysosome degradation of the pathogen which we find is important for systemic infection in vivo. Through these strategies, E. piscicida successfully establishes an intracellular lifestyle that could contribute to its survival and dissemination during infection.
Results
Intracellular replication of E. piscicida in non-phagocytic cells depends on its T3SS but not T6SS
E. piscicida prefers an intracellular lifestyle upon infection in either epithelial [8] or phagocytic cells [23]. However, the virulence factors involved in such an intracellular process remain undefined. Considering that T3SS and T6SS are the most important virulence factors for E. piscicida, we first monitored the survival and replication of both the wild-type EIB202 and the isogenic T3SS or T6SS mutant strains in three different non-phagocytic cells, HeLa, Caco-2 and ZF4. Both EIB202 and ΔT6SS infection of nonphagocytic cells, followed by gentamicin-induced death of extracellular bacteria, revealed a progressive increase in intracellular bacterial numbers over time (Fig 1A). In contrast, no replication was observed in the ΔT3SS mutant, indicating that a functional T3SS, but not a T6SS, is required for intracellular survival of E. piscicida in non-phagocytic cells. In order to visualize bacterial invasion and intracellular replication, HeLa cells were infected with green fluorescent protein (GFP)-tagged E. piscicida strains, confocal microscopy images were acquired and the number of intracellular bacteria in EIB202-infected cells were scored over time. About 15% of HeLa cells containing hyper-replicating bacteria were observed after EIB202 and ΔT6SS infection for 8 h, but not with the ΔT3SS mutant strain (Fig 1B and 1C). Collectively, these results suggest that E. piscicida can survive and replicate in non-phagocytic cells via a mechanism mediated by the T3SS, but not T6SS.
E. piscicida prevents endosome maturation and lysosome fusion
Once inside host cells, invasive bacteria either replicate within the endosome or escape the vacuole and replicate in the cytoplasm. Consistent with our previous study [21], E. piscicida located within vacuolar compartment (s) after infection which we named the E. piscicida-contained vacuoles (ECVs) (S1A Fig). Galectin-3 is a β-galactoside binding protein that is specifically recruited to disrupted pathogen-containing vacuoles [24]. To further investigate if E. piscicida remains inside the vacuoles during its whole intracellular lifecycle, the presence of galectin-3 around the ECV was assessed using fluorescence microscopy. Less than 10% of vacuoles harboring WT E. piscicida co-localized with galectin-3 in HeLa cells over time (S1B Fig). These data suggest that E. piscicida EIB202 resides and replicates inside pathogen-containing vacuoles throughout the course of infection.
Next, we wished to understand the strategies used by this pathogen to establish and maintain its intracellular life cycle. Following internalization, foreign particles and many bacteria are usually found within membrane-bound compartments that sequentially develop into early and late endosomes for ultimate fusion with lysosomes, where the particles are degraded. We monitored the acquisition of endosomal markers and lysosomal fusion in E. piscicida-containing vesicles over time using confocal microscopy. Early after invasion (1 h after infection), over 70% of both intracellular wild-type and ΔT3SS mutant were found enclosed within vacuoles that co-localized with the early endosomal protein Rab5 (Fig 2A and 2B), indicating interactions with early endosomes. These interactions were transient, as Rab5 colocalization rapidly decreased to 5% and 3% by 4 h post-gentamicin incubation after infection with both wild-type EIB202 and the ΔT3SS mutant strains (Fig 2A and B). As Rab5 colocalization was progressively lost, an increasing number of ΔT3SS mutant bacteria colocalized with the late endosomal markers Rab7 and lysosome-associated membrane protein 1 (Lamp-1) over time, which is consistent with vacuolar maturation (Fig 2B-E). In contrast, the majority of the vacuoles containing wild-type EIB202 were negative for both Rab7 and Lamp1 (Fig 2B-E). These results suggest wild-type E. piscicida transiently interacts with early endosomes, but avoids endosome maturation during infection.
