Burkholderia pseudomallei BopE suppresses the Rab32-dependent defense pathway to promote its intracellular replication and virulence

ABSTRACT

Melioidosis is a serious infectious disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Recently, Rab32-dependent immune vesicles emerge as a critical defense pathway to restrict the intracellular B. pseudomallei. However, B. pseudomallei can evade host immune vesicles and survive in the cytoplasm, although this mechanism is not well understood. In this study, we found Rab32-dependent vesicles could effectively combat B. pseudomallei infection, but not all intracellular B. pseudomallei were encapsulated in Rab32-positive vesicles. To explore how B. pseudomallei counteracted the Rab32-dependent defense pathway, transcriptomic profiling of B. pseudomallei was performed to characterize the response dynamics during infection. We found that the type III secretion system of B. pseudomallei was activated, and a variety of effector proteins were highly upregulated. Among them, BopE, BprD, and BipC were shown to interact with Rab32. Interestingly, BopE directly interacts with host Rab32, potentially suppressing Rab32 function by interfering with nucleotide exchange, which in turn restricts the recruitment of Rab32 to bacterial-containing vesicles. Knocking out of BopE can increase the proportion of Rab32-positive vesicles, suppressing the intracellular replication and virulence of B. pseudomallei. Collectively, our findings have demonstrated that BopE may be an important effector for B. pseudomallei to evade from the Rab32-dependent killing vesicles into the cytosol for survival and replication. Therefore, a deeper understanding of the interaction between BopE and the host Rab32-dependent restriction pathway may provide an effective therapeutic strategy for the elimination of intracellular B. pseudomallei.

IMPORTANCE

B. pseudomallei is facultative intracellular bacterium that has evolved numerous strategies to evade host immune vesicles and survive in the cytoplasm. Rab32-dependent vesicles are one of these immune vesicles, but the mechanism by which B. pseudomallei escape Rab32-dependent vesicles remains elusive. Here, we find B. pseudomallei infection leading the activation of the type III secretion system (T3SS-3) and increasing the expression of various effectors. Specifically, we identify that BopE, an effector secreted by T3SS-3, triggers vesicle escape to promote B. pseudomallei pathogenicity and survival. Mechanistically, BopE suppresses the activation of Rab32 by interfering with nucleotide exchange, ultimately triggering vesicle escape and intracellular survival. We also find knocking out the bopE gene can increase the proportion of Rab32-positive vesicles that trap B. pseudomallei, dampening the survival of B. pseudomallei both in vitro and in vivo. Taken together, our findings provide insights into the molecular mechanisms of pathogen effector-induced vesicle escape, indicating a potential melioidosis treatment via blocking B. pseudomallei BopE-host Rab32 interaction.

INTRODUCTION

Melioidosis, a tropical illness, which can be acquired from the environment, is caused by Burkholderia pseudomallei infection. Currently, community-acquired clinical cases of melioidosis mainly result from injurious inoculation, ingestion, and inhalation of aerosolized bacteria (1, 2). It is estimated that there were currently 165,000 annual cases of melioidosis and 89,000 deaths worldwide (3). The clinical symptoms of melioidosis are diverse, including asymptomatic infection, chronic localized pathology, acute sepsis, or a latent infection that can reactivate decades later (4). Owing to the serious biological hazard, B. pseudomallei, the causative agent, has been classified as a tier-1 select agent by the Center for Disease Control (CDC) of the United States (2). Melioidosis is highly endemic in Southeast Asia and northern Australia, and the endemic regions are believed to be expanding globally, such as in southeast Queensland, Australia, and regions of the southern United States, with a variety of factors including climate change, environmental disruption, and increased human activity (5). Although melioidosis is of growing concern, the therapeutic resources are extremely limited (6). Elucidating the pathogenic mechanism of B. pseudomallei infection is essential for clinical diagnosis, treatment, and prevention of melioidosis.

B. pseudomallei can invade a broad range of host cells, particularly through a process where it is phagocytosed into phagosomes by macrophages. And following internalization, B. pseudomallei is encapsulated in macrophage endocytic vesicles. The maturation of phagocytic vesicle transport is crucial for the clearance of pathogens within macrophages (7). It has established the role of Rab32 in the regulation of phagosome maturation and intracellular survival of various pathogens, such as Salmonella typhi (8) and L. monocytogenes (9). Functionally, Rab32 participates in the regulation of the cyclic transition of vesicles by facilitating the cyclic transition from an inactive state (bound to GDP) to an active state (bound to GTP). The activation and recruitment of Rab32 to bacterial-containing vesicles lead to the degradation of engulfed bacteria (9, 10). Rab32 is also able to promote the transport of the antibacterial agent itaconate, to vesicles resulting in engulfed bacterial elimination (11–13). Notably, our previous research has demonstrated that Rab32 is recruited to B. pseudomallei-containing phagosome vesicle (BCV) and promotes the fusion of BCV with lysosomes and activates lysosomal acid hydrolase, thus limiting the intracellular growth of B. pseudomallei in murine macrophages (14). However, growing evidence suggests B. pseudomallei has evolved numerous strategies to evade BCV, by escaping in the cytoplasm (15–17). The mechanism by which B. pseudomallei escapes from Rab32-dependent vesicles remains to be elucidated.

