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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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Lower intracellular content of Mfa1+Pg in MoDCs treated with Rapamycin.A) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* P<0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey’s test for multiple comparisons. B) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P. gingivalis infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections). C) Quantifications of the fluorescent intensity of CFSE-labeled P. gingivalis and LC3-II signals within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey’s test for multiple comparisons (* P<0.001).
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ppat.1004647.g003: Lower intracellular content of Mfa1+Pg in MoDCs treated with Rapamycin.A) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* P<0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey’s test for multiple comparisons. B) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P. gingivalis infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections). C) Quantifications of the fluorescent intensity of CFSE-labeled P. gingivalis and LC3-II signals within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey’s test for multiple comparisons (* P<0.001).

Mentions: To investigate the role of autophagy as a putative survival mechanism utilized by Mfa1+Pg, the viability of P. gingivalis strains in MoDCs was monitored after induction of autophagy by the mTOR inhibitor, Rapamycin. Initial studies established that the viability of MoDCs and of P. gingivalis alone were not Rapamycin-sensitive at the concentrations used. Entry into MoDCs resulted in a significant increase in survival of Mfa1+Pg at 24 hours (p<0.001) (Fig. 3A). Rapamycin treatment of Mfa1+Pg-infected MoDCs (Mfa1+Pg+Rapa) significantly decreased P. gingivalis survival by ~48% (p <0.001) (Fig. 3A). Increased autophagy induction was confirmed by immuno-labeling of LC3-II in MoDCs treated with Rapamycin for 11 hours (1 hour after P. gingivalis infections). Rapamycin treatment increased the LC3-II signal in cells infected with all fimbriated strains as well as in un-infected (Cont.) (Fig. 3B and C).


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)

Lower intracellular content of Mfa1+Pg in MoDCs treated with Rapamycin.A) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* P<0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey’s test for multiple comparisons. B) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P. gingivalis infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections). C) Quantifications of the fluorescent intensity of CFSE-labeled P. gingivalis and LC3-II signals within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey’s test for multiple comparisons (* P<0.001).
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004647.g003: Lower intracellular content of Mfa1+Pg in MoDCs treated with Rapamycin.A) Pg381 and mutant strains count after 24 hr incubation with human MoDCs with/without Rapamycin treatment. The survived bacteria were measured after maintaining the lysed MoDCs suspension in anaerobic broth for 5 days. The plot represents the means ±standard deviation of CFU within MoDCs harvested from three healthy individuals (* P<0.001). The analysis of readings used One-way AVOVA analysis of different groups and Tukey’s test for multiple comparisons. B) Epifluorescence microscopy images of MoDCs treated with Rapamycin 1 hour after P. gingivalis infections. LC3-II (red-fluorescent dye) and the bacterial strains (green CFSE) were studied in MoDCs 11 hours after Rapamycin treatment (12 hours after infections). C) Quantifications of the fluorescent intensity of CFSE-labeled P. gingivalis and LC3-II signals within infected MoDCs using NIS-Elements BR software. One-way ANOVA analysis was used to compare the means of intensity of different groups and Tukey’s test for multiple comparisons (* P<0.001).
Mentions: To investigate the role of autophagy as a putative survival mechanism utilized by Mfa1+Pg, the viability of P. gingivalis strains in MoDCs was monitored after induction of autophagy by the mTOR inhibitor, Rapamycin. Initial studies established that the viability of MoDCs and of P. gingivalis alone were not Rapamycin-sensitive at the concentrations used. Entry into MoDCs resulted in a significant increase in survival of Mfa1+Pg at 24 hours (p<0.001) (Fig. 3A). Rapamycin treatment of Mfa1+Pg-infected MoDCs (Mfa1+Pg+Rapa) significantly decreased P. gingivalis survival by ~48% (p <0.001) (Fig. 3A). Increased autophagy induction was confirmed by immuno-labeling of LC3-II in MoDCs treated with Rapamycin for 11 hours (1 hour after P. gingivalis infections). Rapamycin treatment increased the LC3-II signal in cells infected with all fimbriated strains as well as in un-infected (Cont.) (Fig. 3B and C).

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