Considering that luminal acidification is another critical characteristic of endosome maturation, we used the fixable acid tropic probe LysoTracker to monitor acidic organelles in infected cells. A major overlap was found between the dye and ΔT3SS mutant strain, but not wild-type EIB202 as early as 2 h (Fig 3A and B). Thus, these results suggest the ECVs formed by the wild-type bacterium avoid vacuolar acidification and maturation by perturbing the fusion with lysosomes. To further understand this process, we assessed ECV co-localization with TR-dextran. Prior to bacterial infection, cells were pulsed with TR-dextran for 6 h followed by overnight chase in dye-free medium to ensure that the probe is delivered from early and recycling endosomes to lysosomes [25]. Confocal immunofluorescence and quantification data showed that the majority of wild-type EIB202-containing vacuoles did not co-localize with TR-dextran (Fig 3C and D). In contrast, when cells were infected with the ΔT3SS strain, more than 70% of the ECVs did co-localize with TR-dextran at 8 h post-infection (Fig 3C and D). Taken together, these results indicate that E. piscicida utilize its T3SS to successfully evade lysosomal fusion and ultimately replicate in nonacidic compartments lacking lysosomal or late endosomal characteristics.
ECVs acquire ER markers during maturation into a replicative organelle
Our results iundicate that wild-type E. piscicida circumvented the classical endocytic pathway to establish a specialized replication-permissive niche. This raises the possibility that the bacterium may interact with other intracellular compartments. The endoplasmic reticulum (ER) was reported to be recruited and hijacked by many intracellular pathogens to create their replication niche [1,26]. We first used a specific ER-tracker dye to assess the intracellular ER structures and analyzed the presence of ER marker in bacterium-containing vacuoles. Surprisingly, ER-tracker labeling was present in the EIB202 replication compartment located at the perinuclear regions of HeLa cells, but this was excluded in ΔT3SS-containing vacuoles (Fig 4A and B). The same result was observed when we studied the distribution of the ER membrane-bound lectin calnexin in EIB202 or ΔT3SS-infected cells (Fig 4C and D). Together, these data suggest that E. piscicida replicates inside ER-enriched vacuoles.
A T3SS effector EseJ is responsible for E. piscicida’s intracellular replication
The finding that the T3SS plays a critical role in the inhibition of endosome maturation and E. piscicida lysosome degradation raised the question of what T3SS effectors are involved in this process. To date, several E. piscicida T3SS effectors including EseG [16], EseJ [17], EseH [18] and EseK [19] have been identified. To assess the role of individual effectors, we tested the ability of WT and isogenic E. piscicida effector mutants to replicate inside non-phagocytic host cells. We found that only the eseJ mutant showed a marked deficiency in intracellular replication compared to wild-type bacteria as assessed by CFU intracellular counts (Fig. 5A). To determine whether the impaired ability of the eseJ mutant to replicate intracellularly was attributed to effects on lysosome fusion and degradation, we investigated the characteristics of the ΔeseJ-containing compartments over time. Coincident with the intracellular fate of ΔT3SS, ΔeseJ-containing vacuoles progressively co-localized with Rab7 (S2A Fig) and Lamp-1(Fig 5B and S2B Fig). Moreover, the mature Lamp-1-positive ΔeseJ-containing vacuoles were found to be fused with lysosomes as assessed using the acidification probe LysoTracker Red DND-99 and pre-loaded Dextran (Fig 5C and 5D). These findings suggest that the effector EseJ is critical for E. piscicida’s replication inside cells by disrupting vacuolar trafficking to the lysosome during infection.
To further assess the function of EseJ, we analyzed the effect of EseJ expression on the transport and the degradation of exogenously added DQ-Red bovine serum albumin (BSA), which emits red fluorescence upon proteolytic degradation and is used as a sensitive indicator of lysosomal activity [14]. Bright punctate signal intensity of DQ-Red BSA were significantly attenuated in cells stably expressing EseJ compared with that observed in cells expressing vector alone (Fig 6A and 6B), indicating that EseJ expression suppresses lysosome function. Consistently, cells infected with wild-type, but not ΔT3SS or ΔeseJ E. piscicida mutants displayed a remarkable decrease in DQ-Red BSA fluorescence intensity (S2C Fig). Next, we investigated the delivery of endosomal cargo to lysosomes by pre-loading cells with dextran 488 prior to transfection and then treated the cells with rhodamine dextran. In line with the results shown above, the dextran derivatives co-localized with a Mander’s coefficient of more than 0.5 in control cells, suggesting significant endosome-lysosome fusion, whereas EseJ-HA expression resulted in significantly less co-localization (Fig 6 C and 6D). Collectively, these results demonstrate that T3SS effector EseJ is both necessary and sufficient to block endocytic trafficking to lysosomes and consequently critical for E. piscicida’s intracellular replication.