The type III secretion system (T3SS) is a highly recognized mechanism employed by pathogens to secrete a variety of effector proteins into the host cell, which enables bacteria to escape from endocytic vesicles, promoting their intracellular survival and pathogenesis (18). B. pseudomallei encodes for three distinct T3SSs (T3SS-1, BPSS1390-1408; T3SS-2, BPSS1613-1629; and T3SS-3, BPSS1520-1554). Among these, T3SS-3 is particularly well characterized for its role in the intracellular virulence and manipulation of host cell processes (19–21). Inactivating T3SS-3 in B. pseudomallei effectively prevents the pathogen from escaping the BCVs, which can be achieved either by altering the apparatus proteins of T3SS-3, such as BsaZ, BsaQ, and BipD (20, 22), or by employing small molecule inhibitors that target the ATPase activity of BsaS, a component of T3SS-3 (23). Notably, it has been reported that Salmonella typhi delivers two T3SS effectors, SopD2 and GtgE, to disrupt host immune response by specifically interacting with Rab32 and deactivating Rab32 (8, 24). Therefore, T3SS-3 in B. pseudomallei may be the significant strategy that interferes with Rab32 function to facilitate B. pseudomallei escape from the Rab32-positive vesicles.

In our study, we found that a proportion of B. pseudomallei was able to escape from Rab32-positive vesicles and replicate within the cytoplasm. Transcriptomic analysis found that the T3SS-3 of B. pseudomallei was activated and a broad range of its effector proteins were highly upregulated during intracellular infection. Interestingly, the effector proteins BipC, BopE, and BprD were shown to interact with Rab32. In particular, BopE can prevent Rab32 recruitment to BCVs, facilitating B. pseudomallei escape into the cytoplasm for replicating, which may be mediated by interfering with Rab32 at the level of nucleotide exchange. Furthermore, we found that BopE-deficient strains not only showed a significant reduction in the number of Rab32-positive vesicles in RAW264.7 cells but also exhibited a decrease in virulence in mice, suggesting that BopE plays a crucial role in modulating B. pseudomallei-host interactions. These findings may shed a light on the pathogenic mechanism by which B. pseudomallei manipulates Rab32-dependent defense pathways for its intracellular replication.

RESULTS

B. pseudomallei is able to escape Rab32-positive vesicles and replicate intracellularly

Our previous research has established that the Rab32-dependent membrane vesicle traffic pathway in macrophages could be a host strategy for the restriction of phagocytosed B. pseudomallei (14). This hypothesis was further substantiated by our observation of a pronounced elevation in Rab32 expression levels following B. pseudomallei infection (Fig. S1). To investigate the effect of Rab32 on the elimination of B. pseudomallei, we overexpressed Rab32 with pcDNA4-Rab32 or knocked down Rab32 with siRNA in RAW264.7 cells (Fig. 1A and B). Subsequently, these cells were infected with B. pseudomallei at an MOI of 10. It was observed that the intracellular B. pseudomallei were efficiently co-localized with Rab32. Interestingly, we found that the proportion of Rab32-positive vesicles in negative control (NC) cells was 62.26% ± 2.92%, which significantly decreased to 15.4% ± 2.65% (t = 20.52, P < 0.0001) after knocking down Rab32 in RAW264.7 cells. Conversely, when Rab32 was overexpressed, the proportion was significantly increased to 83.26% ± 4.80% (t = 6.47, P = 0.0029), as seen in Fig. 1C. However, not all intracellular bacteria were contained in Rab32-positive vesicles. Visibly, Rab32-positive vesicle-free B. pseudomallei were ~37.74% in the NC group, while the percentage was decreased to ~16.74% when overexpressing Rab32 in RAW264.7 cells (Fig. 1D). This observation was also recorded by live cell video microscopy (Movie S1). These findings suggest that while Rab32-dependent defense vesicles have the ability to encapsulate B. pseudomallei, the bacteria can still manage to escape from these vesicular enclosures.

Fig 1.

B. pseudomallei is able to escape Rab32 vesicles and replicate intracellularly. (A) Western blot analysis of Rab32 in RAW264.7 cells. Rab32 was detected by its antibody; cells without treatment were used as the NC. (B) The relative expression levels of Rab32 were determined by quantifying band intensities presented in panel A. The ratio of the intensities of all Rab32 bands to that of β-actin has been calculated and adjusted for normalization against the group of NC. (C) Quantification of Rab32 co-localized with B. pseudomallei. Fifty infected RAW264.7 cells in 10 fields of view were counted in each group after 2 hours of infection from three independent experiments. All assessments were performed in a blinded manner. *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant (one-way ANOVA). (D) Representative immunofluorescence images of co-localization of Rab32 and B. pseudomallei in RAW264.7 cells after 2 hours of infection by confocal microscopy. RAW264.7 cells were infected with B. pseudomallei (MOI = 10:1) and imaged at the indicated time points of 2 hpi. Rab32 was stained with an anti-Rab32 antibody (green); B. pseudomallei was stained with anti-B. pseudomallei antibody (red). Scale bar, 5 µm. (E and F) Intracellular growth of B. pseudomallei was detected by CFU assay. Plate count of B. pseudomallei at 8 hpi (E). Intracellular B. pseudomallei burdens in RAW264.7 cells at the indicated times after infection (F). NC is negative control group, siRab32 is Rab32-siRNA group, and pRab32 is Rab32 overexpression group. The graphed data represent mean ± SD of three biological replicates. *P < 0.05, **P < 0.01, and ***P < 0.001; ns, not significant (one-way ANOVA).

To further estimate the intracellular survival of B. pseudomallei, bacterial survival assays were conducted at different infection periods. We found the expression level of Rab32 did not affect the bacterial load of B. pseudomallei at the early stage, as there was no statistically significant difference in the amount of intracellular B. pseudomallei at 2 hours post-infection. But after 8 hours of infection, the intracellular survival of B. pseudomallei was significantly increased to 7.34 ± 0.13 log₁₀CFU/mL in Rab32 knockdown cells, compared with 6.55 ± 0.11 log₁₀CFU/mL in the NC group (P = 0.0004), while it significantly decreased to 6.14 ± 0.09 log₁₀CFU/mL when Rab32 overexpressed (P = 0.013), and siRab32 group rose more than 10 times relative to the pRab32 group (P < 0.0001), shown in Fig. 1E and F. These indicate that Rab32 overexpression can efficiently suppress B. pseudomallei intracellular replication. Collectively, our findings indicate that Rab32 can be immediately recruited to the BCV membrane, thereby limiting B. pseudomallei intracellular replication. In response, B. pseudomallei is presumed to have evolved some specific strategies to avoid containment within Rab32-positive vesicles for intracellular replication.

