0042, unpaired two tailed t-test). KU-57788 As expected, the fliI mutant derivatives of EPEC E2348/69 secreting FliC via the LEE-encoded T3SS were non-motile (Fig. 5C), due to the absence of an intact

Cell Cycle inhibitor flagella export apparatus. Figure 5 A. Representative immunoblot of secreted proteins prepared from derivatives of EPEC E2348/69 grown in hDMEM and detected with anti-H6 FliC antibodies. Lane 1: E2348/69; lane 2: ΔfliC; lane 3: ΔfliC (pFliC); lane 4: ΔfliI (pFliC); lane 5: ΔfliI/escF (pFliC); lane 6: ΔfliI/escF (pFliCEscF). B. NF-kappa B-dependent luciferase reporter activity in HEK293 cells stimulated with secreted proteins prepared from derivatives of EPEC E2348/69. 1. EPEC E2348/69; 2. ΔfliC; 3. ΔfliC (pFliC); 4. ΔfliI (pFliC); 5. ΔfliI/escF (pFliC); 6. ΔfliI/escF (pFliCEscF); 7. hDMEM alone. Results are expressed as the mean fold increase ± SEM with respect to the unstimulated control (fold = 1) and are representative of three independent experiments performed in triplicate C. Motility of derivatives of EPEC E2348/69 shown in (A) in 0.2% hDMEM agar. 1. EPEC E2348/69; 2. ΔfliC; 3. ΔfliC (pFliC); 4. ΔfliI (pFliC); 5. ΔfliI/escF (pFliC); 6. ΔfliI/escF (pFliCEscF). Discussion

Many Gram-negative pathogens utilize a T3SS to deliver diverse effector proteins directly into eukaryotic cells. The structure of the T3SS apparatus is conserved among different pathogens and shares structural Nepicastat similarity with the flagella basal body. The reported ancestral relationship between the two secretion systems is based on low sequence similarity between some

components as well as functional conservation [33]. Under certain conditions, virulence effector proteins may be secreted, but not translocated by the flagella T3SS [34–37]. The preferential secretion of effector proteins by their cognate T3SS rather than the flagella export apparatus depends largely on a system of chaperones that confer pathway specificity. In Salmonella mafosfamide enterica serovar Typhimurium, truncated forms of the effectors SptP and SopE that lack the chaperone binding domain for secretion by the T3SS are instead secreted by the flagella export apparatus [35, 38]. This suggests that not only do the T3SS system chaperones confer pathway specificity, but also that the flagella export system is the default secretion pathway for unchaperoned proteins [35]. Recently, Miao et al (2006) showed that flagellin from S. Typhimurium present in the cytosol of infected macrophages stimulated IL1-β release in macrophages through activation of the intracellular NACHT-leucine-rich repeat protein, Ipaf. The activation of Ipaf by cytosolic flagellin was dependent on the SPI1-encoded T3SS and not the flagella biosynthesis locus [39].