Role of EseJ in E. piscicida’s infection in vivo
E. piscicida T3SS was reported to act as a critical virulence factor in disease pathogenesis in both mouse and fish infection models [23, 27]. To assess the role of the T3SS effector EseJ in animal infection, C57BL/6 wild-type mice were orally infected with wild-type and isogenic ΔeseJ E. piscicida strains. Compared to wild-type EIB202, the ΔeseJ strain showed reduced bacterial burdens in the cecum and intestinal lumen as well as systemic sites including the liver, spleen and kidneys (Fig 7A). Likewise, reduced colonization of the ΔeseJ mutant was observed in zebrafish larvae after infection when compared to the wild-type bacterium (Fig 7B). Notably, zebrafish infected with wild-type E. piscicida showed marked mortality, with ~75% of the animals succumbing by day 3–4 post-infection whereas only ~50% of fish succumbed to infection with the eseJ mutant (Fig. 7C). Collectively, these data suggest that the effector EseJ contributes to E. piscicida colonization and virulence in vivo.
Discussion
E. piscicida was recently shown to invade and proliferate within many non-phagocytic cells [8], but the mechanism by which the bacterium its own survival inside host cells remained unclear. Here we report a comprehensive description of how E. piscicida turns the intracellular environment into a hospitable niche that allows for efficient bacterial replication. Subversion of the phagocytic pathway by intracellular bacteria is a general mechanism to establish an appropriate replication niche. Pathogens are known to adopt diverse strategies to disrupt the maturation process at different stages and to prevent its delivery into a phagolysosome. For example, Mycobacterium remains within an early endosomal compartment [7] that excludes the vacuolar ATPase, thus inhibiting the acidification of the bacterial phagosome. The maturation of the SCV is arrested at a late endosome-like stage, selectively excluding proteins such as mannose 6-phosphate receptors (MPR) and lysosomal cathepsin proteins [13]. In the present study, we tracked the acquisition of endosomal markers and lysosomal fusion in E. piscicida-containing vesicles over time using confocal microscopy and demonstrated that E. piscicida bypassed the classical endosome pathway after transiently interactions with early endosomes. Our studies indicate that using this strategy, E. piscicida disrupts endosomal maturation and evades lysosome degradation.
Our study characterized an important contribution of the T3SS effector EseJ in regulating endocytic trafficking of E. piscicida within host cells. Intracellular expression of EseJ was found necessary and sufficient to block endocytic progression to lysosomes (Fig 5 and Fig 6). Notably, the eseJ mutant was greatly impaired in intracellular replication when compared to the wild-type bacterium, indicating that EseJ is an important factor for intracellular survival and replication of E. piscicida. However, it remais unclear how EseJ evades fusion with lysosomes to evade degradation. One possibility is that EseJ interacts with host small guanine nucleotide binding proteins (GTPases), phospholipids or other host proteins that are enriched and central for endocytic trafficking. The strategy of interacting with endosome-bounded proteins is an efficient tactic used by other pathogens to combat the host’s bactericidal defenses. For example, Mycobacterium tuberculosis (Mtb) secretes SapM, a phosphatase that removes PI(3)P from Mtb-containing vacuoles by converting it to PI, thereby arresting endosomal maturation [28]. Legionella pneumophila secreted VipD to interact with early endosomal protein Rab5 to protect from endosomal fusion [29]. Another question is whether EseJ act in concert with other virulence factors involved in the regulation of ECV trafficking which needs to be investigated in future studies.
Orchestration with other intracellular compartments and routing into a specialized compartment favorable for replication is another important mechanism for the survival of bacterial pathogens inside host cells. For example, biogenesis of Legionella-replicative compartments depends upon a rapid interception of COPI-dependent vesicular trafficking from endoplasmic ER exit sites [12]. Formation of SCVs is associated with the Golgi apparatus and induces endosomal tubulations that extend towards the cell periphery [30]. Interestingly, we observed obvious ER characteristic associated with ECVs. However, how E. piscicida recruits and interacts with ER remains to be elucidated.