The T3SS-3 of B. pseudomallei is activated during infection

In order to investigate the strategies employed by B. pseudomallei to evade Rab32-positive vesicles, we performed comparative transcriptomic analyses between B. pseudomallei cultured in vitro (in vitro B. ps) and during intracellular infection (intracellular B. ps) to obtain a global view of virulence factors involved in B. pseudomallei infection. Applying a cut-off level of twofold change and a P value < 0.05 on the mRNA level, a total of 1,137 differentially expressed genes (DEGs) were detected (Table S3). In these genes, 771 DEGs (67.81%) of B. pseudomallei were upregulated and 366 genes (32.19%) were downregulated (Fig. S2). Pathway enrichment analysis demonstrates that protein export and the bacterial secretion system are enriched during B. pseudomallei infection (Fig. 2A and B). Interestingly, we found that 97.43% of DEGs (38/39) enriched in the type III secretion system (T3SS) was upregulated, which included T3SS-3 regulators (e.g., BspR, BprP, and BsaN), chaperones (e.g., BicA, BicP, and BapB), and apparatus components (e.g., BsaP, BsaZ, and BsaQ). Of particular interest were eight T3SS-3 effectors, including BipC, BipD, BopC, BprD, BopE, BapC, BopA, and BapA, which were emphasized with red highlighting in Fig. 2C. Additionally, a two-component system, such as VirAG (25), and ribosome and flagellar assembly, such as FliG (26), have also shown significant enrichment. Our transcriptomic data suggest that B. pseudomallei T3SS-3 is activated during the infection process.

Fig 2.

Transcriptomic analyses reveals type III secretion system was activated during B. pseudomallei infection. (A) Enriched terms of the DEGs visualized in cirFunMap from the KOBAS tool. Each node represents an enriched term, and the node color represents different clusters for enriched pathways; the node size represents six levels of enriched P value, node sizes from small to large: 0.05 ≤ P value ≤ 1, 0.01 ≤ P value < 0.05, 0.00 ≤ P value < 0.01, 0.0001 ≤ P value < 0.001, 1e⁻¹⁰ ≤ P value < 0.0001, 0 ≤ P value < 1e⁻¹⁰. (B) Enriched terms of DEGs visualized in cirFunMap. Each row represents an enriched pathway, and the length of the bar represents the enrich ratio, which is calculated as “mapped gene number”/“background gene number.” The color of the bar is the same as the color in the circular network above (A), which represents different clusters. For each cluster, if there are more than five terms, top five with the highest enrich ratio were displayed. (C) The heatmap of the DEGs enriched in T3SS-3 with a cutoff threshold of 2.0-fold difference between in vitro B. ps and intracellular B. ps. T3SS-3 effector proteins were marked as red.

T3SS-activated B. pseudomallei highly expressed effector proteins during infection

The T3SS is essential for the intracellular survival strategies of B. pseudomallei, with its effectors commonly deployed to subvert host cell immune responses (27). BsaN, together with the chaperone BicA, acts as a central regulator that plays an essential role in activating the functionality of T3SS-3 in B. pseudomallei (28, 29). To ascertain the role of the T3SS-3 during B. pseudomallei infection, the transcriptional levels of BsaN and BicA were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and the results demonstrated a marked increase in the expression levels of these two genes (Fig. 3A). This upregulation is consistent with the RNA sequencing data and points to a probable active role of the T3SS-3 in facilitating the infection process. We further detected the expression dynamics of T3SS-3 effectors among the DEGs and found a significant upregulation of eight effectors during the infection (Fig. 3B). These results suggest that T3SS-3 is activated during B. pseudomallei infection, and the eight upregulated effectors may be crucial for B. pseudomallei to persist within host cells.

Fig 3.

B. pseudomallei highly expresses T3SS-3 effector proteins during infection. (A) Quantification of the expression levels of BicA and BsaN in intracellular B. pseudomallei by RT-qPCR analysis. B. pseudomallei that infected RAW264.7 cells for 4 hpi serve as the intracellular B. ps group, while B. pseudomallei cultured in vitro (in vitro B. ps) is used as control group. (B) The mRNA levels of B. pseudomallei T3SS-3 effectors were analyzed by RT-qPCR. rpoB of B. pseudomallei was used as a reference gene. All data are presented as mean ± SD, and from at least three independent experiments, ***P < 0.001 (Student’s t-test).

B. pseudomallei T3SS effector protein BopE interacts with Rab32

Heterologous expression of prokaryotic proteins within eukaryotic cellular systems is a widely utilized approach across the fields of cell biology and microbiological research (30, 31). To identify potential effectors that interact with Rab32, we developed an array of expression plasmids with a Flag tag by cloning eight highly expressed B. pseudomallei effector genes into the pcDNA4-Basic vector. These constructs were confirmed to be correct by sequencing and enzymatic digestion, followed by expression verification (Fig. S3). We subsequently executed co-immunoprecipitation assays utilizing HEK293 cells, which were co-transfected with effector-expressing plasmids and pEGFP-Rab32, a vector encoding Rab32 tagged with GFP. SopD2, identified as a GTPase-activating protein (GAP) for Rab32 in Salmonella Typhimurium (24), served as a positive control in our experiments. Notably, we found that BprD, BipC, and BopE were capable of interacting with Rab32, as demonstrated in Fig. 4A.

