Autophagy facilitates intracellular survival of pathogenic rickettsiae in macrophages via evasion of autophagosomal maturation and reduction of microbicidal pro-inflammatory IL-1 cytokine responses

Oliver H Voss; Hodalis Gaytan; Saif Ullah; Mohammad Sadik; Imran Moin; M Sayeedur Rahman; Abdu F Azad

doi:10.1128/spectrum.02791-23

. 2023 Oct 11;11(6):e02791-23. doi: 10.1128/spectrum.02791-23

Autophagy facilitates intracellular survival of pathogenic rickettsiae in macrophages via evasion of autophagosomal maturation and reduction of microbicidal pro-inflammatory IL-1 cytokine responses

Oliver H Voss ^1,^✉, Hodalis Gaytan ¹, Saif Ullah ¹, Mohammad Sadik ¹, Imran Moin ¹, M Sayeedur Rahman ¹, Abdu F Azad ^1,^✉

Editor: Artem S Rogovskyy²

PMCID: PMC10715094 PMID: 37819111

ABSTRACT

The genus Rickettsia is comprised of obligate intracellular bacterial parasites of a wide range of arthropod and vertebrate hosts. Some Rickettsia species (spp.) are responsible for serious human diseases globally. One interesting feature of these stealthy group of pathogens is their ability to exploit host cytosolic defense responses to their benefits. However, the precise mechanism by which pathogenic Rickettsia spp. elude host immune defense responses remains to be determined. Here, we observed that pathogenic Rickettsia typhi and Rickettsia rickettsii (Sheila Smith [SS]), but not non-pathogenic Rickettsia montanensis, become ubiquitinated and induce autophagy upon entry into bone marrow-derived macrophages. Moreover, unlike R. montanensis, both R. typhi and R. rickettsii (SS) colocalized with LC3B and not with Lamp2 upon host cell entry. Finally, we observed that both R. typhi and R. rickettsii, but not R. montanensis, reduce pro-inflammatory interleukin-1 (IL-1) cytokine responses, likely via an autophagy-mediated mechanism. In summary, we identified a previously unappreciated pathway by which both pathogenic R. typhi and R. rickettsii (SS), but not the non-pathogenic R. montanensis, become ubiquitinated, induce autophagy, avoid autolysosomal destruction, and reduce microbicidal IL-1 cytokine responses to establish an intracytosolic niche in macrophages.

IMPORTANCE

Rickettsia spp. are intracellular bacterial parasites of a wide range of arthropod and vertebrate hosts. Some rickettsiae are responsible for several severe human diseases globally. One interesting feature of these pathogens is their ability to exploit host cytosolic defense responses to their benefits. However, the precise mechanism by which pathogenic Rickettsia spp. elude host defense responses remains unclear. Here, we observed that pathogenic Rickettsia typhi and Rickettsia rickettsii (Sheila Smith [SS]), but not non-pathogenic Rickettsia montanensis, become ubiquitinated and induce autophagy upon entry into macrophages. Moreover, unlike R. montanensis, R. typhi and R. rickettsii (SS) colocalized with LC3B but not with Lamp2 upon host cell entry. Finally, we observed that both R. typhi and R. rickettsii (SS), but not R. montanensis, reduce pro-inflammatory interleukin-1 (IL-1) responses, likely via an autophagy-mediated mechanism. In summary, we identified a previously unappreciated pathway by which both pathogenic R. typhi and R. rickettsii (SS) become ubiquitinated, induce autophagy, avoid autolysosomal destruction, and reduce microbicidal IL-1 cytokine responses to establish an intracytosolic niche in macrophages.

KEYWORDS: R. typhi, R. rickettsii, R. montanensis, autophagy, IL-1α, IL-1β, macrophages, bacterial host dissemination

INTRODUCTION

A plethora of intracytosolic bacteria utilize sophisticated strategies to circumvent host defense responses to promote their survival within the host (1, 2). Rickettsiae are arthropod-borne obligate intracellular bacteria with both symbiotic and pathogenic lifestyles. Rickettsia prowazekii, Rickettsia typhi, Rickettsia rickettsii (Sheila Smith [SS]), and Rickettsia conorii, etiological agents for epidemic typhus, murine typhus, Rocky Mountain spotted fever, and Boutonneuse fever, respectively, are well-known human pathogens (3, 4). Apart from their historical record, the global impact of rickettsial infections is illustrated by the resurgence of known, as well as the rise of newly identified rickettsial pathogens (5). Infections of humans with R. rickettsii (SS) continue to cause severe public health threats in South and Central America (6). The resurgence of R. conorii in Europe, the Middle East, and Africa further highlights current threats of rickettsial diseases globally (7). In addition, arthropod-borne rickettsial diseases are also on the rise in the USA, as exemplified by recent outbreaks of R. rickettsii (SS) in Arizona (8) and of R. typhi in California (9) and Texas (10).

