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Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

El-Awady AR, Miles B, Scisci E, Kurago ZB, Palani CD, Arce RM, Waller JL, Genco CA, Slocum C, Manning M, Schoenlein PV, Cutler CW - PLoS Pathog. (2015)

Bottom Line: Survival was decreased by activation of TLR2 and/or autophagy.Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1.These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Periodontics, Georgia Regents University, Augusta, Georgia, United States of America.

ABSTRACT
Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

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Blocking of DC-SIGN inhibits Mfa1+Pg uptake and restores basal LC3-II signal in MoDCs.A) Immuno-fluorescence images of LC3-II and P. gingivalis within MoDCs pre-treated with GP120 (DC-SIGN blocker) after 12 hours of infection. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE. B) Fluorescent intensity of LC3-II and P. gingivalis strains signal (in triplicates) were statistically analyzed (* and # p<0.001). C) Flow cytometry of MoDCs treated with siRNA for DC-SIGN (si-DC-SIGN) after 12 hours. Left panels show the decrease of DC-SIGN in Cont. (un-infected), Pg381 and Mfa1+Pg-infected MoDCs. D) Epifluorescence microscopy images of MoDCs (si-DC-SIGN) infected with Pg381 and Mfa1+Pg. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE. E) The figure shows the CFU counts of the P. gingivalis strains with MoDCs that lack DC-SIGN (si-DC-SIGN). The analysis of readings used One-way ANOVA analysis of different groups and Tukey’s test for multiple comparisons (* p<0.001).
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ppat.1004647.g006: Blocking of DC-SIGN inhibits Mfa1+Pg uptake and restores basal LC3-II signal in MoDCs.A) Immuno-fluorescence images of LC3-II and P. gingivalis within MoDCs pre-treated with GP120 (DC-SIGN blocker) after 12 hours of infection. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE. B) Fluorescent intensity of LC3-II and P. gingivalis strains signal (in triplicates) were statistically analyzed (* and # p<0.001). C) Flow cytometry of MoDCs treated with siRNA for DC-SIGN (si-DC-SIGN) after 12 hours. Left panels show the decrease of DC-SIGN in Cont. (un-infected), Pg381 and Mfa1+Pg-infected MoDCs. D) Epifluorescence microscopy images of MoDCs (si-DC-SIGN) infected with Pg381 and Mfa1+Pg. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE. E) The figure shows the CFU counts of the P. gingivalis strains with MoDCs that lack DC-SIGN (si-DC-SIGN). The analysis of readings used One-way ANOVA analysis of different groups and Tukey’s test for multiple comparisons (* p<0.001).

Mentions: Infection of MoDCs by Mfa1+Pg has previously been shown to depend on engagement of DC-SIGN [14]. We confirmed this result by blocking DC-SIGN with HIV gp120, prior to infection (Fig. 6A and 6B). Blocking DC-SIGN increased the LC3-II signal in MoDCs prior to addition of Mfa1+Pg (Fig. 6B). Moreover, blocking DC-SIGN restored the basal expression of LC3-II in MoDCs (Fig. 6B). To confirm the HIV gp120 blocking experiments, we additionally knocked down DC-SIGN using siRNA. DC-SIGN knockdown inhibited uptake of Mfa1+Pg but not Pg381 (Fig. 6C and 6D) and restored LC3-II signals in MoDCs (Fig. 6C and 6D). Furthermore, DC-SIGN knockdown significantly decreased survival of Mfa1+Pg in MoDCs (Fig. 6E). A scrambled sequence control did not inhibit uptake or effect LC3-II signal. To confirm the contribution of actin-mediated endocytic trafficking in LC3-II induction, MoDCs were treated with cytochalasin-D (CytD) prior to infection. CytD significantly inhibited intracellular localization of both Pg381 and Mfa1+Pg by MoDCs and restored the LC3-II signals to the basal level in MoDCs (S7 Fig. C and D).


Porphyromonas gingivalis evasion of autophagy and intracellular killing by human myeloid dendritic cells involves DC-SIGN-TLR2 crosstalk.

El-Awady AR, Miles B, Scisci E, Kurago ZB, Palani CD, Arce RM, Waller JL, Genco CA, Slocum C, Manning M, Schoenlein PV, Cutler CW - PLoS Pathog. (2015)

