After incubation, the number of viable cells was counted by plating the sample on Luria–Bertani agar plates. Escherichia

coli strains were cultured in Luria–Bertani medium to OD600 = 0.3. Bacterial cells were collected by centrifugation and suspended in PBS. Ten microliters of the bacterial suspension was mixed with fresh swine serum (NihonBiotest Co, Tokyo, Japan) and incubated at 37 °C for 90 min without shaking. After incubation, the selleck compound number of viable cells was counted by plating the sample on Luria–Bertani agar plates. First, we compared the virulence of the EHEC O157:H7 Sakai strain and laboratory E. coli strain W3110 in silkworms. Injection of the Sakai strain into silkworm hemolymph and incubation at 37 °C for 20 h killed the silkworms (Fig. 1a). The LD50 of the Sakai strain was 4.3 × 106 CFU per larva (Table 1). The LD50 of W3110 was 90 times

higher than that of Sakai (Table 1). Next, to identify the genes of EHEC O157:H7 required to kill silkworms, we investigated whether the supposed virulence factors of EHEC O157:H7 Sakai contribute to killing silkworms. The killing ability of double-deletion mutants of the stx1 and stx2 genes that encode Shiga toxin 1 and 2, respectively, in silkworms was indistinguishable from that of the parent strain, SKI5142 (Table 2). Moreover the deletion of ehxA, which encodes enterohemolysin, killed silkworms with an LD50 similar ZVADFMK to that of the parent strain (Table 2). Similarly, the killing ability of the mutant with a deletion of eae, which encodes intimin and plays an essential role in bacterial adhesion to host cells, was indistinguishable from that of the parent strain (Table 2). Deletion of flhDC, which encodes a master regulator of flagellar genes, and deletion of the lrhA gene, which encodes a transcription factor of enterohemolysin,

flagellar genes, and LEE genes, did not attenuate PD184352 (CI-1040) the silkworm-killing ability of EHEC O157:H7 (Table 2). These results suggest that Shiga toxins, enterohemolysin, functions of LEE, and flagellar genes are not required by EHEC O157:H7 to kill silkworms, but some other factors are necessary. We focused our attention on the LPS O-antigen of the outer membrane as a factor involved in the high virulence of EHEC O157:H7 against silkworms. We constructed a deletion mutant of the rfbE gene in the Sakai background, which encodes perosamine synthase, a monosaccharide component of the O-antigen that is specific for O157:H7. We also constructed a deletion mutant of the waaL gene that encodes a ligase of the O-antigen to core-lipid A (Fig. S1a and b). To confirm the absence of the LPS O-antigen in these mutants, we immunostained LPS fractions of these mutants using anti-O157 immunoglobulin. The findings indicated that both deletion mutants, rfbE and waaL, lacked the LPS O-antigen (Fig. S1c). We further confirmed that introducing rfbE or waaL into the respective mutant restored the LPS O-antigen (Fig. S1c).