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Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion.

Rennoll-Bankert KE, Rahman MS, Gillespie JJ, Guillotte ML, Kaur SJ, Lehman SS, Beier-Sexton M, Azad AF - PLoS Pathog. (2015)

Bottom Line: RalF is expressed early during infection, with its inactivation via antibody blocking significantly reducing R. typhi host cell invasion.Collectively, our characterization of RalF as an invasin suggests that, despite carrying a similar Arf-GEF unknown from other bacteria, different intracellular lifestyles across Rickettsia and Legionella species have driven divergent roles for RalF during infection.Furthermore, our identification of lineage-specific Arf-GEF utilization across some rickettsial species illustrates different pathogenicity factors that define diverse agents of rickettsial diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Bacterial Sec7-domain-containing proteins (RalF) are known only from species of Legionella and Rickettsia, which have facultative and obligate intracellular lifestyles, respectively. L. pneumophila RalF, a type IV secretion system (T4SS) effector, is a guanine nucleotide exchange factor (GEF) of ADP-ribosylation factors (Arfs), activating and recruiting host Arf1 to the Legionella-containing vacuole. In contrast, previous in vitro studies showed R. prowazekii (Typhus Group) RalF is a functional Arf-GEF that localizes to the host plasma membrane and interacts with the actin cytoskeleton via a unique C-terminal domain. As RalF is differentially encoded across Rickettsia species (e.g., pseudogenized in all Spotted Fever Group species), it may function in lineage-specific biology and pathogenicity. Herein, we demonstrate RalF of R. typhi (Typhus Group) interacts with the Rickettsia T4SS coupling protein (RvhD4) via its proximal C-terminal sequence. RalF is expressed early during infection, with its inactivation via antibody blocking significantly reducing R. typhi host cell invasion. For R. typhi and R. felis (Transitional Group), RalF ectopic expression revealed subcellular localization with the host plasma membrane and actin cytoskeleton. Remarkably, R. bellii (Ancestral Group) RalF showed perinuclear localization reminiscent of ectopically expressed Legionella RalF, for which it shares several structural features. For R. typhi, RalF co-localization with Arf6 and PI(4,5)P2 at entry foci on the host plasma membrane was determined to be critical for invasion. Thus, we propose recruitment of PI(4,5)P2 at entry foci, mediated by RalF activation of Arf6, initiates actin remodeling and ultimately facilitates bacterial invasion. Collectively, our characterization of RalF as an invasin suggests that, despite carrying a similar Arf-GEF unknown from other bacteria, different intracellular lifestyles across Rickettsia and Legionella species have driven divergent roles for RalF during infection. Furthermore, our identification of lineage-specific Arf-GEF utilization across some rickettsial species illustrates different pathogenicity factors that define diverse agents of rickettsial diseases.

No MeSH data available.