Entry into eukaryotic host cells, followed by intracytosolic growth and dissemination to neighboring cells has long been considered as a conserved process of Rickettsia spp. However, it is important to note (and often overlook) that many described rickettsiae are naturally maintained in arthropods and are considered non-pathogenic, as they cause no or a very mild disease in humans (3, 11, 12). Rickettsia spp. are introduced into the host’s dermis by infected arthropods and the first host defense cells encountered are macrophages and dendritic cells (4). In particular, macrophages are crucial in either terminating an infection at an early stage or succumbing to pathogen colonization, thereby contributing to the dissemination of Rickettsia spp. to distant organs of the host (4). Upon host cell entry, rickettsiae encounter cytosolic host defense responses initiated after sensing the bacteria or associated danger signals. Commonly invading bacteria encounter autophagy and inflammasome responses, two functionally interconnected pathways (13, 14), which typically provide the appropriate defense measures against intracellular pathogens (15, 16). Importantly, recent reports suggest that autophagy acts on intracellular microbes upstream of the inflammasome (17 – 19). Autophagy is triggered by ubiquitination of the intracellular bacterium (20). Ubiquitin (Ub) coats the bacterial surface and recruits autophagy adaptors (p62, NDP52) and induces autophagosome formation with autophagy machinery consisting of Unc-51-like autophagy-activating kinases (ULK) complex, phosphoinositide 3-kinase (PI3K) complex (Beclin1, VSP34, ATG14L) and the ATG16L1 complex (ATG16L1, ATG5, LC3) (21). It is becoming increasingly evident that several intracellular bacteria develop unique mechanisms to modulate autophagy, in particular to avoid autolysosomal destruction, to facilitate host colonization (15, 20). In the case of rickettsiae, the role of autophagy in regulating host colonization remains inconclusive (22 – 25). For instance, Rickettsia australis, a virulent member of the transitional group (TRG), benefited from ATG5-dependent autophagy and suppression of pro-inflammatory cytokine responses to colonize host cells (22, 23). In contrast, reports on Rickettsia parkeri, a mild-virulent member of the spotted fever group (SFG), demonstrated that its surface protein OmpB is critical for protecting against autophagic recognition, and further showed that evasion of autophagy was critical for invasion of bone marrow-derived macrophages (BMDMΦ) and wild-type (WT) mice (24, 25). Intriguingly, we reported that pathogenic members of SFG and typhus group (TG) rickettsiae secrete effectors to promote host colonization by modulating endoplasmic reticulum structures or by hijacking the autophagic defense pathway, respectively (26 – 31). In fact, our findings on the flea-transmitted R. typhi, a pathogenic member of the TG, showed that this intracytoplasmic pathogen is ubiquitinated upon host entry and escapes autolysosomal fusion to establish an intracytosolic niche in non-phagocytic cells (30). Thus, in agreement with reports on R. australis (22, 23), these findings suggest a mechanism by which highly pathogenic Rickettsia spp., including members of TG, TRG, and possibly other SFG rickettsiae, activate autophagy, but subsequently evade autolysosomal destruction, to promote host colonization. These unexpected findings on how SFG, TRG, and TG Rickettsia differentially promote their host dissemination prompted us to test the hypothesis that survival of both pathogenic R. typhi and R. rickettsii (SS), but not the non-pathogenic Rickettsia montanensis, involves evasion of autophagosomal maturation and reduction of microbicidal pro-inflammatory interleukin-1 (IL-1) cytokine responses to establish a replication niche in phagocytic host immune defense cells, like macrophages.

RESULTS

Pathogenic rickettsiae are ubiquitinated and induce autophagy upon entry into macrophages

Preceding findings suggest that intracellular pathogens, like rickettsiae, not only encounter inflammasome-dependent defense mechanisms but are also confronted by another cytosolic defense pathway, autophagy (22 – 25, 30, 32 – 35). Both responses not only are key to mount the appropriate host defense responses, but are also functionally interconnected (15, 16). In one of our preceding reports, we showed that R. typhi is ubiquitinated upon host entry and induces autophagy, but escapes autophagosomal maturation for intracellular colonization in non-phagocytic cells (30). More recently, we demonstrated that unlike R. montanensis (a non-pathogenic SFG member), R. rickettsii (SS) and R. typhi, two highly pathogenic Rickettsia spp., preferentially targeted the non-canonical inflammasome-IL-1α signaling axis in macrophages to support their replication (32). Given these reports by others and our recent findings, we first evaluated the ubiquitination status of R. typhi, R. rickettsii (SS), or R. montanensis during invasion of bone marrow-derived macrophages isolated from C57BL/6J WT mice. Similar to infection studies using R. australis (23), we observed that both R. rickettsii (SS) and R. typhi spp. were ubiquitinated during the course of invasion of WT BMDMΦ (Fig. 1A and B). In contrast, but similar to R. parkeri infections, R. montanensis was not ubiquitinated in WT BMDMΦ (Fig. 1A and B). These data suggest that the life cycle of R. montanensis is restricted by phagolysosomal fusion allowing for only a small number of bacteria to escape into cytosol of macrophages and support the findings from another laboratory using a human monocytic Tohoku Hospital Pediatrics-1 (THP-1) cell line (36, 37). To test that hypothesis, we evaluated the bacterial burdens of R. typhi, R. rickettsii (SS), or R. montanensis during invasion of WT BMDMΦ and demonstrated that, unlike R. montanensis, both R. rickettsii (SS) and R. typhi replicated in macrophages (Fig. 1C), which is in agreement with our previous published work (32). Furthermore, we evaluated the status of autophagy markers p62 (also known as sequestosome-1 [SQSTM1]) and autophagic vesicle formation marker LC3B (38) during infection of WT BMDMΦ by Western blot analyses. Our data revealed that, unlike R. montanensis, infection with R. rickettsii (SS) and R. typhi induced autophagy, as evidenced by an enhanced induction of LC3B and the simultaneous down-regulation of p62 expression (Fig. 1D and E). Given these findings, we performed similar infection studies in the presence of bafilomycin A1 (Baf A1), a late-stage autophagy inhibitor, and found that treatment with Baf A1 resulted in a further increase in LC3B levels providing additional evidence for a direct modulation of autophagy during the invasion of pathogenic rickettsiae (Fig. S1).

