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PMID:23950720
| Citation |
Myeni, S, Child, R, Ng, TW, Kupko, JJ 3rd, Wehrly, TD, Porcella, SF, Knodler, LA and Celli, J (2013) Brucella modulates secretory trafficking via multiple type IV secretion effector proteins. PLoS Pathog. 9:e1003556 |
|---|---|
| Abstract |
The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. |
| Links |
PubMed PMC3738490 Online version:10.1371/journal.ppat.1003556 |
| Keywords |
Animals; Bacterial Proteins/genetics; Bacterial Proteins/metabolism; Bacterial Secretion Systems/physiology; Brucella abortus/genetics; Brucella abortus/metabolism; Brucellosis/metabolism; Brucellosis/pathology; Female; HeLa Cells; Humans; Liver/metabolism; Liver/microbiology; Liver/pathology; Macrophages/metabolism; Macrophages/microbiology; Macrophages/pathology; Membrane Proteins/genetics; Membrane Proteins/metabolism; Mice; Mice, Inbred BALB C; Protein Transport/physiology; Vacuoles/genetics; Vacuoles/metabolism; Vacuoles/microbiology |
Significance
Annotations
| Gene product | Qualifier | GO Term | Evidence Code | with/from | Aspect | Extension | Notes | Status |
|---|---|---|---|---|---|---|---|---|
| GO:0044220: host cell perinuclear region of cytoplasm |
ECO:0000314: |
C |
Figure 3. Confocal micrographs of HeLa cells transfected with Brucella abortus effector protein BspE labeled with HA. BspE was observed forming discrete vesicles in the perinuclear area. |
complete | ||||
| GO:0042000: translocation of peptides or proteins into host |
ECO:0000314: |
P |
Figure 1. TEM1 b-lactamase protein translocation reporter assay. Panel A and B shows percentage of shift in fluorescence emission that correlates with protein Brucella putative effector BAB1_1671 being translocated into 774A.1 host cell (murine cell line). Panel C and D shows representative fluorescence micrographs of protein translocation. Also look Figure S1 and S2. Figure 2. VirB-dependent translocation of Brucella BsP effectors into J774.A1 cells. Panel A. B. abortus strain 2308 (black bars) or virB9 mutant strain (white bars) expressing the CyaA fusions to the indicated Brucella effector proteins were used to infect J774.A1 cells, and the cAMP level of infected cells was determined. |
complete | ||||
| GO:0044165: host cell endoplasmic reticulum |
ECO:0000314: |
C |
Figure 3. Confocal micrographs of HeLa cells transfected with Brucella abortus effector protein BspA expressing GFP. BspA localizes in the host ER. Figure 4. Shows co-localization of BspA with ER resident chaperone protein calnexin. |
complete | ||||
| GO:0052058: modification by symbiont of host morphology or physiology via substance secreted by type IV secretion system |
ECO:0000314: |
P |
Figure 2. VirB-dependent translocation of Brucella BsP effectors into J774.A1 cells. Shows effector protein BspA (BAB1_0678) translocated by T4SS. Figure 7. Contribution of BspA (BAB1_0678) to inhibition of host cell secretion during infection. |
complete | ||||
| GO:0044068: modulation by symbiont of host cellular process |
ECO:0000314: |
P |
Figure 2. VirB-dependent translocation of Brucella BsP effectors into J774.A1 cells. Shows effector protein BspA (BAB1_0678) translocated by T4SS. Figure 7. Contribution of BspA (BAB1_0678) to inhibition of host cell secretion during infection. |
complete | ||||
| GO:0044164: host cell cytosol |
ECO:0000314: |
C |
Figure 3. Confocal micrgraphs of HeLa transfected cells with Brucella abortus effector proten BspH expressing hemagglutinin display a cytosolic localization |
complete | ||||
| GO:0044164: host cell cytosol |
ECO:0000314: |
C |
Figure 3. Confocal micrographs of HeLa cells transfected with Brucella abortus effector protein BspI expressing hemagglutinin displayed cytosolic localization. |
complete | ||||
| GO:0044164: host cell cytosol |
ECO:0000314: |
C |
Figure 3. Confocal micrographs of HeLa cells transfected with Brucella abortus effector protein BspF expressing hemagglutinin displayed a cytosol localization |
complete | ||||
| GO:0020002: host cell plasma membrane |
ECO:0000314: |
C |
Figure 3. Confocal micrographs of HeLa cells transfected with Brucella abortus effector protein BspF expressing hemagglutinin displayed a host plasma membrane localization. |
complete | ||||
| GO:0052208: modification of morphology or physiology of other organism via substance secreted by type IV secretion system involved in symbiotic interaction |
ECO:0000314: |
P |
