After the construction, all the mutants remained sensitive to P22 and did not show any obvious defects when grown in nutrient rich LB medium or glucose minimal medium. The mutants were also as resistant as the wild-type strain to the action of blood serum, egg white, bile salts, polymyxin (as a representative of antimicrobial peptides), Protein Tyrosine Kinase inhibitor hydrogen peroxide or pH 4 (not shown). Table 3 List of strains used in this study. Strain SPI present SPI absent Reference S. MI-503 purchase Enteritidis 147 Nal wild type 1, 2, 3, 4, 5 none [25] S. Enteritidis 147 Nal ΔSPI1 2,3,4,5 1 this study S. Enteritidis 147 Nal ΔSPI2 1,3,4,5 2 this study S. Enteritidis

147 Nal ΔSPI3 1,2,4,5 3 this study Cyclosporin A order S. Enteritidis 147 Nal ΔSPI4 1,2,3,5 4 this study S. Enteritidis 147 Nal ΔSPI5 1,2,3,4 5 this study S. Enteritidis 147 Nal ΔSPI1-5 none 1,2,3,4,5 this study S. Enteritidis 147 Nal SPI1o 1 2,3,4,5 this study S. Enteritidis 147 Nal SPI2o 2 1,3,4,5 this study S. Enteritidis 147 Nal SPI3o 3 1,2,4,5 this study S. Enteritidis 147 Nal SPI4o 4 1,2,3,5 this study S. Enteritidis 147 Nal SPI5o 5 1,2,3,4 this study S. Enteritidis 147 Nal ΔSPI1&2 3,4,5 1,2 this study S. Enteritidis 147 Nal SPI1&2o 1,2 3,4,5 this study

Infection of chickens In the first experimental infection, day-old chickens (Ross breed, 10 birds/group) were infected orally with 5 × 107 CFU of either the wild-type strain or the SPI mutants. In the second infection, four groups, each of 10 chickens, were infected with the wild type strain, or ΔSPI1&2, Farnesyltransferase SPI1&2o and SPI1-5 mutants. Counts of the strains in caeca, liver and spleen were determined in 5 birds on day 5 and in remaining 5 birds on day 12 of life i.e. 4 and 11 days post infection, respectively. The last experimental infection was focused on cytokine signaling and in this case, besides 3 non-infected control chickens, three additional chickens per group were infected with wild type strain, ΔSPI1, ΔSPI2, and ΔSPI1&2 mutants. In

all euthanised birds, S. Enteritidis counts in the caeca, liver and spleen were determined after tissue homogenisation in peptone water and plating tenfold serial dilutions on XLD, BGA or Bromothymol-blue agars (Merck) supplemented with nalidixic acid. Samples negative after the direct plating were subjected to pre-enrichment in RV broth supplemented with nalidixic acid for qualitative S. Enteritidis determination. Counts of S. Enteritidis positive after the direct plating were logarithmically transformed. In the case of samples positive only after the pre-enrichment, these were assigned a value of 1 and the negative samples were assigned a value of 0. Samples from the caeca and liver were also fixed in 10% formaldehyde and subjected to haematoxylin and eosin staining.