Fig 1 — *R. rickettsii* (SS) and *R. typhi*, but not *R. montanensis*, are ubiquitinated and induced autophagy in macrophages. (A) BMDMΦ from WT mice were infected with *R. montanensis*, *R. rickettsii* (SS), or *R. typhi* at amultiplicity of infection (MOI) of 20 (for 0.5 and 2 h) and 5 (for 24 and 48 h). Samples were fixed with 4% paraformaldehyde and *Rickettsia* spp. were detected using specific Alexa Fluor 488-conjugated *Rickettsia* (SFG or TG) antibodies, while Ub status was assessed using Alexa Fluor 594-conjuagted anti-Ub antibody. Images represent *Rickettsia*-infected macrophages after 2 h post-infection. DNA was stained using 4´,6-diamidino-2-phenylindole (blue). Colocalization between *Rickettsia* and Ub was analyzed using Coloc 2 plugin Fiji software. Bars in panel A, 10 µm. (B) Graph shows the percentage of Ub positive stained *Rickettsia* at indicated time points (0.5, 2, 24, and 48 h) of infection. Approximately 100–200 bacteria were counted per strain and time point. (C) Bacterial burdens in *Rickettsia*-infected BMDMΦ were evaluated 0.5, 2, 24, and 48 h post-infection by *GltA* reverse transcription quantitative real-time PCR (RT-qPCR). Relative copy number (RCN) of *GltA* expression was normalized by the expression of the *GAPDH*. (D) *Rickettsia*-infected WT BMDMΦ, as described in panel A, were lysed and samples were immunoblotted with anti-p62, anti-LC3B, anti-elongation factor Ts (EF-Ts), and anti-GAPDH antibodies. Immunoblot data are a representative of three independent experiments. (E) Densitometry analysis from samples shown in panel D was performed using Fiji software and data represent the fold change between the ratios of LC3B-II/GAPDH, LC3B-II/LC3B-I, or p62/GAPDH. Error bars (B, C, E) represent means ± standard error of the mean (SEM) from five independent experiments; NS, non-significant; *P ≤ 0.05; ***P ≤ 0.005; ****P ≤ 0.001.

Pathogenic rickettsiae avoid autolysosomal destruction to establish a replicative niche in macrophages

Autophagy is an intracellular process that delivers autophagosomes to the lysosomes for degradation and is considered as one of the host defense pathways to combat bacterial infection, including other cellular functions (19, 20, 39). Given our above presented data that pathogenic, but not non-pathogenic, Rickettsia spp. are ubiquitinated upon host cell entry (Fig. 1A and B), we next evaluated the status of autophagy marker, LC3B, and lysosomal marker, Lamp2, during infection of WT BMDMΦ by immunofluorescence assay (IFA). We observed that, unlike R. montanensis, R. typhi and R. rickettsii (SS) colocalized with autophagy marker, LC3B, over the course of infection (Fig. 2A through D). As we previously demonstrated that R. typhi was able to avoid autolysosomal destruction in non-phagocytic cells (30), we next assessed the colocalization pattern of all three Rickettsia spp. with Lamp2. We observed that, unlike R. montanensis, both R. typhi and R. rickettsii (SS) spp. did not colocalize with Lamp2 in WT BMDMΦ (Fig. 2A through E), suggesting that both pathogenic Rickettsia spp. induce autophagy and escape autolysosomal destruction to facilitate their intracytosolic survival.

Fig 2 — *R. rickettsii* (SS) and *R. typhi* evade autolysosomal destruction to establish a replication niche in macrophages. (**A–D**) BMDMΦ from WT mice were infected with *R. typhi*, *R. rickettsii* (SS), or *R. montanensis* at a multiplicity of infection (MOI) of 20 (for 0.5 and 2 h) and 5 (for 24 and 48 h). Cells were fixed with 4% paraformaldehyde and *Rickettsia* spp. were detected using the SFG or TG *Rickettsia*-specific antibodies conjugated with Alexa Fluor 488, while LC3B and Lamp2 expressions were assessed using Alexa Fluor 594-conjuagted anti-LC3B or anti-Lamp2 antibodies, respectively. Images represent *Rickettsia*-infected macrophages after 2 h post-infection. DNA was stained using 4´,6-diamidino-2-phenylindole (blue). Colocalization between *Rickettsia* and LC3B or Lamp2 was analyzed using Coloc 2 plugin Fiji software. Bars in panels A–D, 10 µm. (E) Graphs show the percentage of LC3B or Lamp2 positive stained *Rickettsia* at indicated time points (0.5, 2, 24, and 48 h) of infection. Approximately 100–200 bacteria were counted per strain and time point. Error bars (E) represent means ± standard error of the mean (SEM) from five independent experiments; NS, non-significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005; ****P ≤ 0.001.

Intracytosolic survival of pathogenic rickettsiae requires induction of autophagy and reduction of microbicidal pro-inflammatory IL-1 cytokine responses

Based on recent findings from other laboratories and ours (22 – 25, 30, 32 – 35, 40), it has become evident that Rickettsia spp. exhibit variable degree of pathogenicity, indicating species-specific strategies to respond to mechanism of host defense surveillance, including autophagy and inflammasome responses. To address the role of autophagy in modulating the intracellular survival of Rickettsia spp., we utilized BMDMΦ from mice genetically lacking the ATG5 gene (ATG5^fl/fl-LysM-Cre) in myeloid cells, a well-accepted mutant model defective in the autophagy pathway (41). Next, we determined the colocalization status of R. montanensis, R. typhi, and R. rickettsii (SS) with LC3B during infection of ATG5^fl/fl or ATG5^fl/fl-LysM-Cre BMDMΦ by IFA. We found that, unlike R. montanensis, both R. typhi and R. rickettsii (SS) strains colocalized with LC3B in ATG5^fl/fl BMDMΦ, and not in ATG5^fl/fl-LysM-Cre BMDMΦ (Fig. 3A through D; Fig. S2). Moreover, we observed that, unlike R. typhi and R. rickettsii (SS), R. montanensis colocalized with Lamp2 in ATG5^fl/fl BMDMΦ (Fig. 3A through D; Fig. S2). However, all three Rickettsia strains colocalized with Lamp2 in ATG5^fl/fl-LysM-Cre BMDMΦ (Fig. 3A through D; Fig. S2). As it has been reported that ATG5 can modulate other host events independently of autophagy (42), we employed BMDMΦ from mice genetically deprived in the Beclin1 gene function (Beclin1^fl/fl-LysM-Cre) in myeloid cells, an alternative mutant model defective in the autophagy pathway (41). Accordingly, we determined the colocalization status of R. montanensis, R. typhi, and R. rickettsii (SS) with LC3B and Lamp2 during infection of Beclin1^fl/fl-LysM-Cre or Beclin1^fl/fl (corresponding WT control) BMDMΦ by IFA. In line with our infection studies using ATG5^fl/fl and ATG5^fl/fl-LysM-Cre BMDMΦ, we found that, unlike R. montanensis, both R. typhi and R. rickettsii (SS) strains colocalized with LC3B in Beclin1^fl/fl BMDMΦ, and not in Beclin1^fl/fl-LysM-Cre BMDMΦ (Fig. S3). Furthermore, we observed that, unlike R. typhi and R. rickettsii (SS), R. montanensis colocalized with Lamp2 in Beclin1^fl/fl BMDMΦ (Fig. S3), while all three Rickettsia spp. colocalized with Lamp2 in Beclin1^fl/fl-LysM-Cre BMDMΦ (Fig. S3), suggesting that the intracytosolic survival of both pathogenic R. typhi and R. rickettsii (SS) strains in macrophages requires the activation of autophagy in an ATG5-/Beclin1-dependent manner and escape from autolysosomal destruction.

