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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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Induction of autophagy impairs the survival of Mfa1+Pg strain within MoDCs.A) Survival of P. gingivalis strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show P. gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines. The effect of rapamycin on Mfa1+Pg, Pg381 and FimA+Pg survival within MoDCs are shown in figures B, C and D respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1+Pg, Pg381 and FimA+Pg) between treated (rapamycin) and untreated were significantly different overtime (p-value <0.=001).
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ppat.1004647.g004: Induction of autophagy impairs the survival of Mfa1+Pg strain within MoDCs.A) Survival of P. gingivalis strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show P. gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines. The effect of rapamycin on Mfa1+Pg, Pg381 and FimA+Pg survival within MoDCs are shown in figures B, C and D respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1+Pg, Pg381 and FimA+Pg) between treated (rapamycin) and untreated were significantly different overtime (p-value <0.=001).

Mentions: To determine the kinetics of survival of P. gingivalis within MoDCs, as well as the involvement of autophagy, intracellular bacteria were monitored after 6, 12, 24 and 48 hours of incubation with MoDCs with or without Rapamycin (Fig. 4). The levels of P. gingivalis Mfa1+Pg within MoDCs (Mfa1+Pg+DC) were the highest at all time points except at 6 hours when we observed similar levels to that observed with Pg381+DCs (Fig. 4A). Levels of Pg381 and FimA+Pg within MoDCs (Pg381+DCs and FimA+Pg+DCs) were nonetheless significantly higher than bacteria without MoDCs, until 12 hours, at which point we observed a significant decrease in survival of both strains at 24 and 48 hours. Moreover, the highest level of Mfa1+Pg was observed at 24 hours, with the numbers of P. gingivalis increasing within MoDCs at 6, 12 and 24 hours (Fig. 4A). Activation of autophagy with Rapamycin significantly inhibited Mfa1+Pg survival at 6, 12 and 24 hours within MoDCs (Mfa1+Pg+DCs+Rapa) (Fig. 4B). However, the numbers of Mfa1+Pg detected within MoDCs treated with Rapamycin (Mfa1+Pg+DCs+Rapa) were still higher than bacteria alone (Mfa1+Pg) at 12 and 24 hours (Fig. 4B). For Pg381, rapamycin also significantly decreased intracellular survival, with no significant differences detected relative to bacteria alone except at 12 hours (Fig. 4C). FimA+Pg exhibited significant intracellular survival (FimA+Pg+DCs) only at 12 hours, which was significantly inhibited with rapamycin treatment (Fig. 4D). The analysis of the data using three-factor repeated measures ANOVA showed that both time and intracellular environment were significant factors (P<0.05) in P. gingivalis survival, with rapamycin significantly impairing P. gingivalis survival with MoDCs.


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)

Induction of autophagy impairs the survival of Mfa1+Pg strain within MoDCs.A) Survival of P. gingivalis strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show P. gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines. The effect of rapamycin on Mfa1+Pg, Pg381 and FimA+Pg survival within MoDCs are shown in figures B, C and D respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1+Pg, Pg381 and FimA+Pg) between treated (rapamycin) and untreated were significantly different overtime (p-value <0.=001).
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Related In: Results  -  Collection

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ppat.1004647.g004: Induction of autophagy impairs the survival of Mfa1+Pg strain within MoDCs.A) Survival of P. gingivalis strains within MoDCs after 6, 12, 24 and 48 hours. Blue lines show P. gingivalis strains survival within MoDCs and their survival in anaerobic condition in the absence of DCs are showed in black lines. The effect of rapamycin on Mfa1+Pg, Pg381 and FimA+Pg survival within MoDCs are shown in figures B, C and D respectively. A three-factor repeated measures ANOVA using mixed models was used to test the effect of strain and rapamycin treatment over time on OD reading. The survival curves for the strains are showed in blue, while the effect of rapamycin treatments are in red. Bacterial survivals in the absence of MoDCs with and without rapamycin are plotted in grey and black, respectively. Statistical analysis showed that the strain by rapamycin treatment overtime interaction indicates the pattern of means in each strain (Mfa1+Pg, Pg381 and FimA+Pg) between treated (rapamycin) and untreated were significantly different overtime (p-value <0.=001).
Mentions: To determine the kinetics of survival of P. gingivalis within MoDCs, as well as the involvement of autophagy, intracellular bacteria were monitored after 6, 12, 24 and 48 hours of incubation with MoDCs with or without Rapamycin (Fig. 4). The levels of P. gingivalis Mfa1+Pg within MoDCs (Mfa1+Pg+DC) were the highest at all time points except at 6 hours when we observed similar levels to that observed with Pg381+DCs (Fig. 4A). Levels of Pg381 and FimA+Pg within MoDCs (Pg381+DCs and FimA+Pg+DCs) were nonetheless significantly higher than bacteria without MoDCs, until 12 hours, at which point we observed a significant decrease in survival of both strains at 24 and 48 hours. Moreover, the highest level of Mfa1+Pg was observed at 24 hours, with the numbers of P. gingivalis increasing within MoDCs at 6, 12 and 24 hours (Fig. 4A). Activation of autophagy with Rapamycin significantly inhibited Mfa1+Pg survival at 6, 12 and 24 hours within MoDCs (Mfa1+Pg+DCs+Rapa) (Fig. 4B). However, the numbers of Mfa1+Pg detected within MoDCs treated with Rapamycin (Mfa1+Pg+DCs+Rapa) were still higher than bacteria alone (Mfa1+Pg) at 12 and 24 hours (Fig. 4B). For Pg381, rapamycin also significantly decreased intracellular survival, with no significant differences detected relative to bacteria alone except at 12 hours (Fig. 4C). FimA+Pg exhibited significant intracellular survival (FimA+Pg+DCs) only at 12 hours, which was significantly inhibited with rapamycin treatment (Fig. 4D). The analysis of the data using three-factor repeated measures ANOVA showed that both time and intracellular environment were significant factors (P<0.05) in P. gingivalis survival, with rapamycin significantly impairing P. gingivalis survival with MoDCs.

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