Related in: MedlinePlus

R. typhi RalFRt interacts with RvhD4 and is expressed early during host cell invasion.(A) Bacterial two-hybrid (B2H) assay reveals an interaction between RalFRt and RvhD4. ralFRtFL and ralFRtΔT4S were cloned into pTRG (prey) and rvhD4 was cloned into pBT (bait) of the B2H system. Constructed bait and prey plasmids were co-transformed into BacterioMatch II reporter electro-competent cells. Transformants were screened on non-selective plate (left) and positive interactions were identified on dual selective screening plate (right). The amino acid sequence deleted from ralFRtΔT4S (positively charged residues are colored blue) is shown at bottom. (B) Quantification of bacterial growth in the B2H assay described in panel A. Percent growth of CFUs of reporter cells harboring recombinant plasmids on dual selective screening medium was calculated relative to CFUs obtained on non-selective medium. Error bars represent mean ± SD of three independent experiments (Student’s two-sided t-test). (C) R. typhi RvhD4 exhibits ATPase activity. A series dilution of purified RvhD4 in assay buffer was incubated with reagent for 30 min at 21°C. The inorganic phosphate (Pi) released from ATP was quantified by measuring absorbance at OD 620 nm. As a negative control, a non-related R. typhi protein (RT0600) was assayed. Error bars represent mean ± SD of three independent experiments. * p = 0.01, **** p<0.0001; Student’s two-sided t-test. (D) Protein immunoblot of recombinant RvhD4 (~64 kDa) used in ATPase activity assays described in panel C. (E) RalFRt is surface exposed. Purified R. typhi was treated with 400 μg/mL or 800 μg/mL Proteinase K or in buffer alone for 1 hr. Lysates were resolved and immunoblotted for RalF or the R. typhi cytoplasmic control protein, elongation factor Ts (EF-Ts). Densitometry was performed using ImageJ and the intensity of RalF was normalized to EF-Ts. Representative image from two independent experiments is shown. Intensity of RalF normalized to EF-Ts and relative to untreated control is shown below the immunoblots. (F) RalF is expressed during early infection. HeLa cells infected with R. typhi for 10 and 30 min were fixed and R. typhi and RalF detected with rat anti-R. typhi (red) and affinity purified rabbit anti-RalFRt (green) antibodies, respectively. DAPI (blue) is shown in the merged image. Boxed regions are enlarged to show detail. Pre-immune (PI) cells were treated with rabbit PI serum in place of anti-RalFRt antibody. (Scale bar: 10 μm). (G) Anti-RalFRt IgG and Fab fragments inhibit R. typhi host cell infection. HeLa cells were infected with partially purified R. typhi pre-absorbed for 30 min with 20μg PI IgG serum, anti-RalFRt IgG, PI Fab fragments or anti-RalFRt Fab fragments. Cells were fixed 2 hrs post infection and R. typhi and the cell membrane detected with anti-R. typhi serum and Alexa Fluor 594 wheat germ agglutinin, respectively. The number of R. typhi per host cell was counted for 100 individual host cells in three independent experiments and normalized to PI serum. Error bars represent mean ± SD (Student’s two-sided t-test).
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ppat.1005115.g001: R. typhi RalFRt interacts with RvhD4 and is expressed early during host cell invasion.(A) Bacterial two-hybrid (B2H) assay reveals an interaction between RalFRt and RvhD4. ralFRtFL and ralFRtΔT4S were cloned into pTRG (prey) and rvhD4 was cloned into pBT (bait) of the B2H system. Constructed bait and prey plasmids were co-transformed into BacterioMatch II reporter electro-competent cells. Transformants were screened on non-selective plate (left) and positive interactions were identified on dual selective screening plate (right). The amino acid sequence deleted from ralFRtΔT4S (positively charged residues are colored blue) is shown at bottom. (B) Quantification of bacterial growth in the B2H assay described in panel A. Percent growth of CFUs of reporter cells harboring recombinant plasmids on dual selective screening medium was calculated relative to CFUs obtained on non-selective medium. Error bars represent mean ± SD of three independent experiments (Student’s two-sided t-test). (C) R. typhi RvhD4 exhibits ATPase activity. A series dilution of purified RvhD4 in assay buffer was incubated with reagent for 30 min at 21°C. The inorganic phosphate (Pi) released from ATP was quantified by measuring absorbance at OD 620 nm. As a negative control, a non-related R. typhi protein (RT0600) was assayed. Error bars represent mean ± SD of three independent experiments. * p = 0.01, **** p<0.0001; Student’s two-sided t-test. (D) Protein immunoblot of recombinant RvhD4 (~64 kDa) used in ATPase activity assays described in panel C. (E) RalFRt is surface exposed. Purified R. typhi was treated with 400 μg/mL or 800 μg/mL Proteinase K or in buffer alone for 1 hr. Lysates were resolved and immunoblotted for RalF or the R. typhi cytoplasmic control protein, elongation factor Ts (EF-Ts). Densitometry was performed using ImageJ and the intensity of RalF was normalized to EF-Ts. Representative image from two independent experiments is shown. Intensity of RalF normalized to EF-Ts and relative to untreated control is shown below the immunoblots. (F) RalF is expressed during early infection. HeLa cells infected with R. typhi for 10 and 30 min were fixed and R. typhi and RalF detected with rat anti-R. typhi (red) and affinity purified rabbit anti-RalFRt (green) antibodies, respectively. DAPI (blue) is shown in the merged image. Boxed regions are enlarged to show detail. Pre-immune (PI) cells were treated with rabbit PI serum in place of anti-RalFRt antibody. (Scale bar: 10 μm). (G) Anti-RalFRt IgG and Fab fragments inhibit R. typhi host cell infection. HeLa cells were infected with partially purified R. typhi pre-absorbed for 30 min with 20μg PI IgG serum, anti-RalFRt IgG, PI Fab fragments or anti-RalFRt Fab fragments. Cells were fixed 2 hrs post infection and R. typhi and the cell membrane detected with anti-R. typhi serum and Alexa Fluor 594 wheat germ agglutinin, respectively. The number of R. typhi per host cell was counted for 100 individual host cells in three independent experiments and normalized to PI serum. Error bars represent mean ± SD (Student’s two-sided t-test).