Overall, our studies demonstrate a complex and deliberate intracellular life cycle of E. piscicida in non-phagocytic cells (see model in Fig. 8). The bacterium not not only invades the host cells, but also subverts trafficking of bacterium-containing vacuoles through the endosomal pathway and translocation to an specialized aggressively replication niche. Moreover, we showed that a T3SS effector EseJ is essential for the intracellular replication by disrupting endosomal maturation and lysosome fusion, which is critical for virulence of E. piscicida in vivo.
Methods
Ethics Statemen
The animal trials in this study were performed according to the Chinese Regulations of Laboratory Animals—The Guidelines for the Care of Laboratory Animals (Ministry of Science and Technology of People’s Republic of China) and Laboratory Animal-Requirements of Environment and Housing Facilities (GB 14925-2010, National Laboratory Animal Standardization Technical Committee). The license number associated with their research protocol was 20170912-08, which was approved by The Laboratory Animal Ethical Committee of East China University of Science and Technology. All surgery was performed under carbon dioxide anesthesia, and all efforts were made to minimize suffering.
Bacterial strains and cell culture
Wild type Edwardsiella piscicida EIB202, the T3SS mutant and the T6SS mutant were constructed and grown as described previously [22]. For constitutive expression of GFP or mCherry, E. piscicida strains were electroporated with pUTt0456GFP or pUTt0456mCherry, respectively. HeLa cells (ATCC number CCL-2), Caco-2 cells (ATCC number HTB-37) and ZF4 cells (ATCC number CRL-2050) were all from China Center for Type Culture Collection. HeLa cells and Caco-2 cells were cultured at 37°C under 5% CO2 atmosphere in Dulbecco’s minimal Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS), called growth medium (GM). ZF4 cells were cultured at 30°C under 5% CO2 atmosphere in GM.
Construction of mutant strains
In-frame deletion mutants of the effector genes including eseG, eseJ, eseH and eseK were generated by the sacB-based allelic exchange as previously described. The fragments upstream and downstream of each effector gene were fused by overlap PCR. These fragments were then cloned into the sacB suicide vector pDMK and linearized with BglII and SphI, and the resulting plasmids were transformed into Escherichia coli (E. coli) CC118 λpir. The correct plasmids were then transformed into E. coli SM10 λpir and then conjugated into EIB202. The trans-conjugants with the plasmids integrated into the chromosome by homologous recombination were selected on tryptic soy agar (TSA) medium containing kanamycin (Km, 50 mg/ml) or colistin (Col, 12.5 mg/ml). To complete the allelic exchange for in-frame deletions, double-crossover events were counter-selected on TSA plates containing 10% sucrose. All of the mutants were confirmed by PCR amplification of the respective DNA loci, and subsequent DNA sequencing of each PCR product.
Infection protocol
HeLa cells, Caco-2 cells or ZF4 cells were infected with E. piscicida strains at a multiplicity of infection (MOI) of 100. E. piscicida was grown overnight in tryptic soy broth (TSB) at 30°C with shaking, then diluted into fresh DMEM with standing at 30°C until OD600 reached 0.8. Harvested bacteria in phosphate-buffer saline (PBS) suspensions were added to cells according to MOI. To synchronize infection, plates were then centrifuged at 600 g for 10 min. At 1 h after incubation, cells were washed three times with PBS and then incubated with growth medium containing 100 μg/ml gentamicin for 1h to kill the extracellular bacteria, after which the gentamicin concentration was decreased to 10 μg/ml for the remainder of the experiment.
Gentamicin protection assay
For enumeration of viable intracellular bacteria, bacteria were added to triplicate wells of HeLa cell monolayers for infection as described above. At each indicated time point, extracellular bacteria were killed with gentamicin. Monolayers were washed with PBS, and cells were lysed by incubation with PBS containing 1% Triton X-100 for 30 min at room temperature. The lysate was serially diluted in PBS and plated onto TSB agar plates. Plates were incubated at 30°C overnight for subsequent CFU enumeration.
