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. 2014 Jun 9;9(6):e99135. doi: 10.1371/journal.pone.0099135

Figure 6. Western immunoblots of L. lactis expressing S. pyogenes pili.

Figure 6

Cell wall extracts from L. lactis cells transformed with plasmids carrying the S. pyogenes M1/T1 strain pilus operon were immunoblotted with antisera against either FctA backbone pilin (anti-Spy0128), FctB minor pilin (anti-Spy0130), or Cpa adhesin (anti-Spy0125) to show the effect of SipA mutations on pilus polymerisation. Cells expressing WT levels of SipA show a laddering of high molecular weight polymers. SipA deletion mutant (ΔSipA) shows no pilin polymerisation, only monomeric FctA or FctB. Mutating aspartic acid D61 (SipA D61A and D61S) or the double mutation of SipA D61A/K98A, or the V99R S3 pocket-occluding mutant have minimal effect on pilus polymerisation, with each mutant producing high molecular weight polymers. Replacement of the M1/T1 SipA (FCT2) with T9 SipA (FCT3) produced pilus polymerisation, but with more lower molecular weight multimers including FctA-B dimers (∼50 kDa) and pili with no cpa. FctA = 32 kDa, FctB = 18 kDa, Cpa = 75 kDa.