Mentions: As predicted Arf-GEFs, we anticipated RalFR proteins to be secreted extracellularly into the host cell. Prior to invasion, L. pneumophila utilizes its dot/icm I-T4SS to translocate RalFL into host cells [48]. Like RalFL, R. typhi RalF (RalFRt) lacks a predicted N-terminal Sec secretion signal [58], trans-membrane spanning regions [59] and a β-barrel structure [60], suggesting its secretion via a Sec-independent pathway, possibly the Rickettsiales vir homolog (rvh) T4SS [61]. Accordingly, in order to determine if RalFRt interacts with the rvh T4SS, we performed a bacterial two-hybrid assay with full length RalFRt (RalFRtFL) and RvhD4, the rvh T4SS coupling protein. T4SS coupling proteins (VirD4 family) are ATPases that function as “gatekeepers” to regulate substrate entry into the T4SS channel [62,63]. Co-transformation of bait (encoding RvhD4) and prey (encoding RalFRtFL) plasmids in BacterioMatch II reporter electrocompetent cells resulted in bacterial growth on selective media (Fig 1A), indicating RalFRtFL and RvhD4 interact, and thus implicating RalFRt as an rvh T4SS effector.


Which Way In? The RalF Arf-GEF Orchestrates Rickettsia Host Cell Invasion.

Rennoll-Bankert KE, Rahman MS, Gillespie JJ, Guillotte ML, Kaur SJ, Lehman SS, Beier-Sexton M, Azad AF - PLoS Pathog. (2015)