Labeling of subcellular compartments with dyes
For the acidification studies, 75 nM LysoTracker Red DND-99 (Invitrogen) was added to the samples 30 min prior to cell fixation. For labeling of lysosomes with Texas Red dextran, HeLa cells were treated with 100 μg/ml of Texas Red dextran (Invitrogen) for 12 h and chased overnight. For ER staining, cells were washed with HBSS and stained with 100 nM ER-tracker (green) for 30 min at indicated time point. For DQ Red BSA assay, after 8 h of infection or cells transfection, cells were incubated for 1 h in growth medium containing DQ Red BSA (0.25 mg/ml), washed with PBS, and incubated in growth medium for 4 h.
Dextran 488 Loading and Rhodamine Dextran Pulse-chase
HeLa cells were seeded on coverslips in 24-well tissue culture plates at 2×105 cells/well then incubated in presence of dextran Alexa Fluor® 488 (0.1 mg/ml) for 8 h. Cells were then washed twice with PBS, incubated with growth medium and transfected with vector or EseJ-HA overnight. The following day cells were incubated for 30 min in the presence of tetramethylrhodamine dextran (0.2 mg/ml), then washed twice with PBS, and the dye was chased for 2 h in regular growth medium.
Immunofiuorescence and confocal microscopy
HeLa cells were seeded onto 24-well plates containing sterile coverslips at a density of 2×105 cells/ml. Following infection with E. piscicida strains and gentamicin incubation for the indicated time, cells were washed with phosphate-buffered saline (PBS) and then fixed in 4% (v/v) paraformaldehyde for 10 min at room temperature. After washing with PBS, cells were blocked and permeabilized in PBS containing 10% (v/v) normal goat serum (NGS) and 1% (v/v) bovine serum albumin (BSA) and 0.1% (w/v) saponin (SS-PBS) for 10 min at room temperature. Primary antibody of LAMP-1(clone H4A3) and secondary antibodies were diluted in SS-PBS at appropriate dilutions and incubated serially for 1 h at room temperature. Between antibody incubations, coverslips were washed three times with PBS containing 0.05% (w/v) saponin for 5 min each time. Nuclei and actin cytoskeleton were stained with Hoechst (Sigma) and rhodamine-phalloidin (Molecular Probes), respectively. Fixed samples were viewed on a Nikon A1R confocal microscope. Images were analyzed using ImageJ (NIH).
Mice infection
C57BL/6J wild-type from the Jackson Lab (6–8 weeks old) were bred under specific pathogen-free conditions. For oral infections, water and food were withdrawn 4 h before per os (p.o.) treatment with 20 mg/100 μL streptomycin per mouse. Afterward, animals were supplied with water and food ad libitum. At 20 h after streptomycin treatment, water and food were withdrawn again for 4 h before the mice were orally infected with 2.5 × 107 CFU/g of EIB202 or ΔeseJ suspension in 200 μL PBS, or treated with sterile PBS (control). Thereafter, drinking water ad libitum was offered immediately and food 2 h post-infection. At the indicated time points, mice were sacrificed and the tissue samples from the intestinal tracts, kidneys, spleens, and livers were removed for analyses.
Zebrafish infection
Three-month old adult zebrafish (about 0.4 g) were randomly divided into groups (n=35) and infected via intramuscular injection with bacterial sample (50 cfu/fish) or PBS as a control. Fish mortality was recorded in each infection group over a period of 4 days. For immersion infection of zebrafish larvae, larvae at 5 days post-fertilization were randomly divided and immersed in PBS or PBS containing 105 cfu/ml E. piscicida wild-type or ΔeseJ for 2 h. Subsequently, they were transferred to 10-cm dishes, with approximately 50 larvae in 15 ml of E3 medium per dish, and incubated at 28 °C. The bacterial colonization of every 5 fishes were then analyzed at different time points. All animal experiments were approved by the Institutional Animal Care and Use Committee of East China University of Science and Technology.
Statistical analysis
All experiments were performed three times (as indicated in the figure legends). Statistical analyses were performed by using the student’s t-test in the SPSS software (Version 11.5, SPSS Inc.). In all cases, the significance level was defined as * p ≤ 0.05,** p ≤ 0.01 and *** p ≤ 0.001.