Fig 3 — Intracytosolic survival of *R. rickettsii* (SS) and *R. typhi* involves autophagy and evasion of autolysosomal destruction. (**A–C**) BMDMΦ from ATG5^fl/fl (WT) or ATG5^fl/fl-LysM-Cre (knockout [KO]) mice were infected with *R. typhi*, *R. rickettsii* (SS), or *R. montanensis* at a multiplicity of infection (MOI) of 20 (for 0.5 and 2 h) and 5 (for 24 and 48 h). Cells were fixed with 4% paraformaldehyde and *Rickettsia* were detected using the SFG or TG *Rickettsia*-specific antibodies conjugated with Alexa Fluor 488, while LC3B and Lamp2 expressions were assessed using Alexa Fluor 594-conjuagted anti-LC3B or anti-Lamp2 antibodies, respectively. Images represent *Rickettsia*-infected macrophages after 2 h post-infection. DNA was stained using 4´,6-diamidino-2-phenylindole (blue). Colocalization between *Rickettsia* and LC3B or Lamp2 was analyzed using Coloc 2 plugin Fiji software. Bars in panels A–C, 10 µm. (D) Graphs show the percentage of LC3B or Lamp2 positive stained *Rickettsia* at indicated time points (0.5, 2, 24, and 48 h) of infection. Approximately 100–200 bacteria were counted per strain and time point. Error bars (D) represent means ± standard error of the mean (SEM) from five independent experiments; NS, non-significant; ****P ≤ 0.001.

Our recent findings identified a previously unappreciated mechanism by which R. typhi and R. rickettsii (SS), unlike R. montanensis, benefited from a reduced IL-1 cytokine response, specifically IL-1α, to support their replication within the host (32). Intriguingly, R. australis benefited from ATG5-dependent autophagy induction and reduction of pro-inflammatory cytokine responses (22, 23); however, a successful host colonization of R. parkeri involved the evasion of autophagy and inflammasome-mediated host cell death (24, 25), leaving the precise mechanism inconclusive. To gain further mechanistic insight, we first evaluated the IL-1α and IL-1β cytokine responses in cultured supernatants of R. montanensis-, R. typhi-, and R. rickettsii (SS)-infected ATG5^fl/fl BMDMΦ, as well as ATG5^fl/fl-LysM-Cre BMDMΦ. We observed that infection of ATG5^fl/fl BMDMΦ with R. typhi or R. rickettsii (SS) produced significantly lower levels of IL-1β or IL-1α cytokine, as compared to that of R. montanensis (Fig. 4A). Infection of ATG5^fl/fl-LysM-Cre BMDMΦ resulted in an increase of IL-1β or IL-1α cytokine levels for both R. typhi and R. rickettsii (SS) (Fig. 4A). Furthermore, we observed that, unlike R. montanensis, both R. typhi and R. rickettsii (SS) replicated in infected ATG5^fl/fl BMDMΦ; however, the replication of both bacteria was significantly impaired in ATG5^fl/fl-LysM-Cre BMDMΦ (Fig. 4B). Next, we validated our IL-1β or IL-1α cytokine and bacterial burden data in R. montanensis-, R. typhi-, or R. rickettsii (SS)-infected Beclin1^fl/fl BMDMΦ and Beclin1^fl/fl-LysM-Cre BMDMΦ. Our assays showed that infection of Beclin1^fl/fl BMDMΦ with R. typhi or R. rickettsii (SS) produced lower IL-1β or IL-1α cytokine levels, as compared to that of R. montanensis (Fig. S4A). Infection of Beclin1^fl/fl-LysM-Cre BMDMΦ with Rickettsia spp. resulted in an increase of IL-1β or IL-1α cytokine levels for both R. typhi and R. rickettsii (SS) (Fig. S4A). Furthermore, we found that, unlike R. montanensis, both R. typhi and R. rickettsii (SS) replicated in infected Beclin1^fl/fl BMDMΦ; however, the replication of both bacteria was impaired in Beclin1^fl/fl-LysM-Cre BMDMΦ (Fig. S4B). Given these data, we sought to determine the biological importance of autophagy induction and IL-1β or IL-1α cytokine responses in modulating the survival of all three Rickettsia spp. in macrophages. In this effort, we neutralized the activity of IL-1β or IL-1α cytokines using anti-IL-1β or anti-IL-1α antibodies and assessed the effect on the bacterial burdens for R. montanensis-, R. typhi-, and R. rickettsii (SS)-infected ATG5^fl/fl BMDMΦ, as well as ATG5^fl/fl-LysM-Cre BMDMΦ. Neutralization of IL-1α and IL-1β (with a lower effectiveness) resulted in an increase in bacterial loads of R. typhi-, R. rickettsii (SS)-, and R. montanensis-infected ATG5^fl/fl BMDMΦ (Fig. 4C). Importantly, antibody neutralization of IL-1α and IL-1β (with a lower effectiveness), increased the bacterial burdens of R. typhi-, and R. rickettsii (SS)-infected ATG5^fl/fl-LysM-Cre BMDMΦ, reaching levels observed in infected ATG5^fl/fl BMDMΦ (Fig. 4C). Of note, concurrent anti-IL-1α and anti-IL-1β antibody treatments in R. typhi- and R. rickettsii (SS)-infected ATG5^fl/fl BMDMΦ or ATG5^fl/fl-LysM-Cre BMDMΦ did not result in a synergistic effect (Fig. 4C). Neutralization of either IL-1α or IL-1β (with a lower effectiveness) cytokine resulted in an increase in the bacterial loads of R. montanensis-infected ATG5^fl/fl BMDMΦ or ATG5^fl/fl-LysM-Cre BMDMΦ (Fig. 4C), while concurrent treatment with both antibodies resulted in synergistic increase in bacteria loads (Fig. 4C). The efficiency of antibody-mediated blocking was validated by comparing the levels of IL-1β and IL-1α cytokines in the supernatants of IgG isotype control, IL-1β and IL-1α antibody-treated Rickettsia-infected ATG5^fl/fl BMDMΦ, as well as ATG5^fl/fl-LysM-Cre BMDMΦ (Fig. S5).