Fig 4.

B. pseudomallei T3SS effector protein BopE directly interacted with Rab32. (A) Co-immunoprecipitation assays of Rab32 and B. pseudomallei T3SS effector proteins. The molecular mass of FLAG-tagged T3SS effector proteins and that of GFP-tagged Rab32 are denoted within corresponding parentheses. “Input” refers to the total protein lysate, while “IP:Flag” indicates the immunoprecipitated protein using an anti-Flag antibody. “IB:GFP/Flag/Tubulin” denotes the immunoblotting with antibodies against GFP (to detect pEGFP-Rab32), Flag (to detect the T3SS effector proteins), and tubulin (as a loading control). (B) The interaction assay between B. pseudomallei T3SS effector proteins and Rab32 using yeast two-hybrid system. The transformants were plated onto low-stringency selection plates (left) to verify the successful integration of the plasmids and high-stringency selection plates (right) to detect any potential interactions between the T3SS effectors and Rab32. (C) Representative immunofluorescence images of co-localization of Rab32 and BopE in HEK293T cells. GFP-BopE and Flag-Rab32 were co-expressed in HEK293T cells. Rab32 was stained with anti-Flag (red), BopE, or SopD2 tagged with GFP (green). Images show maximum-intensity projections of confocal Z-stacks. The empty vector of pEGFP-C1 was used as a negative control, and SopD2 was used as a positive control. Scale bars, 5 µm. (D) Confocal microscopy of BopE and Rab32 in panel C with fluorescence intensity plotted along the arrows.

To further substantiate the interactions with Rab32, we employed the yeast two-hybrid system, a well-established method for assessing protein-protein interactions, known as the Y2H assay. We found that only the Y2HGold strain, co-transformed with the pGBKT7-BopE and pGADT7-Rab32 expression vectors, exhibited growth under both low-stringency conditions (Leu⁻ and Trp⁻) and high-stringency conditions (Ade⁻, His⁻, Leu⁻, and Trp⁻), which indicates BopE can interact with Rab32 in vivo (Fig. 4B). Confocal microscopy analysis also confirmed significant co-localization of BopE with Rab32 (Fig. 4C and D). Collectively, our findings provide evidence supporting the interaction between BopE and Rab32 in vivo.

BopE suppresses Rab32 in a manner unrelated to its GEF activity

A previous study suggested BopE can activate small GTPases Rac1 and Cdc42 through its guanine nucleotide exchange factor (GEF) function (32). We then hypothesis that BopE can also interfere in the activation of Rab32. Surprisingly, through an in vitro nucleotide exchange assay using N-methylanthraniloyl-guanosine diphosphate (Mant-GDP) (33), we found BopE can suppress the GTP exchange activation for Rab32 in a dose-dependent manner, while it can activate Rac1 as previously described (Fig. 5A; Fig. S4B). It suggested BopE has an additional function to suppress Rab32 in a manner unrelated to its GEF activity. To further investigate this hypothesis, we introduce a catalytically inactive mutant (BopE^R207E/N216P) and a highly active mutant (BopE^N224P/R230Q) as previously described (34). However, neither those two mutants can reverse the suppression of BopE to Rab32 (Fig. 5B). The T3SS effector SopD from Salmonella Typhimurium has been shown to activate Rab8 through direct interaction and is unrelated to its GAP activity (35). Therefore, we examine the interaction between BopE and its mutants with Rab32. As show in Fig. 5C and D, interference with GEF activity of BopE does not alter its co-localization with Rab32. Taken together, these results suggest that BopE suppresses Rab32 via a mechanism independent of its GEF function.

Fig 5.

BopE interferes with the nucleotide exchange process of Rab32. (A) Nucleotide exchange assay for Rab32. ΔFluorescence is equal to fluorescence intensity measured at each specified time point minus the baseline fluorescence intensity recorded at the onset of the experiment. All dots are presented as mean ± SD and derived from three independent experiments. ***P < 0.001 (two-way ANOVA with Tukey’s method). (B) BopE inhibits the activation of Rab32 independent of the GEF activity. The mutant BopE^N224P/R230Q exhibits high catalytic activity, while the mutant BopE^R207E/N216P has lost its catalytic activity. Fluorescence spectrometry assay data were showed mean ± SD from three independent experiments. ***P < 0.001 (two-way ANOVA with Tukey’s method). (C) BopE interacts with Rab32 independent of its GEF activity. Representative immunofluorescence images of co-localization of Rab32 and the GEF activity mutants of BopE in HEK293T cells. Rab32 was stained with an anti-Flag antibody (red), BopE, or its mutants tagged with GFP (green). Scale bar, 5 µm. (D) Confocal microscopy of BopE and Rab32 in panel C with fluorescence intensity plotted along the arrows.

BopE targets the Rab32 pathogen-restriction pathway to enhance B. pseudomallei intracellular replication and virulence

To examine the potential effects of the BopE-Rab32 interaction on the Rab32 pathogen-restriction pathway. We developed BopE-deficient (ΔbopE) and complemented (ΔbopE/bopE) strains of B. pseudomallei (Fig. S5). Confocal microscopy analysis was performed at various time points post-infection. We observed that Rab32 was efficiently recruited to BCVs within 2 hpi, followed by a decline in recruitment at 4 hpi. Notably, the number of Rab32-positive BCVs was significantly higher in the ΔbopE-infected monolayers than in the wild-type and complement infected cells (Fig. 6A and B). Our findings suggest that BopE plays a role in suppressing the recruitment of Rab32 to BCVs.

Fig 6.