Blocking of DC-SIGN inhibits Mfa1+Pg uptake and restores basal LC3-II signal in MoDCs.A) Immuno-fluorescence images of LC3-II and P. gingivalis within MoDCs pre-treated with GP120 (DC-SIGN blocker) after 12 hours of infection. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE. B) Fluorescent intensity of LC3-II and P. gingivalis strains signal (in triplicates) were statistically analyzed (* and # p<0.001). C) Flow cytometry of MoDCs treated with siRNA for DC-SIGN (si-DC-SIGN) after 12 hours. Left panels show the decrease of DC-SIGN in Cont. (un-infected), Pg381 and Mfa1+Pg-infected MoDCs. D) Epifluorescence microscopy images of MoDCs (si-DC-SIGN) infected with Pg381 and Mfa1+Pg. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE. E) The figure shows the CFU counts of the P. gingivalis strains with MoDCs that lack DC-SIGN (si-DC-SIGN). The analysis of readings used One-way ANOVA analysis of different groups and Tukey’s test for multiple comparisons (* p<0.001).
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4352937&req=5

ppat.1004647.g006: Blocking of DC-SIGN inhibits Mfa1+Pg uptake and restores basal LC3-II signal in MoDCs.A) Immuno-fluorescence images of LC3-II and P. gingivalis within MoDCs pre-treated with GP120 (DC-SIGN blocker) after 12 hours of infection. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE. B) Fluorescent intensity of LC3-II and P. gingivalis strains signal (in triplicates) were statistically analyzed (* and # p<0.001). C) Flow cytometry of MoDCs treated with siRNA for DC-SIGN (si-DC-SIGN) after 12 hours. Left panels show the decrease of DC-SIGN in Cont. (un-infected), Pg381 and Mfa1+Pg-infected MoDCs. D) Epifluorescence microscopy images of MoDCs (si-DC-SIGN) infected with Pg381 and Mfa1+Pg. LC3-II was detected in red-fluorescent dye and the bacterial strains were pre-labeled with green CFSE. E) The figure shows the CFU counts of the P. gingivalis strains with MoDCs that lack DC-SIGN (si-DC-SIGN). The analysis of readings used One-way ANOVA analysis of different groups and Tukey’s test for multiple comparisons (* p<0.001).
Mentions: Infection of MoDCs by Mfa1+Pg has previously been shown to depend on engagement of DC-SIGN [14]. We confirmed this result by blocking DC-SIGN with HIV gp120, prior to infection (Fig. 6A and 6B). Blocking DC-SIGN increased the LC3-II signal in MoDCs prior to addition of Mfa1+Pg (Fig. 6B). Moreover, blocking DC-SIGN restored the basal expression of LC3-II in MoDCs (Fig. 6B). To confirm the HIV gp120 blocking experiments, we additionally knocked down DC-SIGN using siRNA. DC-SIGN knockdown inhibited uptake of Mfa1+Pg but not Pg381 (Fig. 6C and 6D) and restored LC3-II signals in MoDCs (Fig. 6C and 6D). Furthermore, DC-SIGN knockdown significantly decreased survival of Mfa1+Pg in MoDCs (Fig. 6E). A scrambled sequence control did not inhibit uptake or effect LC3-II signal. To confirm the contribution of actin-mediated endocytic trafficking in LC3-II induction, MoDCs were treated with cytochalasin-D (CytD) prior to infection. CytD significantly inhibited intracellular localization of both Pg381 and Mfa1+Pg by MoDCs and restored the LC3-II signals to the basal level in MoDCs (S7 Fig. C and D).

Bottom Line: Survival was decreased by activation of TLR2 and/or autophagy.Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1.These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

View Article: PubMed Central - PubMed

Affiliation: Department of Periodontics, Georgia Regents University, Augusta, Georgia, United States of America.

ABSTRACT
Signaling via pattern recognition receptors (PRRs) expressed on professional antigen presenting cells, such as dendritic cells (DCs), is crucial to the fate of engulfed microbes. Among the many PRRs expressed by DCs are Toll-like receptors (TLRs) and C-type lectins such as DC-SIGN. DC-SIGN is targeted by several major human pathogens for immune-evasion, although its role in intracellular routing of pathogens to autophagosomes is poorly understood. Here we examined the role of DC-SIGN and TLRs in evasion of autophagy and survival of Porphyromonas gingivalis in human monocyte-derived DCs (MoDCs). We employed a panel of P. gingivalis isogenic fimbriae deficient strains with defined defects in Mfa-1 fimbriae, a DC-SIGN ligand, and FimA fimbriae, a TLR2 agonist. Our results show that DC-SIGN dependent uptake of Mfa1+P. gingivalis strains by MoDCs resulted in lower intracellular killing and higher intracellular content of P. gingivalis. Moreover, Mfa1+P. gingivalis was mostly contained within single membrane vesicles, where it survived intracellularly. Survival was decreased by activation of TLR2 and/or autophagy. Mfa1+P. gingivalis strain did not induce significant levels of Rab5, LC3-II, and LAMP1. In contrast, P. gingivalis uptake through a DC-SIGN independent manner was associated with early endosomal routing through Rab5, increased LC3-II and LAMP-1, as well as the formation of double membrane intracellular phagophores, a characteristic feature of autophagy. These results suggest that selective engagement of DC-SIGN by Mfa-1+P. gingivalis promotes evasion of antibacterial autophagy and lysosome fusion, resulting in intracellular persistence in myeloid DCs; however TLR2 activation can overcome autophagy evasion and pathogen persistence in DCs.

Show MeSH
Related in: MedlinePlus