Fig 4 — *R. rickettsii* (SS) and *R. typhi*, but not *R. montanensis*, utilize autophagy induction and subsequent inhibition of microbicidal pro-inflammatory IL-1 responses to establish a replication niche. (A) Culture supernatants of uninfected and *R. typhi*-, *R. rickettsii* (SS)-, or *R. montanensis*-infected ATG5^fl/fl (WT) and ATG5^fl/fl-LysM-Cre [Knockout (KO)] BMDMΦ were analyzed 24 h post-infection to determine the level of IL-1β and IL-1α cytokines using Legendplex kits (BioLegend) followed by flow cytometry. (B) Bacterial burdens in infected BMDMΦ were evaluated 0.5, 2, 24, and 48 h post-infection by *GltA* reverse transcription quantitative real-time PCR (RT-qPCR). Expression of the host housekeeping gene *GAPDH* was used for normalization. (C) Bacterial burdens in *Rickettsia*-infected ATG5^fl/fl or ATG5^fl/fl-LysM-Cre treated with anti-IL-1α, IL-1β, both anti-IL-1α and anti-IL-1β antibodies, or IgG isotype controls. Samples were evaluated at 24 h post-infection by *GltA* RT-qPCR. Expression of the host housekeeping gene *GAPDH* was used for normalization. Error bars (**A–C**) represent means ± standard error of the mean (SEM) from five independent experiments; NS, non-significant; *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.005. (D) Proposed working model on how pathogenic *Rickettsia* spp. initiate autophagy, evade autolysosomal destruction, and suppress IL-1 cytokine responses to establish an intracytosolic replication niche in macrophages. Of note, a sizable amount of non-pathogenic *Rickettsia* is likely destroyed by phagolysosomal fusion, while a subpopulation may escape lysosomal fusion, ultimately resulting in the induction of IL-1 responses.

Collectively, the data presented here are in agreement with prior reports from our laboratory and others (22, 23, 30, 32), further confirming that the intracytosolic survival of R. typhi and R. rickettsii (SS), but not R. montanensis, in macrophages requires the activation of autophagy in an ATG5-/Beclin1-dependent manner, escape from autolysosomal destruction, and inhibition of IL-1α and IL-1β cytokines responses (Fig. 4D).

DISCUSSION

Various intracellular bacterial pathogens employ sophisticated mechanisms to hijack host cellular processes to facilitate their host survival. Such strategies entail reprogramming host phosphoinositide metabolism, which can facilitate uptake into host cells, modify phagosomes, undercut apoptosis, and interfere with other cellular defense mechanisms, including inflammasomes and autophagy. However, in the case of rickettsiae, the mechanisms by which these intracytosolic pathogens modulate both inflammasome and autophagy responses to facilitate their replication in endothelial cells and immune cells, like macrophages, are only now emerging (22 – 25, 30, 32 – 35, 40, 43). In fact, recent findings from others and our laboratory have provided some intriguing findings that may suggest that Rickettsia spp. exhibit species-specific inflammasome-mediated immune responses to establish an intracellular niche (22 – 24, 32, 34). However, the precise role of autophagy in regulating host colonization by Rickettsia spp. remains to be determined (22 – 25). For instance, R. australis benefited from autophagy induction and reduction of pro-inflammatory cytokine responses (22, 23), while R. parkeri evaded autophagic responses to colonize the host (24, 25). Our recent reporting on R. typhi showed that ubiquitination followed by autophagy induction, and the escape from autolysosomal destruction, were crucial steps for R. typhi to colonize non-phagocytic cells (30). Based on these findings from others and our laboratory, we tested the hypothesis that both pathogenic R. typhi and R. rickettsii (SS), but not the non-pathogenic R. montanensis, induce autophagy, evade autolysosomal destruction, and reduce microbicidal pro-inflammatory IL-1 responses to establish an intracytosolic replication niche in host immune defense cells, like macrophages.

Our data revealed that, using partially purified rickettsiae, R. montanensis is not ubiquitinated, while both R. typhi and R. rickettsii (SS) are ubiquitinated, during infection of macrophages. Ubiquitination of R. typhi and R. rickettsii (SS) is in agreement with infection studies using the Renografin-purified R. australis spp. (23). In contrast, no ubiquitination of partially purified R. montanensis was observed in our assays, which is in agreement with reports using the mild-pathogenic R. parkeri spp., where a MD-76R (overlaid or step gradient)-purified bacterium was used for ubiquitination studies (24, 25). Collectively, these findings by our laboratory and others suggest that variations in the intracellular behavior of Rickettsia spp. are likely due to species-specific strategies employed by the bacteria and not because of differences in the purification methodologies. However, the precise mechanism of these phenotypic differences on rickettsial ubiquitination requires further investigation. In addition, our data demonstrated that, unlike R. montanensis, both R. typhi and R. rickettsii (SS) spp. induced autophagy and evaded lysosomal destruction during macrophage invasion. Importantly, our presented data using ATG5-/Beclin1-deficient macrophages further strengthen our hypothesis that R. typhi and R. rickettsii (SS) utilize autophagy for establishing an intracytosolic replication niche in macrophages (Fig. 4D).