BopE targets the Rab32-dependent defense pathway allowing B. pseudomallei to survive in the host. (A) Representative immunofluorescence images of co-localization of Rab32 and B. pseudomallei mutant strains in RAW264.7 cells at the indicated times after infection. Scale bar, 50 µm. (B) Quantification of B. pseudomallei in Rab32-positive vesicles in panel A. Ten random fields were counted in each slice, and three different experiments were conducted in every group. All assessments were performed in a blinded manner. *P < 0.05, **P < 0.01, and ***P < 0.001 (one-way ANOVA). (C and D) Intracellular B. pseudomallei burdens in RAW264.7 cells at 8 hpi. WT is the wild-type strain of B. pseudomallei, ΔbopE is the bopE deficient strain, and ΔbopE/bopE is the bopE complementation strain. The data were showed as means ± SD. Three different experiments were conducted in each group. *P < 0.05 and **P < 0.01 (one-way ANOVA). (E) Survival rate of WT, ΔbopE, or ΔbopE/bopE B. pseudomallei-infected BALB/c mice. Mice models were infected with 4 × 10⁵ CFU of B. pseudomallei WT, ΔbopE, or ΔbopE/ bopE and then continued to be observed until day 5. χ² = 4.101, P = 0.0429 (ΔbopE vs. WT); χ² = 5.141, P = 0.0234 (ΔbopE vs. ΔbopE/bopE), 12 mice per group (log-rank test). (F) The graphical model illustrates that BopE, a T3SS-3 effector of B. pseudomallei, suppresses the Rab32-dependent defense pathway in macrophage, thereby facilitating intracellular replication.

Furthermore, we explored the effects of BopE on the intracellular survival and the virulence of B. pseudomallei. We observed a significant reduction in intracellular bacterial loads within the BopE-deficient strain infection group, in contrast to the groups infected with the wild-type or BopE complement strains, at 8 hpi as shown in Fig. 6C and D. We then sought to determine the role of BopE in the Rab32-dependent host pathogen-restriction pathway during B. pseudomallei infection in vivo. Consistent with our hypothesis, the BopE-deficient mutant strain of B. pseudomallei showed obviously attenuated virulence and reduced mortality in infected mice, compared with wild-type strain- and BopE complement strain-infected groups (Fig. 6E). Taken together, our results demonstrate that BopE interacts with Rab32 and likely suppresses its activation at the nucleotide exchange level, thereby preventing Rab32’s recruitment to BCVs, thus counteracting the host pathogen-restriction pathway and facilitating intracellular replication and virulence of B. pseudomallei in the host, as illustrated in Fig. 6F.

DISCUSSION

Our immune system is engaged in a continuous battle against invading pathogens to avoid infection. As an important part of the innate immune system, macrophages can actively phagocytose pathogens, form bacteria-containing phagocytic vesicles, then initiate membrane traffic, and transport bacteria-filled vesicles to degradative intracellular compartments. Recently, numerous studies emphasize the importance of Rab32-dependent membrane trafficking in the elimination of intracellular bacteria in macrophages (9, 12, 24). We have also previously described that the Rab32-dependent pathway can restrict the intracellular replication of B. pseudomallei (14). However, it is believed that B. pseudomallei has the ability to escape from defensive BCVs and replicate in the cytoplasm (15). After invading host cells, B. pseudomallei often employ several strategies to escape the host cell immune system. It can circumvent autophagy either by modulating ATG10 or by manipulating the host’s lipid metabolism for its intracellular survival (36, 37). Additionally, B. pseudomallei can trigger the PERK-mediated unfolded protein response, a mechanism that supports its survival both in vivo and in vitro, thereby maintaining its survival and proliferation within a variety of host cells (38, 39). However, the mechanism by which B. pseudomallei escapes from Rab32-dependent vesicles remains to be elucidated.

In this study, the overexpression of Rab32 led to the accumulation of Rab32-positive vesicles and increased the probability of containing B. pseudomallei, thereby inhibiting B. pseudomallei intracellular survival. While interference with Rab32 expression showed the opposite effect. This is consistent with our previous findings that macrophages can specifically detect B. pseudomallei, triggering an upregulation of Rab32 as part of the host immune response (14). Interestingly, we found not all intracellular B. pseudomallei were encompassed by Rab32-positive vesicles despite Rab32 overexpression. This implies that B. pseudomallei may employ certain strategies to counteract the bactericidal effects of the Rab32-dependent pathway. In a parallel study, Rab32 is strongly recruited to human-adapted pathogen Salmonella enterica serovar Typhi (S. Typhi)-containing phagosome vesicle and restrict the replication of S. Typhi, but not the broad host range Salmonella enterica serovar Typhimurium (S. Typhimurium), as these serovars have evolved potent bacterial effectors, SopD2 and GtgE, to neutralize the Rab32-dependent defense pathway (24). Pathogens that enter the endocytic network must subvert membrane trafficking to avoid further transportation to degradative intracellular compartments. Many pathogenic bacteria accomplish this subversion with effector proteins delivered by specialized secretion systems, which can directly modulate membrane traffic (40, 41).

To identify potential effectors in B. pseudomallei that target the Rab32-mediated host defense pathway, specifically by preventing Rab32 recruitment to BCVs, we conducted a transcriptomic analysis of B. pseudomallei’s response to macrophage-mediated immune clearance. This analysis revealed that genes associated with T3SS of B. pseudomallei were significantly enriched among the DEGs. Our findings align with the results obtained through the innovative “TRANSITomic” approach, which has detailed the transcriptomes of individual B. pseudomallei cells at distinct stages of host cell infection, demonstrating that B. pseudomallei strategically deploys its T3SSBsa to escape the vesicle to gain entry into the host cell cytoplasm (16). We also observed a significant upregulation of DEGs in the type VI secretion system (T6SS) of B. pseudomallei at 4 hpi, including genes such as hcp1, tssA, and tssB, as detailed in Table S3. This is consistent with a previous report that highlighted the significant modulation of B. pseudomallei T6SS over 6 hours of infection (42).