R. typhi RalFRt interacts with RvhD4 and is expressed early during host cell invasion.(A) Bacterial two-hybrid (B2H) assay reveals an interaction between RalFRt and RvhD4. ralFRtFL and ralFRtΔT4S were cloned into pTRG (prey) and rvhD4 was cloned into pBT (bait) of the B2H system. Constructed bait and prey plasmids were co-transformed into BacterioMatch II reporter electro-competent cells. Transformants were screened on non-selective plate (left) and positive interactions were identified on dual selective screening plate (right). The amino acid sequence deleted from ralFRtΔT4S (positively charged residues are colored blue) is shown at bottom. (B) Quantification of bacterial growth in the B2H assay described in panel A. Percent growth of CFUs of reporter cells harboring recombinant plasmids on dual selective screening medium was calculated relative to CFUs obtained on non-selective medium. Error bars represent mean ± SD of three independent experiments (Student’s two-sided t-test). (C) R. typhi RvhD4 exhibits ATPase activity. A series dilution of purified RvhD4 in assay buffer was incubated with reagent for 30 min at 21°C. The inorganic phosphate (Pi) released from ATP was quantified by measuring absorbance at OD 620 nm. As a negative control, a non-related R. typhi protein (RT0600) was assayed. Error bars represent mean ± SD of three independent experiments. * p = 0.01, **** p<0.0001; Student’s two-sided t-test. (D) Protein immunoblot of recombinant RvhD4 (~64 kDa) used in ATPase activity assays described in panel C. (E) RalFRt is surface exposed. Purified R. typhi was treated with 400 μg/mL or 800 μg/mL Proteinase K or in buffer alone for 1 hr. Lysates were resolved and immunoblotted for RalF or the R. typhi cytoplasmic control protein, elongation factor Ts (EF-Ts). Densitometry was performed using ImageJ and the intensity of RalF was normalized to EF-Ts. Representative image from two independent experiments is shown. Intensity of RalF normalized to EF-Ts and relative to untreated control is shown below the immunoblots. (F) RalF is expressed during early infection. HeLa cells infected with R. typhi for 10 and 30 min were fixed and R. typhi and RalF detected with rat anti-R. typhi (red) and affinity purified rabbit anti-RalFRt (green) antibodies, respectively. DAPI (blue) is shown in the merged image. Boxed regions are enlarged to show detail. Pre-immune (PI) cells were treated with rabbit PI serum in place of anti-RalFRt antibody. (Scale bar: 10 μm). (G) Anti-RalFRt IgG and Fab fragments inhibit R. typhi host cell infection. HeLa cells were infected with partially purified R. typhi pre-absorbed for 30 min with 20μg PI IgG serum, anti-RalFRt IgG, PI Fab fragments or anti-RalFRt Fab fragments. Cells were fixed 2 hrs post infection and R. typhi and the cell membrane detected with anti-R. typhi serum and Alexa Fluor 594 wheat germ agglutinin, respectively. The number of R. typhi per host cell was counted for 100 individual host cells in three independent experiments and normalized to PI serum. Error bars represent mean ± SD (Student’s two-sided t-test).
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ppat.1005115.g001: R. typhi RalFRt interacts with RvhD4 and is expressed early during host cell invasion.(A) Bacterial two-hybrid (B2H) assay reveals an interaction between RalFRt and RvhD4. ralFRtFL and ralFRtΔT4S were cloned into pTRG (prey) and rvhD4 was cloned into pBT (bait) of the B2H system. Constructed bait and prey plasmids were co-transformed into BacterioMatch II reporter electro-competent cells. Transformants were screened on non-selective plate (left) and positive interactions were identified on dual selective screening plate (right). The amino acid sequence deleted from ralFRtΔT4S (positively charged residues are colored blue) is shown at bottom. (B) Quantification of bacterial growth in the B2H assay described in panel A. Percent growth of CFUs of reporter cells harboring recombinant plasmids on dual selective screening medium was calculated relative to CFUs obtained on non-selective medium. Error bars represent mean ± SD of three independent experiments (Student’s two-sided t-test). (C) R. typhi RvhD4 exhibits ATPase activity. A series dilution of purified RvhD4 in assay buffer was incubated with reagent for 30 min at 21°C. The inorganic phosphate (Pi) released from ATP was quantified by measuring absorbance at OD 620 nm. As a negative control, a non-related R. typhi protein (RT0600) was assayed. Error bars represent mean ± SD of three independent experiments. * p = 0.01, **** p<0.0001; Student’s two-sided t-test. (D) Protein immunoblot of recombinant RvhD4 (~64 kDa) used in ATPase activity assays described in panel C. (E) RalFRt is surface exposed. Purified R. typhi was treated with 400 μg/mL or 800 μg/mL Proteinase K or in buffer alone for 1 hr. Lysates were resolved and immunoblotted for RalF or the R. typhi cytoplasmic control protein, elongation factor Ts (EF-Ts). Densitometry was performed using ImageJ and the intensity of RalF was normalized to EF-Ts. Representative image from two independent experiments is shown. Intensity of RalF normalized to EF-Ts and relative to untreated control is shown below the immunoblots. (F) RalF is expressed during early infection. HeLa cells infected with R. typhi for 10 and 30 min were fixed and R. typhi and RalF detected with rat anti-R. typhi (red) and affinity purified rabbit anti-RalFRt (green) antibodies, respectively. DAPI (blue) is shown in the merged image. Boxed regions are enlarged to show detail. Pre-immune (PI) cells were treated with rabbit PI serum in place of anti-RalFRt antibody. (Scale bar: 10 μm). (G) Anti-RalFRt IgG and Fab fragments inhibit R. typhi host cell infection. HeLa cells were infected with partially purified R. typhi pre-absorbed for 30 min with 20μg PI IgG serum, anti-RalFRt IgG, PI Fab fragments or anti-RalFRt Fab fragments. Cells were fixed 2 hrs post infection and R. typhi and the cell membrane detected with anti-R. typhi serum and Alexa Fluor 594 wheat germ agglutinin, respectively. The number of R. typhi per host cell was counted for 100 individual host cells in three independent experiments and normalized to PI serum. Error bars represent mean ± SD (Student’s two-sided t-test).
Mentions: As predicted Arf-GEFs, we anticipated RalFR proteins to be secreted extracellularly into the host cell. Prior to invasion, L. pneumophila utilizes its dot/icm I-T4SS to translocate RalFL into host cells [48]. Like RalFL, R. typhi RalF (RalFRt) lacks a predicted N-terminal Sec secretion signal [58], trans-membrane spanning regions [59] and a β-barrel structure [60], suggesting its secretion via a Sec-independent pathway, possibly the Rickettsiales vir homolog (rvh) T4SS [61]. Accordingly, in order to determine if RalFRt interacts with the rvh T4SS, we performed a bacterial two-hybrid assay with full length RalFRt (RalFRtFL) and RvhD4, the rvh T4SS coupling protein. T4SS coupling proteins (VirD4 family) are ATPases that function as “gatekeepers” to regulate substrate entry into the T4SS channel [62,63]. Co-transformation of bait (encoding RvhD4) and prey (encoding RalFRtFL) plasmids in BacterioMatch II reporter electrocompetent cells resulted in bacterial growth on selective media (Fig 1A), indicating RalFRtFL and RvhD4 interact, and thus implicating RalFRt as an rvh T4SS effector.