Our findings also raised another intriguing question as to why both R. typhi and R. rickettsii (SS) spp. seem to possess the ability to actively induce autophagy, in contrast to R. montanensis. Furthermore, our data suggest that both R. typhi and R. rickettsii (SS) spp. can efficiently escape from phagolysosomal destruction, become ubiquitinated, and induce autophagy. However, R. montanensis localized with Lamp2⁺-lysosomes and failed to evade lysosomal destruction, a phenotype previously observed in THP-1 cells (36, 37), suggesting that the phagolysosomal maturation likely contributes to the lack of ubiquitination and autophagic recognition of R. montanensis. Also, as Rickettsia-specific immunodominant outer membrane proteins (e.g., Scas) are predicted to be expressed on either pathogenic or non-pathogenic Rickettsia spp. (44), another factor to consider is the presence of an effector repertoire that differs depending on the rickettsial virulence. In fact, we recently reported that R. typhi induces autophagy upon infection of non-phagocytic cells, while subsequently avoiding autolysosomal destruction, via the function of secreted effectors, including phosphatidylinositol 3-kinase Risk1 and phospholipases Pat1 and Pat2 (30, 44). This mechanism of host invasion seems consistent with rickettsiae close relatives, such as Anaplasma phagocytophilum and Ehrlichia chaffeensis (45 – 48), or other intracellular pathogens such as Shigella (49, 50). So, it is conceivable that effectors (e.g., Risk1 and Pat1/2 [30, 44]), either alone or in combination with other currently yet to be identified effectors, could account for the observed phenotypic differences in host colonization among Rickettsia spp. (R. montanensis, R. typhi, and R. rickettsii [SS]). The precise mechanism and composition of the effector repertoire for each Rickettsia spp. are currently a matter of active research and still remain to be determined.

Our preceding report suggest that IL-1 signaling responses played a role in limiting rickettsial infection in vivo and in vitro (32). Specifically, we observed that both R. typhi and R. rickettsii (SS), but not R. montanensis, blocked cell death and reduced non-canonical inflammasome-dependent IL-1α responses, in order to establish an intracytosolic replication niche in macrophages. In line with these findings, we now report that the R. montanensis, but not R. typhi and R. rickettsii (SS), is efficiently cleared by macrophages, a mechanism supported by previously published findings with various SFG Rickettsia spp. using THP-1 cells (36, 37). Another intriguing observation is that the survival of both R. typhi and R. rickettsii (SS) was restricted in ATG5- or Beclin1-deficient macrophages, as evident by their localization with destructive Lamp2⁺-lysosomes and the increase in pro-inflammatory IL-1 cytokine response. These findings suggests that activation of autophagy is critical for the intracellular survival of both R. typhi and R. rickettsii (SS), likely via an autophagy-mediated proteasomal degradation of pro-inflammatory IL-1 cytokines and/or signaling components/molecules involved in their induction (17 – 19), a mechanism supporting the report on R. australis (23). However, the precise mechanism on how the intracellular survival of R. typhi and R. rickettsii (SS) benefits from the activation of an ATG5-/Beclin1-dependent autophagy pathway to limit pro-inflammatory IL-1 cytokine responses remains unclear and requires further investigation. Given the data presented in this manuscript, our recent report (30, 32), and published reports by others (13, 14, 17, 19, 22, 23), we now present a working model of host invasion by which both pathogenic R. typhi and R. rickettsii (SS) become ubiquitinated, induce autophagy, avoid autolysosomal destruction, and reduce microbicidal IL-1 cytokine responses to establish an intracytosolic niche in macrophages (Fig. 4D).

MATERIALS AND METHODS

Antibodies and reagents

Mono- and polyubiquitinylated conjugated antibody (clone: FK1) (anti-Ub) was obtained from Enzo Life Sciences. Anti-LC3B (E5Q2K) antibody was from Cell Signaling Technology. Antibodies against whole Rickettsia (SFG or TG) were raised in-house. Antibody against rickettsial cytoplasmic protein elongation factor Ts (EF-Ts) was obtained from Primm Biotech as previously described (30). The p62/SQSTM1 antibody was purchased from Sigma. Lamp2 (H4B4) and GAPDH (FL-335) antibodies were from Santa Cruz Biotechnology. ProLong Gold antifade mounting medium with DAPI (4´,6-diamidino-2-phenylindole), Halt protease and phosphatase inhibitor cocktail, paraformaldehyde (PFA), and Alexa 488/594-conjugated secondary antibodies were purchased from Thermo Fisher Scientific.

Bacterial strains, cell culture, and infection

Vero76 cells (African green monkey kidney, American Type Culture Collection [ATCC], RL-1587) were maintained in minimal Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37°C with 5% CO₂. R. montanensis (M5/6) and R. rickettsii (SS) strains were obtained from Dr. Ted Hackstadt (Rocky Mountain Laboratories, NIH, MT, USA), while the R. typhi strain (Wilmington) was obtained from CDC. All Rickettsia strains were propagated in Vero76 cells grown in DMEM supplemented with 5% FBS at 34°C with 5% CO₂. All Rickettsia strains were partially purified as described previously (27, 51). Partially purified R. montanensis, R. rickettsii (SS), and R. typhi were used for infection of BMDMΦ at 34°C. At early stages of infection (before the doubling time [8 to 10 h]) of rickettsiae), a higher multiplicity of infection (MOI) of 20 (for 0.5 and 2 h post-infection [hpi]) was used to ensure the presence of sufficient number of bacteria, as compared to MOI of 5 at later time points (24 and 48 hpi), to determine the biological functions of the bacteria during host infection (26 – 28, 30, 32).