T3SS is the key virulence for B. pseudomallei by injecting bacterial effector proteins into host cells. Once inside host cells, the effector proteins subvert host cell processes in favor of the bacteria (21, 43). Among the DEGs, we found that a variety of effector proteins of B. pseudomallei, such as BprD, BipC, and BopE, were highly expressed in the state of infection. Specifically, BopE displays significant homology to Salmonella effector proteins SopE and SopE2 (Fig. S6). It has been characterized as GEFs for the Rho GTPases Cdc42 (cell division cycle 42) and Rac1, which promote the invasion of B. pseudomallei into host cells (32). Moreover, the amino acids 78–261 of BopE have been reported as a GEF catalytic domain; the mutant BopE^N224P/R230Q showed high catalytic activity, but BopE^R207E/N216P lost its catalytic activity (34). Interestingly, in our study, we highlighted BopE interacting with Rab32 indeed has an inhibitory effect on the activation of Rab32 at the level of nucleotide exchange. Crucially, this inhibition is independent of the GEF activity of BopE, as demonstrated by the mutants BopE^N224P/R230Q and BopE^R207E/N216P. Our results suggest that BopE may have distinct effects on various small GTPases in the host. It is similar to Salmonella effector SopE2, which acts as a GEF for Rho GTPase Cdc42 but not Rac1 (44). Likewise, Salmonella effector SopD is known as a GAP that deactivates Rab8, while in the late stages of infection, SopD activates Rab8 by promoting its dissociation from guanine nucleotide dissociation inhibitors (35). Besides, BopE also appears to localize to the host-cell membrane; this could be a part of the bacterial strategy to manipulate host cell processes to facilitate its infection and survival. But how BopE localizes to the host cell membrane, which proteins on the host cell membrane it interacts with, and how it affects the infection process are questions that require extensive in vitro and in vivo experimentation for further clarification. Collectively, we hypothesize that the dual functionality of BopE facilitates the invasion of B. pseudomallei into host cells while simultaneously suppressing the Rab32-dependent defense pathway, enhancing its intracellular survival. Thereby, a comprehensive understanding of the role of BopE in modulating host cell functions necessitates further research to elucidate.

In summary, the current study builds upon the foundational work of Hu et al., who established the critical role of Rab32 in the intracellular survival of B. pseudomallei. This study not only validates our previous observations but also provides a significant extension to this body of knowledge. We demonstrate that B. pseudomallei employs the T3SS-3 effector BopE to subvert the Rab32-dependent defense pathway, thereby enhancing its intracellular replication and virulence. This discovery elucidates a critical aspect of the evasion strategy of B. pseudomallei and underscores the importance of Rab32 in the pathogenesis of melioidosis. Moreover, our findings may offer valuable insights for the development of targeted therapeutic interventions.

MATERIALS AND METHODS

Bacterial strains, plasmids, and culture conditions

B. pseudomallei (BPC006, NC_018529.1, and NC_018527.1) and Escherichia coli were cultured on Luria broth (LB) agar plates or in liquid LB media at 37°C with shaking at 200 rpm. E. coli DH5α strain was used for plasmid construction and cloning. E.coli S17-1/λpir strain was used as plasmid donor strain for conjugation experiments. Trimethoprim (Tp; 200 µg/mL) or kanamycin (Kan; 250 µg/mL) was utilized for the screening of B. pseudomallei. Live B. pseudomallei was carried out under standard laboratory conditions (biosafety level 3 [BSL-3]). Bacterial strains and plasmids are listed in Table S1.

Cell culture and infection

HEK293T and RAW264.7 were cultured in Dulbecco’s modified Eagle medium (DMEM; Thermo Fisher C11995500BT) supplemented with 10% fetal bovine serum (FBS; Gibco 10091148) at 37°C in a humidified atmosphere with 5% CO₂. Cell infection with B. pseudomallei was carried out as previously described (14). Briefly, cells were seeded overnight and infected with the B. pseudomallei at a multiplicity of infection (MOI) of 10 for 1.5 hours. Then, cells were washed twice with phosphate-buffered saline (PBS) and incubated in fresh medium containing 250 µg/mL kanamycin to kill the extracellular bacteria. At the selected time points, macrophage cells were lysed using RIPA for western blot or TRIzol for RT-qPCR or fixed in 4% paraformaldehyde for immunofluorescence or lysed using 0.1% Triton X-100 for plate counting to determine the intracellular bacterial counts. The number of bacteria was estimated by measuring the absorbance of the bacterial suspension at 600 nm.

Gene silence and overexpression

Rab32 was knocked down by a small interfering (si)RNA that targets the Rab32 gene (NM_026405) in RAW264.7 cells and overexpressed by the plasmid of pcDNA4-Rab32. Briefly, RAW264.7 cells were seeded till they reached 70%–80% confluence; then, siRNA oligo nucleotides targeting mouse Rab32 (siRab32; Santa Cruz, sc-152636) or pcDNA4-Rab32 (pRab32) were directly mixed with Advanced DNA RNA Transfection Reagent (ZETA LIFE, USA) according to the manufacturer’s instructions. Following incubation at room temperature for 10 min, the complex was then added to cell culture plates. After 24 hours of incubation, cells were harvested and the expression levels of Rab32 were detected by western blotting. Cells that received no treatment were used as NC. All experiments were performed in triplicate.

Bacterial survival assays

Intracellular survival of B. pseudomallei in RAW264.7 cells was estimated as previously described with some modifications (45). Briefly, cells were infected with either WT or bopE mutant of B. pseudomallei as described above. At the indicated time points, the infected cells were washed with PBS three times and lysed with 0.1% Triton X-100 (Sigma, T8787). Cell lysates were serially diluted 10-fold with PBS and plated onto LB agar. CFU were then measured after 36 hours of incubation at 37°C. We performed the experiment three replicates independently.

