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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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High intracellular content of Mfa1+Pg within human MoDCs.A) Transmission electron microscopy (TEM) of MoDCs infected with P. gingivalis for 2, 12 and 24 hours (left, middle and right panels). The sections show the intra-and extra-cellular contents of Cont. (un-infected), Pg381, Mfa1+Pg and FimA+Pg infected MoDCs for the different time points. B) The figure shows the intracellular content of P. gingivalis strains after 24 hours of infection. MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re-suspended and maintained in anaerobic broth for 5 days. The data represents CFU within MoDCs harvested from three healthy individuals. The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey’s test for multiple group comparisons within 3 different experiments (* statistical significance at the p<0.001). C) Growth curves of Pg381 and mutant strains (Mfa1+Pg and Fima+Pg) in anaerobic condition in the absence of DCs. The means ±standard deviations were analyzed by One-way ANOVA of different groups and Tukey’s test for multiple group comparisons within 3 different experiments.
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ppat.1004647.g001: High intracellular content of Mfa1+Pg within human MoDCs.A) Transmission electron microscopy (TEM) of MoDCs infected with P. gingivalis for 2, 12 and 24 hours (left, middle and right panels). The sections show the intra-and extra-cellular contents of Cont. (un-infected), Pg381, Mfa1+Pg and FimA+Pg infected MoDCs for the different time points. B) The figure shows the intracellular content of P. gingivalis strains after 24 hours of infection. MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re-suspended and maintained in anaerobic broth for 5 days. The data represents CFU within MoDCs harvested from three healthy individuals. The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey’s test for multiple group comparisons within 3 different experiments (* statistical significance at the p<0.001). C) Growth curves of Pg381 and mutant strains (Mfa1+Pg and Fima+Pg) in anaerobic condition in the absence of DCs. The means ±standard deviations were analyzed by One-way ANOVA of different groups and Tukey’s test for multiple group comparisons within 3 different experiments.

Mentions: At 2, 12 and 24h after bacterial co-culture with MoDCs, the MoDCs were imaged for intracellular P. gingivalis by epifluorescence microscopy and transmission electron microscopy (TEM). All P. gingivalis strains except the double fimbriae negative P. gingivalis strain MFB (Table 1) were taken up by MoDCs. There were marked differences in the P. gingivalis content of MoDCs at 2, 12 and 24 hours, particularly when comparing double fimbriae positive strain Pg381 to Mfa1+Pg (Fig. 1A). We observed a higher number of Mfa1+Pg within MoDCs (Fig. 1A) (S1 Fig.) This difference was most apparent after 24 hours, with large numbers of intra-and extra-cellular bacteria present. In contrast, MoDCs infected with Pg381 showed minimal bacterial content after 24 hours. Survival of intracellular bacteria was then assessed quantitatively by lysing MoDCs and growing bacteria in broth cultures and on anaerobic blood agar plates. Mfa1+Pg was recovered at higher numbers from MoDCs lysates in broth and on blood agar compared to Pg381 Fig. 1B). No significant difference was detected in the growth or death patterns of all strains in the media under anaerobic conditions in the absence of DCs (Fig. 1C).


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)

High intracellular content of Mfa1+Pg within human MoDCs.A) Transmission electron microscopy (TEM) of MoDCs infected with P. gingivalis for 2, 12 and 24 hours (left, middle and right panels). The sections show the intra-and extra-cellular contents of Cont. (un-infected), Pg381, Mfa1+Pg and FimA+Pg infected MoDCs for the different time points. B) The figure shows the intracellular content of P. gingivalis strains after 24 hours of infection. MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re-suspended and maintained in anaerobic broth for 5 days. The data represents CFU within MoDCs harvested from three healthy individuals. The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey’s test for multiple group comparisons within 3 different experiments (* statistical significance at the p<0.001). C) Growth curves of Pg381 and mutant strains (Mfa1+Pg and Fima+Pg) in anaerobic condition in the absence of DCs. The means ±standard deviations were analyzed by One-way ANOVA of different groups and Tukey’s test for multiple group comparisons within 3 different experiments.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004647.g001: High intracellular content of Mfa1+Pg within human MoDCs.A) Transmission electron microscopy (TEM) of MoDCs infected with P. gingivalis for 2, 12 and 24 hours (left, middle and right panels). The sections show the intra-and extra-cellular contents of Cont. (un-infected), Pg381, Mfa1+Pg and FimA+Pg infected MoDCs for the different time points. B) The figure shows the intracellular content of P. gingivalis strains after 24 hours of infection. MoDCs infected with Pg381 or isogenic mutants strains were lysed and the survived intracellular bacteria were re-suspended and maintained in anaerobic broth for 5 days. The data represents CFU within MoDCs harvested from three healthy individuals. The means ±standard deviation (in triplicates) were analyzed by One-way ANOVA of different groups and Tukey’s test for multiple group comparisons within 3 different experiments (* statistical significance at the p<0.001). C) Growth curves of Pg381 and mutant strains (Mfa1+Pg and Fima+Pg) in anaerobic condition in the absence of DCs. The means ±standard deviations were analyzed by One-way ANOVA of different groups and Tukey’s test for multiple group comparisons within 3 different experiments.
Mentions: At 2, 12 and 24h after bacterial co-culture with MoDCs, the MoDCs were imaged for intracellular P. gingivalis by epifluorescence microscopy and transmission electron microscopy (TEM). All P. gingivalis strains except the double fimbriae negative P. gingivalis strain MFB (Table 1) were taken up by MoDCs. There were marked differences in the P. gingivalis content of MoDCs at 2, 12 and 24 hours, particularly when comparing double fimbriae positive strain Pg381 to Mfa1+Pg (Fig. 1A). We observed a higher number of Mfa1+Pg within MoDCs (Fig. 1A) (S1 Fig.) This difference was most apparent after 24 hours, with large numbers of intra-and extra-cellular bacteria present. In contrast, MoDCs infected with Pg381 showed minimal bacterial content after 24 hours. Survival of intracellular bacteria was then assessed quantitatively by lysing MoDCs and growing bacteria in broth cultures and on anaerobic blood agar plates. Mfa1+Pg was recovered at higher numbers from MoDCs lysates in broth and on blood agar compared to Pg381 Fig. 1B). No significant difference was detected in the growth or death patterns of all strains in the media under anaerobic conditions in the absence of DCs (Fig. 1C).

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