Differentiation of bone marrow-derived macrophages

Femurs and tibias from 8- to 10-week-old female C57BL/6J WT mice were obtained as described previously (32, 52). Femurs and tibias from female C57BL/6J conditional gene knockout mice, Atg5^fl/fl-LysM-Cre and Beclin-1^fl/fl-LysM-Cre mice, in which the Atg5 or Beclin1 gene was deleted from myeloid cells (mainly monocyte/macrophages), as well as their corresponding WT littermates (ATG5^fl/fl or Beclin1^fl/fl), were provided by Dr. Christina Stallings (Washington University School of Medicine, MO, USA) (41). Isolated bone marrow cells were differentiated in RPMI 1640 medium supplemented with 10% FBS and 30% L929-conditioned medium (a source of macrophage colony stimulating factor) and cultured for 7 days as described previously (32, 52).

Measurement of cytokines and chemokines

IL-1 cytokine concentrations in supernatants from cultured BMDMΦ were assessed using the Legendplex mouse inflammation kit (BioLegend) following the manufacturer’s instructions as described previously (32, 52).

RNA isolation and quantitative real-time PCR

To determine viable bacterial number during the course of host infection, we performed Real time-quantitative polymerase chain reaction (RT-qPCR) assay on isolated RNA (26, 53, 54). In this effort, BMDMΦ samples were collected at 0.5, 2, 24, and 48 h post-infection. RNA was extracted from 1 × 10⁶ BMDMΦ using the Quick-RNA miniprep kit (Zymo Research). The iScript Reverse Transcription Supermix kit (Bio-Rad; 1708841) was used to synthesize cDNAs from 200 ng of RNA according to the manufacturer’s instructions. Quantitative real-time PCR was performed using SYBR Green (Thermo Fisher Scientific), 2 µL cDNA, and 1 µM each of the following oligonucleotides for rickettsial (housekeeping) citrate synthase gene (GltA), and host (housekeeping) GAPDH gene. Relative copy number (RCN) of GltA expression was normalized by the expression of the GAPDH and calculated with the equation RCN = E ^−ΔCt, where E = efficiency of PCR and Ct = Ct target − Ct GAPDH. Melting curve analyses were performed at the end of each run to ensure that only one product was amplified as described previously (32, 55).

Immunofluorescence

Eight-well chamber slides were seeded with BMDMΦ (30–50 × 10⁴ cells per well) and infected with partially purified pathogenic and non-pathogenic Rickettsia spp. (MOI = 20 [0.5 and 2 h] and MOI = 5 [24 and 48 h]) as described previously (26 – 28, 30, 32). Briefly, partially purified rickettsiae were added to BMDMΦ and incubated for various lengths of time at 34°C with 5% CO₂. Following incubation, cells were washed three times with 1× phosphate-buffered saline (PBS) and fixed with 4% PFA for 20 min at room temperature. Cells were than permeabilized in blocking buffer (0.3% saponin and 0.5% normal goat serum in 1× PBS) for 30 min and incubated for 1 h with the following primary antibodies diluted in antibody dilution buffer (0.3% saponin in 1× PBS): anti-Rickettsia (1:100 or 1:500), anti-Ub (1:100), anti-LC3B (1:100), and anti-Lamp2 (1:100). Cells were then washed with 1× PBS and incubated for 1 h with anti-Alexa Fluor 488 or anti-Alexa Fluor 594 secondary antibodies diluted 1:1,500 in antibody dilution buffer. Next, cells were washed with 1× PBS and mounted with ProLong Gold antifade mounting medium containing DAPI. Images were acquired using the Nikon W-1 spinning disk confocal microscope (University of Maryland Baltimore, Confocal Core Facility) and degree of colocalization (yellow⁺-stained bacteria) between Rickettsia and ubiquitin; LC3B or Lamp2 was analyzed using Fiji software as described previously (30). Quantification of the mean percentage of cellular marker positive bacteria was calculated from approximately 100 host cells for each experiment, while each experiment was repeated five times. The percentage of internalized bacteria (approximately 100–200 bacteria were counted per strain and time point) was calculated by dividing the number of extracellular bacteria by the total number of bacteria, multiplying by 100, and then subtracting this number from 100% to get the percentage of intracellular bacteria.

Extract preparation and Western blot analysis

Rickettsia-infected BMDMΦ cells were lysed for 2 h at 4°C in ice-cold lysis buffer (50 mM HEPES [pH 7.4], 137 mM NaCl, 10% glycerol, 1 mM EDTA, 0.5% NP-40, and supplemented with protease and phosphatase inhibitory cocktails) as described previously (30, 32). Equal amounts of protein were loaded for SDS-PAGE and membranes were probed with anti-p62, anti-LC3B, anti-GAPDH, and anti-EF-Ts antibodies, followed by enhanced chemiluminescence with secondary antibodies conjugated to horseradish peroxidase.

Neutralization of IL-1α and IL-1β in vitro

For in vitro neutralization of IL-1α and IL-1β, R. typhi-, R. rickettsii (SS)-, or R. montanensis-infected BMDMΦ from C57BL/6J ATG5^fl/fl or ATG5^fl/fl-LysM-Cre mice were treated with 100 nM anti-IL-1α (clone ALF-161; BioXCell), anti-IL-1β (clone B122; BioXCell), or IgG isotype control (Armenian hamster IgGs; BioXCell) antibodies for 24 h, and bacterial burden was assessed by quantitative real-time PCR.

Statistical analysis

The statistical significance was assessed using analysis of variance with Bonferroni’s procedure and Student’s t-test (GraphPad Prism Software, version 8). Data are presented as mean ± standard error of the mean (SEM), unless stated otherwise. Alpha level was set to 0.05.