Bottom Line: RalF is expressed early during infection, with its inactivation via antibody blocking significantly reducing R. typhi host cell invasion.Collectively, our characterization of RalF as an invasin suggests that, despite carrying a similar Arf-GEF unknown from other bacteria, different intracellular lifestyles across Rickettsia and Legionella species have driven divergent roles for RalF during infection.Furthermore, our identification of lineage-specific Arf-GEF utilization across some rickettsial species illustrates different pathogenicity factors that define diverse agents of rickettsial diseases.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland, United States of America.

ABSTRACT
Bacterial Sec7-domain-containing proteins (RalF) are known only from species of Legionella and Rickettsia, which have facultative and obligate intracellular lifestyles, respectively. L. pneumophila RalF, a type IV secretion system (T4SS) effector, is a guanine nucleotide exchange factor (GEF) of ADP-ribosylation factors (Arfs), activating and recruiting host Arf1 to the Legionella-containing vacuole. In contrast, previous in vitro studies showed R. prowazekii (Typhus Group) RalF is a functional Arf-GEF that localizes to the host plasma membrane and interacts with the actin cytoskeleton via a unique C-terminal domain. As RalF is differentially encoded across Rickettsia species (e.g., pseudogenized in all Spotted Fever Group species), it may function in lineage-specific biology and pathogenicity. Herein, we demonstrate RalF of R. typhi (Typhus Group) interacts with the Rickettsia T4SS coupling protein (RvhD4) via its proximal C-terminal sequence. RalF is expressed early during infection, with its inactivation via antibody blocking significantly reducing R. typhi host cell invasion. For R. typhi and R. felis (Transitional Group), RalF ectopic expression revealed subcellular localization with the host plasma membrane and actin cytoskeleton. Remarkably, R. bellii (Ancestral Group) RalF showed perinuclear localization reminiscent of ectopically expressed Legionella RalF, for which it shares several structural features. For R. typhi, RalF co-localization with Arf6 and PI(4,5)P2 at entry foci on the host plasma membrane was determined to be critical for invasion. Thus, we propose recruitment of PI(4,5)P2 at entry foci, mediated by RalF activation of Arf6, initiates actin remodeling and ultimately facilitates bacterial invasion. Collectively, our characterization of RalF as an invasin suggests that, despite carrying a similar Arf-GEF unknown from other bacteria, different intracellular lifestyles across Rickettsia and Legionella species have driven divergent roles for RalF during infection. Furthermore, our identification of lineage-specific Arf-GEF utilization across some rickettsial species illustrates different pathogenicity factors that define diverse agents of rickettsial diseases.

No MeSH data available.


Related in: MedlinePlus