Directed evolution and targeted mutagenesis to murinize listeria monocytogenes internalin A for enhanced infectivity in the murine oral infection model

Table 2 Supplementary information for Figure 6.

Clone	1	2	3	4	5	6	7	8
(iii) Low	T273I	Q190L	*Q190L*	Q190L	Q190L T229P	G303E	Q190L	Q190L N386I
Fold increase vs Wt	9.44	5.82	*6.98*	4.15	13.23	12.12	6.10	7.94
(iv) Medium	T164A K301I G303E T399I	L86F N143K P159A Q196L K218M V224A G303E Q306H	Q190L L329Q S470C	T164A K301I G303E	N259Y T399I	Q190L G248R	F193Y K301E N413Y K507I	*T164A* *K301I* *G303E*
Fold increase vs Wt	3.25	9.31	7.79	6.85	8.14	6.57	4.05	*10.08*
(v) High	L149M N259Y	Q190L S223C N252Y I351T	S173I G303E T446A D449H	S173I T268I G303E T446A D449H	Q190L S223C N252Y I351T	*N259Y*	N239D S311C N325D	*S173I* *L185F* *L188I*
Fold increase vs Wt	23.21	15.89	8.64	19.31	9.08	*16.36*	8.24	*15.42*
(vi) Very High	Q190L A270G K301G	V123A Q190L P290Q N349D	Q190L	Q196K P290S L404S N413Y D457V	N130I F150V L203F Y369F N381I S487N	L294V S308R Y369S N381I S487N	L122I S292T E330V I458V	Q190L D199V S377N P444S K495N
Fold increase vs Wt	4.14	9.33	6.96	8.71	9.56	7.12	7.51	9.33

Mutations identified in the Bg lII/Bst XI fragment of pNZBinlA (iii-vi) and the invasion increase into CT-26 cells versus L. lactis InlA^WT. The amino acid mutations identified which involved in the interaction between InlA^WT and hCDH1 are highlighted in bold.
Details highlighted in bold and italics are mutations recombined in the chromosome of EGD-e.
L. lactis InlA site directed mutants with fold invasion increase into CT-26 cells vs L. lactis InlA^WT in brackets: S192N (21), Y369 S (20), S192N+Y369 S (30).
Below: Amino acids in InlA^WT which interact with hCDH1 and amino acid changes identified from error prone PCR screen. R85, N104: D Q*, N107, F150: V, E170, E172: T*, Q190: L, S192, R211, D213, I235, T237, E255, N259: Y, K301: I E G, N321: Y, E323, N325: D, E326, Y343, T345, Y347, F348, R365, F367, Y369: F S, W387, S389. * N104 and E172 mutations were found from additional screens and sequencing.

ISSN: 1471-2180