Paracoccidioides brasiliensis downmodulates α3 integrin levels in human lung epithelial cells in a TLR2-dependent manner

Paracoccidioidomycosis (PCM) is the most prevalent systemic mycosis in Latin America and may be caused by the species Paracoccidioides brasiliensis. In the lungs, this fungus interacts with epithelial cells, activating host cell signalling pathways, resulting in the production of inflammatory mediators. This event may be initiated through the activation of Pattern-Recognition Receptors such as Toll-like Receptors (TLRs). By interacting with cell wall components, TLR2 is frequently related to fungal infections. In this work, we show that, after 24 h post-infection with P. brasiliensis, A549 lung epithelial cells presented higher TLR2 levels, which is important for IL-8 secretion. Besides, integrins may also participate in pathogen recognition by host cells. We verified that P. brasiliensis increased α3 integrin levels in A549 cells after 5 h of infection and promoted interaction between this receptor and TLR2. However, after 24 h, surprisingly, we verified a decrease of α3 integrin levels, which was dependent on direct contact between fungi and epithelial cells. Likewise, we observed that TLR2 is important to downmodulate α3 integrin levels after 24 h of infection. Thus, P. brasiliensis can modulate the host inflammatory response by exploiting host cell receptors and cell signalling pathways.


Supplementary Figure 1. Images of multiple exposure times of Western blot membranes showed in Figure 1a
For membrane 1a, it was used a 6% polyacrylamide gel. To evaluate -actin, membrane 1a was cut in order to analyse only samples with the observed TLR2 bands. For this membrane, it was used the molecular weight marker "Precision plus protein standards dual color" (BIO-RAD code #161-0374). To analyse -actin, PVDF membrane was cut between the 75 kDa and 50 kDa markers.

Continuation of the Supplementary Figure 1. Images of multiple exposure times of Western blot membranes showed in Figure 1b
For membrane 1b, it was used a 10% polyacrylamide gel. As we did not observe separate -actin bands in the original membrane [(1) -actin 45 kDa], we submitted aliquots of some of these samples to another -actin analysis by Western blot [(2) -actin 45 kDa]. For these membranes, it was used the molecular weight marker "Precision plus protein standards dual color" (BIO-RAD code #161-0374). To analyse -actin, PVDF membranes were cut between the 75 kDa and 50 kDa markers. For membrane 1c, it was used an 8% polyacrylamide gel. For membrane 1d, it was used a 6% polyacrylamide gel. For both membranes, it was used the molecular weight marker "Spectra Multicolor broad range protein ladder" (Thermo Scientific code #26634). To analyse -actin, PVDF membranes were cut near the 70 kDa and 50 kDa markers.

Supplementary Figure 2. Images of multiple exposure times of Western blot membrane showed in Figure 2a
For this membrane, it was used an 8% polyacrylamide gel. It was used the molecular weight marker "Precision plus protein standards dual color" (BIO-RAD code #161-0374). To analyse -actin, PVDF membrane was cut between the 75 kDa and 50 kDa markers.

Continuation of the Supplementary Figure 2. Images of multiple exposure times of Western blot membrane showed in Figure 2c
For this membrane, it was used an 8% polyacrylamide gel. It was used the molecular weight marker "Precision plus protein standards dual color" (BIO-RAD code #161-0374). To analyse -actin, PVDF membrane was cut between the 75 kDa and 50 kDa markers.

Supplementary Figure 3. Images of multiple exposure times of Western blot membranes showed in Figures 3a and 3b
For membrane 3a, it was used a 10% polyacrylamide gel and the molecular weight marker "Precision plus protein standards dual color" (BIO-RAD code #161-0374). For membrane 3b, it was used a 6% polyacrylamide gel and the molecular weight marker "TrueColor High Range Protein Marker" (Sinapse Inc code #S2600). Membrane 3a was cut near the 75 kDa marker before incubation with TLR2 antibody.

Supplementary Figure 4. Images of multiple exposure times of Western blot membranes showed in Figures 4a, 4b and 4c
For these membranes, it was used a 6% polyacrylamide gel. It was used the molecular weight marker "Precision plus protein standards dual color" (BIO-RAD code #161-0374). To analyse -actin, PVDF membranes were cut between the 75 kDa and 50 kDa markers.

Continuation of the Supplementary Figure 4. Images of multiple exposure times of Western blot membranes showed in Figure 4d
For membrane 4d, it was used a 6% polyacrylamide gel. It was used the molecular weight marker "Precision plus protein standards dual color" (BIO-RAD code #161-0374). To analyse -actin, PVDF membrane was cut between 75 kDa and 50 kDa.

Continuation of the Supplementary Figure 4. Images of multiple exposure times of Western blot membranes showed in Figure 4e
For membrane 4e, it was used a 10% polyacrylamide gel. It was used the molecular weight marker "Precision plus protein standards dual color" (BIO-RAD code #161-0374). To analyse -actin, PVDF membrane was cut between 75 kDa and 50 kDa.

Supplementary Figure 5. Images of multiple exposure times of Western blot membrane showed in Figure 5a
For this membrane, it was used a 10% polyacrylamide gel. It was used the molecular weight marker "Precision plus protein standards dual color" (BIO-RAD code #161-0374). To analyse -actin, PVDF membranes were cut between 75 kDa and 50 kDa. Figure 5. Images of multiple exposure times of Western blot membrane showed in Figure 5b For this membrane, it was used a 10% polyacrylamide gel. It was used the molecular weight marker "Precision plus protein standards dual color" (BIO-RAD code #161-0374). To analyse -actin, PVDF membranes were cut between 75 kDa and 50 kDa. Figure 6. Images of multiple exposure times of Western blot membrane showed in Figure 6 For this membrane, it was used a 10% polyacrylamide gel. It was used the molecular weight marker "Precision plus protein standards dual color" (BIO-RAD code #161-0374). To analyse -actin, PVDF membranes were cut between 75 kDa and 50 kDa.