Western blot

The expression of target proteins was detected by western blot as previously described with some modifications (39). Briefly, equal amounts of denatured proteins were separated by SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore, IPVH00010). The membranes were blocked with 5% (wt/vol) skimmed milk for 2 hours, followed by an overnight incubation at 4°C with Rab32 (Proteintech 10999-1-AP; 1:1,000), Flag tag (ZENBIO R24091; 1:1,000), GFP tag (Bioss bsm-33019M; 1:1,000) antibody, or anti-BopE rabbit serum (1:500). Membranes were subsequently incubated with HRP-linked anti-rabbit IgG (CST 7074S; 1:5,000) or anti-mouse IgG (CST 91,196S; 1:5,000) secondary antibody at the room temperature for 2 hours. Blots were scanned with the ChemiDoc Touch System (Bio-Rad, USA) and analyzed using Image Lab Software (version 6.0.0, build 25, Bio-Rad, USA). GAPDH (CST 8884; 1:1,000) or DnaK (CUSABIO CSB-PA633459HA01EGW; 1:2,000) was used as loading control, and three independent replicates were performed.

Immunofluorescence

Cells were seeded on coverslips (NEST, 801011) in 24-well plates and transfected with the indicated plasmids or siRNA. After incubation for 24 hours, cells were washed twice with PBS and fixed in 4% paraformaldehyde (Beyotime P0099) for 10 min; subsequently, cells were permeabilized with 0.3% Triton X-100 (amresco, 0694-1L) and incubated with Flag tag antibody (ZENBIO R24091; 1:500), Rab32 antibody (Proteintech 10999-1-AP; 1:300), or a mouse polyclonal antibody of B. pseudomallei (1:200) and subsequently with the Alexa Fluor 647 anti-mouse secondary antibody (Thermo Fisher A-31571; 1:2,000) or Alexa Fluor 488 anti-rabbit secondary antibody (Thermo Fisher A-11008; 1:2,000). Nucleus was stained with DAPI (Thermo Fisher 62248; 300 nM). The coverslips were mounted onto glass slides using anti-fade mounting medium (Invitrogen, S36967). Confocal images were taken with the Leica SP8 confocal microscope (Leica Microsystems) using a 60× lens objective.

Intracellular bacteria isolation and RNA-seq

The BPC006 strain was cultured on the LB agar plate 36 hours at 37°C. A single colony was selected to inoculate into 5 mL of LB broth and incubated at 37°C for 12 hours. The cultures were divided into two equal parts (in vitro B.ps and intracellular B. ps) based on the bacterial count with an MOI of 100. Intracellular B. ps was used on infected RAW264.7 cells, and intracellular bacteria were isolated as previously described (46). Briefly, after 4 hours of infection, cells were washed extensively with PBS and lysed in 20 mM Tris-HCl (pH 7.6) buffer containing 150 mM NaCl and 0.5% Triton X-100. To recover intracellular bacteria, collected cell lysates were centrifuged at 600 × g for 5 min to remove nuclei and cell debris, and then, the supernatant was centrifuged at 4,000 × g for 20 min. The pellets were immediately washed with RIPA buffer (Beyotime P0013D) to remove residual host RNA and centrifuged at 6,000 × g for 5 min to get the final bacterial pellets. In vitro B.ps were cultivated in DMEM medium, without the presence of RAW264.7 cells, and were recovered as the group of intracellular B. ps.

The total RNA was extracted from the harvested bacteria using a RNAprep Pure Cell/Bacteria Kit (Tiangen DP340) following the manufacturers’ protocols and sent to the Sangon Biotech (Shanghai, China) company for quality control and complementary DNA (cDNA) library construction, followed by sequencing. Trimmomatic (version 0.36) was used to trim low-quality reads and remove adapters of raw data. The Bowtie2 (version 2.3.2) was used to align the trimmed sequence to B. pseudomallei C006 and matched to the Burkholderia pseudomallei K96243 genome (NC_006350.1and NC_006351.1). The DEGs in which |log₂FoldChange| ≥ 1 and P value < 0.05 were analyzed using DESeq2 software (version 1.12.4). KOBAS 3.0 (http://bioinfo.org/kobas/) was used to determine the main biological functions of DEGs enriched in Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses (47).

Real-time qPCR

To validate the data generated from GO analysis and pathway analysis, eight DEGs of effectors were selected from T3SS-1 for RT-qPCR analysis. The respective primers were listed in Table S2. The total RNA used for RT-PCR was extracted by TRIzol (Invitrogen Life Technologies, USA) according to the manufacturer’s recommendation. Next, cDNA was retro-transcribed from 1 µg of total RNA using a PrimeScript RT Reagent Kit with gDNA Eraser (Takara, Dalian, China). The cDNA samples were then amplified using SYBR Premix Ex Taq II (Takara, Dalian, China) on a CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA). Six replicates were respectively conducted in the infected group (I) and non-infected group (NI). Relative fold expression for target genes was calculated by the 2^−ΔΔCt method relative to rpoB as reference gene.

Co-immunoprecipitation

The interactions between T3SS effectors and Rab32 were assessed using immunoprecipitation assays. Briefly, HEK293T cells were co-transfected with a combination of plasmids, including pEGFP-Rab32 (encoding Rab32 with a GFP tag) and pcDNA4-Flag-effectors (encoding T3SS effectors BapA, BipC, BopA, BopC, BopE, BprD, BapC, or BipD, each fused with a Flag tag). Additionally, pcDNA4-Flag-SopD2 was included as a positive control for the interaction (24). After 24 hours of transfection, cells were collected and lysed in lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, and protease inhibitor cocktails) on ice. The supernatants of the cell lysates were isolated by centrifugation at 12,000 × g for 30 min at 4°C, and the supernatants were then incubated with anti-Flag M2 Affinity Gel (Sigma-Aldrich, A2220) overnight at 4°C. The resins were washed three times with lysis buffer followed by elution with 3× Flag peptide for 2 hours at 4°C. The IP samples were detected by western blot.