Figure 1. TEM1 b-lactamase protein translocation reporter assay. Shows translocation of effector protein BspF (BAB1_1948) Figure 2. VirB-dependent translocation of Brucella BsP effectors into J774.A1 cells. Shows effector protein BspF (BAB1_01948) translocated by T4SS. Figure 4. A subset of Bsp effectors target the host secretory pathway. Shows that effector protein BspF (BAB1_1948) targets secretory pathway by colocalization with calnexin a marker of ER. Figure 5. Ectopic expression of BspF (BAB1_1948) in HeLa cells interferes with the host secretory pathway. Figure 7. Contribution of BspF (BAB1_1948) to inhibition of host cell secretion during infection. |
complete | ||||
| GO:0044165: host cell endoplasmic reticulum |
ECO:0000314: |
C |
Figure 3. Confocal micrographs of HeLa cells transfected with Brucella abortus effector protein BspB expressing hemagglutinin displayed a localization in the ER. Figure 4. Shows co_localization of BspB with ER-resident chaperone protein calnexin confirmed its ER localization. |
complete | ||||
| GO:0052058: modification by symbiont of host morphology or physiology via substance secreted by type IV secretion system |
ECO:0000314: |
P |
Figure 2. VirB-dependent translocation of Brucella BsP effectors into J774.A1 cells. Shows effector protein BspB (BAB1_0712) translocated by T4SS. Figure 7. Contribution of BspB (BAB1_0712) to inhibition of host cell secretion during infection. |
complete | ||||
| GO:0044068: modulation by symbiont of host cellular process |
ECO:0000314: |
P |
Figure 7. Contribution of BspB (BAB1_0712) to inhibition of host cell secretion during infection. |
complete | ||||
| GO:0044165: host cell endoplasmic reticulum |
ECO:0000314: |
C |
Figure 3. Confocal micrographs of HeLa cells transfected with Brucella abortus effector protein BspD expressing GFP displayed a ER localization. Figure 4. Shows co-localization of BspD with ER-resident chaperon, calnexin confirming its localization. |
complete | ||||
| GO:0044165: host cell endoplasmic reticulum |
ECO:0000314: |
C |
Figure 3. Confocal micrographs of HeLa cells transfected with Brucella abortus effector protein BspK expressing hemagglutinin displayed a ER localization. Figure 4. Shows co-localiztion of BspK with ER-resident chaperone, calnexin that confirmed its ER localization. |
complete | ||||
| GO:0034976: response to endoplasmic reticulum stress |
ECO:0000314: |
P |
Figure S1. Panel B and C shows that, when ectopically expressed in mammalian cells (HEK 293T), BAB2_0541 expression induces significant ER stress to levels similar or higher than a positive control VceC and upregulation of BiP, but not CHOP. |
complete | ||||
| GO:0044177: host cell Golgi apparatus |
ECO:0000314: |
C |
Figure 3. Confocal micrographs of HeLa cells transfected with Brucella abortus effector protein BspC expressing GFP displayed a golgi apparatus localization. Figure 4. Shows co-localization of BspC with a protein resident in golgi GM130 that confirmed BspC golgi localization. |
complete | ||||
| GO:0042000: translocation of peptides or proteins into host |
ECO:0000314: |
P |
Figure 1. TEM1 b-lactamase protein translocation reporter assay. Panel A and B shows percentage of shift in fluorescence emission that correlates with protein Brucella putative effector BAB1_0847 being translocated into J774A.1 host cell (murine cell line). Panel C and D shows representative fluorescence micrographs of protein translocation. Also look Figure S1 and S2. Figure 2. VirB-dependent translocation of Brucella BsP effectors into J774.A1 cells. Panel A. B. abortus strain 2308 (black bars) or virB9 mutant strain (white bars) expressing the CyaA fusions to the indicated Brucella effector proteins were used to infect J774.A1 cells, and the cAMP level of infected cells was determined. |
complete | ||||
| GO:0034976: response to endoplasmic reticulum stress |
ECO:0000314: |
P |
Figure S1. Panel B and C shows that, when ectopically expressed in mammalian cells (HEK 293T), BAB1_0847 expression induces significant ER stress to levels similar or higher than a positive control VceC and upregulation of BiP, but not CHOP. |
complete | ||||
| GO:0034976: response to endoplasmic reticulum stress |
ECO:0000314: |
P |
Figure S1. Panel B and C shows that, when ectopically expressed in mammalian cells (HEK 293T), BAB1_0227 expression induces significant ER stress to levels similar or higher than a positive control VceC and upregulation of BiP, but not CHOP. |
complete | ||||
Notes
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