ACKNOWLEDGMENTS

We gratefully acknowledge Christina Stallings (Washington University School of Medicine, MO, USA) and Ted Hackstadt (Rocky Mountain Laboratories, NIH, MT, USA) for generously providing us with essential biological specimens and reagents, including femurs from various knockout mice and rickettsial strains. We would like to thank Magda Beier-Sexton for her administrative, technical, organizational, and editorial contributions to the manuscript.

This work was supported with funds from the NIAID/NIH grants (R01AI017828 and R01AI126853 to A.F.A., and R21AI166821 to O.H.V. and M.S.R.).

O.H.V., M.S.R., and A.F.A. planned the research, analyzed, and interpreted the data; O.H.V., H.G., M.S., S.U., I.M., and M.S.R. performed the experiments; A.F.A. and O.H.V. contributed to the overall project administration and supervision; O.H.V., M.S.R., and A.F.A. wrote the manuscript; and all authors participated in editing the manuscript.

The funding sources had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

Contributor Information

Oliver H. Voss, Email: ovoss@som.umaryland.edu.

Abdu F. Azad, Email: aazad@som.umaryland.edu.

Artem S. Rogovskyy, Texas A&M University, College Station, Texas, USA

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/spectrum.02791-23.

Fig. S1. spectrum.02791-23-s0001.eps.

Treatment with a late-stage autophagy inhibitor, bafilomycin A1, results in an increase in LC3B level during rickettsiae invasion.

Click here for additional data file.^{(2.7MB, eps)}

DOI: 10.1128/spectrum.02791-23.SuF1

Fig. S2. spectrum.02791-23-s0002.eps.

LC3B and Lamp2 staining in uninfected ATG5^fl/fl and ATG5^fl/fl-LysM-Cre macrophages.

Click here for additional data file.^{(8.5MB, eps)}

DOI: 10.1128/spectrum.02791-23.SuF2

Fig. S3. spectrum.02791-23-s0003.eps.

Beclin1-dependent autophagy is involved in the intracytosolic survival of both R. rickettsii (SS) and R. typhi species in macrophages.

Click here for additional data file.^{(14.3MB, eps)}

DOI: 10.1128/spectrum.02791-23.SuF3

Fig. S4. spectrum.02791-23-s0004.eps.

Assessment of IL-1α and IL-1β cytokine responses and intracellular survival of R. rickettsii (SS), R. typhi, and R. montanensis species in Beclin1^fl/fl and Beclin1^fl/fl-LysM-Cre macrophages.

Click here for additional data file.^{(2.2MB, eps)}

DOI: 10.1128/spectrum.02791-23.SuF4

Fig. S5. spectrum.02791-23-s0005.eps.

Assessment of IL-1α and IL-1β cytokine responses in Rickettsia-infected ATG5^fl/fl and ATG5^fl/fl-LysM-Cre macrophages treated with IL-1α, IL-1β, or IgG-isotype control antibodies.

Click here for additional data file.^{(2.3MB, eps)}

DOI: 10.1128/spectrum.02791-23.SuF5

ASM does not own the copyrights to Supplemental Material that may be linked to, or accessed through, an article. The authors have granted ASM a non-exclusive, world-wide license to publish the Supplemental Material files. Please contact the corresponding author directly for reuse.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. spectrum.02791-23-s0001.eps.

Treatment with a late-stage autophagy inhibitor, bafilomycin A1, results in an increase in LC3B level during rickettsiae invasion.

Click here for additional data file.^{(2.7MB, eps)}

DOI: 10.1128/spectrum.02791-23.SuF1

Fig. S2. spectrum.02791-23-s0002.eps.

LC3B and Lamp2 staining in uninfected ATG5^fl/fl and ATG5^fl/fl-LysM-Cre macrophages.

Click here for additional data file.^{(8.5MB, eps)}

DOI: 10.1128/spectrum.02791-23.SuF2

Fig. S3. spectrum.02791-23-s0003.eps.

Beclin1-dependent autophagy is involved in the intracytosolic survival of both R. rickettsii (SS) and R. typhi species in macrophages.

Click here for additional data file.^{(14.3MB, eps)}

DOI: 10.1128/spectrum.02791-23.SuF3

Fig. S4. spectrum.02791-23-s0004.eps.

Click here for additional data file.^{(2.2MB, eps)}

DOI: 10.1128/spectrum.02791-23.SuF4

Fig. S5. spectrum.02791-23-s0005.eps.

Assessment of IL-1α and IL-1β cytokine responses in Rickettsia-infected ATG5^fl/fl and ATG5^fl/fl-LysM-Cre macrophages treated with IL-1α, IL-1β, or IgG-isotype control antibodies.

Click here for additional data file.^{(2.3MB, eps)}

DOI: 10.1128/spectrum.02791-23.SuF5

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PERMALINK

Autophagy facilitates intracellular survival of pathogenic rickettsiae in macrophages via evasion of autophagosomal maturation and reduction of microbicidal pro-inflammatory IL-1 cytokine responses

Oliver H Voss

Hodalis Gaytan

Saif Ullah

Mohammad Sadik

Imran Moin

M Sayeedur Rahman

Abdu F Azad

Roles

ABSTRACT

IMPORTANCE

INTRODUCTION

RESULTS

Pathogenic rickettsiae are ubiquitinated and induce autophagy upon entry into macrophages

Fig 1.

Pathogenic rickettsiae avoid autolysosomal destruction to establish a replicative niche in macrophages

Fig 2.

Intracytosolic survival of pathogenic rickettsiae requires induction of autophagy and reduction of microbicidal pro-inflammatory IL-1 cytokine responses

Fig 3.

Fig 4.

DISCUSSION

MATERIALS AND METHODS

Antibodies and reagents

Bacterial strains, cell culture, and infection

Differentiation of bone marrow-derived macrophages

Measurement of cytokines and chemokines

RNA isolation and quantitative real-time PCR

Immunofluorescence

Extract preparation and Western blot analysis

Neutralization of IL-1α and IL-1β in vitro

Statistical analysis

ACKNOWLEDGMENTS

Contributor Information

SUPPLEMENTAL MATERIAL

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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