Yeast two-hybrid assay

Yeast two-hybrid analyses were conducted using the Matchmaker Gold Yeast Two-Hybrid System. Strain Y2HGold was co-transformed with the bait Gal4-BD vector pGBKT7 where B. pseudomallei bprD, bopE, and bipC genes were inserted in frame (pGBKT7-BprD, pGBKT7-BopE, and pGBKT7-BipC) and the prey AD vector pGADT7 where the gene of Rab32 was also inserted in frame (pGADT7-Rab32). Expression of BD and AD fusion proteins was confirmed by western blotting using anti-c-Myc antibody and anti-HA antibody, respectively. The transformants were plated onto low-stringency (lacking leucine and tryptophan) and high-stringency (lacking adenine, histidine, leucine, and tryptophan) selection plates for detection. The interaction between receptor-interacting protein kinase 1 (RIPK1) and the Fas-associated death domain (FADD) was well-documented (48); we used pGBKT7-mRIPK1 and pGADT7-mFADD co-transformed Y2HGold strain as a positive control in this study.

Construction of B. pseudomallei bopE mutant and complementation strain

The construction of B. pseudomallei bopE mutant (ΔbopE) and complementation strain (ΔbopE/bopE) was performed as previously described with some modifications (25). The BPSS1525 (bopE) mutant was constructed in a B. pseudomallei C006 background by double-crossover allelic exchange using the λpir-dependent pK18mobsacB vector. The clean deletion construct was confirmed by Sanger sequencing. For complementation, the full-length of the bopE gene was amplified from the genomic DNA of B. pseudomallei C006. After double enzyme digestion with Xba I and Hind III, the product was cloned into the broad-host-range vector pUCP28T by T4 DNA Ligase (Tiangen Biotech Co. Ltd., Beijing, China) to generate pUCP28T-BopE, which expressed BopE under the control of the 16S promoter of B. pseudomallei. Then, the recombinant plasmid pUCP28T-BopE was electroporated into B. pseudomallei C006. Expression of BopE was confirmed by western blot. The plasmids and primer sequences used in this study were listed in Tables S1 and S2.

Nucleotide exchange assay

BopE and the mutant BopE^N224P/R230Q or BopER^207E/N216P were expressed in Escherichia coli BL21(DE3) with the pET28a vector under isopropyl-β-d-thiogalactoside induction. Subsequently, the fusion proteins were purified through a HisTrap FF (5 mL) column (Cytiva, 17-5255-01) on an Akta pure 25 system. Nucleotide exchange assays were conducted using fluorescent N-methylanthraniloyl-GDP (Mant-GDP, Molecular Probes), following a modified method from a previous report (33). Briefly, Rab32 (Origene, TP502577) or Rac1 (Origene, TP527648) equilibrated in loading buffer (20 mmol/L HEPES-NaOH [pH 7.5], 50 mmol/L NaCl, 0.5 mmol/L MgCl₂, 5 mmol/L EDTA, 1 mmol/L DTT) was loaded with 20-fold excess of Mant-GDP (Jena Bioscience, NU-204S) at room temperature for 1.5 hours. The loading reaction was terminated with 10 mM MgCl₂, diluted with desalting buffer (20 mM HEPES [pH 7.5], 50 mM NaCl, 5 mM MgCl₂, and 1 mM DTT), and desalted using a NAP-5 column (Cytiva, 17085301). Exchange reactions (200 μL total volume) were performed in 20 mM HEPES (pH 7.5), 50 mM NaCl, 5 mM MgCl₂, and 1 mM DTT and contained 30 µM Rab32 in the presence or absence of BopE or BSA. After a 10-min pre-incubation on ice, reactions were initiated with 200 µM GTP (Macklin, G810427) and data were acquired at 25°C using a Molecular Devices SpectraMaxiD5 microplate spectrofluorometer with excitation at 360 nm and emission at 440 nm.

Infection-induced mouse melioidosis model

Specified pathogen-free BALB/c mice at approximately 6–8 weeks were purchased from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China) and were maintained under barrier conditions in a BSL-3 biohazard animal room and provided with free water and diet and a 12-hour light/dark cycle. After 1 week of adaptive feeding, mice were intraperitoneally injected with 4 × 10⁵ CFU of B. pseudomallei (WT, ΔbopE, and ΔbopE/bopE, 12 mice per group) and continuously observed for 5 days.

Statistical analysis

The quantified data with statistical analysis were performed using GraphPad Prism (v9.5.1) software. Unpaired two-sided Student’s t-test, one-way analysis of variance (ANOVA), or two-way ANOVA followed by multiple comparisons was used for statistical analyses. Significance was indicated in the figures and figure legends. All experimental data from the assays were obtained from at least three separate biological samples, and the results were presented as the average values with their standard deviations (means ± SD). The statistical tests and the numbers of samples were indicated in the corresponding figure legends.

ETHICS APPROVAL

All animal experiments were conducted in strict accordance with the recommendations in the Guide for the Laboratory Animal Welfare and Ethics Committee of the Army Medical University (AMUWEC20230035). All efforts were made to minimize animal suffering and to reduce the number of animals used in the study.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/msphere.00453-24.

Supplemental material. msphere.00453-24-s0001.pdf.

Movie legend, Figures S1 to S6, and Tables S1 to S3.

Movie S1. msphere.00453-24-s0002.avi.

Recruitment of Rab32 vesicles to capture intracellular B. pseudomallei.

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