Escherichia coli TOP10 (Invitrogen) was used for construction and purification of the plasmids. Yersinia enterocolitica ΔHOPEMT (MRS40 pIML421 [yopH Δ1-352, yopO Δ65-558, yopP 23 , yopE 21 , yopM 23 , yopT 135 ]), deficient for the Yersinia T3S effectors YopH, O, P, E, M, and T, but T3S-proficient [44] and Verubecestat datasheet T3S-deficient Y. enterocolitica ΔHOPEMT ΔYscU (MRS40 pFA1001 [yopH Δ1-352, yopO Δ65-558, yopP 23 , yopE 21 , yopM 23 , yopT 135 , yscU Δ1-354 ) [45] were used for T3S assays. The yscU gene encodes an essential component of the Y. enterocolitica T3S system, and

the yscU Δ1-354 mutation is non-polar [46]. E. coli or Y. enterocolitica were routinely grown in liquid or solid Luria-Bertani (LB) medium (NZYtech) with the appropriate

antibiotics and supplements. Plasmids were introduced into E. coli or Y. enterocolitica by electroporation. DNA manipulations, plasmids, and primers The plasmids used in this work and their main characteristics are detailed in Additional file 1: Table S1. The DNA primers used in their construction are shown in Additional file 2: Table S2. Plasmids were constructed and purified with proof-reading Phusion DNA polymerase (Finnzymes), restriction enzymes (MBI Fermentas), T4 DNA Ligase (Invitrogen), DreamTaq DNA polymerase (MBI Fermentas), DNA clean & concentrator™-5 Kit and Zymoclean™ Gel DNA Recovery kit (Zymo Research), and purified with GeneElute Plasmid Miniprep kit (Sigma), according to the instructions of the manufacturers. In brief, to analyze T3S signals we constructed {Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleck Anti-infection Compound Library|Selleck Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Selleckchem Anti-infection Compound Library|Selleckchem Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|Anti-infection Compound Library|Antiinfection Compound Library|buy Anti-infection Compound Library|Anti-infection Compound Library ic50|Anti-infection Compound Library price|Anti-infection Compound Library cost|Anti-infection Compound Library solubility dmso|Anti-infection Compound Library purchase|Anti-infection Compound Library manufacturer|Anti-infection Compound Library research buy|Anti-infection Compound Library order|Anti-infection Compound Library mouse|Anti-infection Compound Library chemical structure|Anti-infection Compound Library mw|Anti-infection Compound Library molecular weight|Anti-infection Compound Library datasheet|Anti-infection Compound Library supplier|Anti-infection Compound Library in vitro|Anti-infection Compound Library cell line|Anti-infection Compound Library concentration|Anti-infection Compound Library nmr|Anti-infection Compound Library in vivo|Anti-infection Compound Library clinical trial|Anti-infection Compound Library cell assay|Anti-infection Compound Library screening|Anti-infection Compound Library high throughput|buy Antiinfection Compound Library|Antiinfection Compound Library ic50|Antiinfection Compound Library price|Antiinfection Compound Library cost|Antiinfection Compound Library solubility dmso|Antiinfection Compound Library purchase|Antiinfection Compound Library manufacturer|Antiinfection Compound Library research buy|Antiinfection Compound Library order|Antiinfection Compound Library chemical structure|Antiinfection Compound Library datasheet|Antiinfection Compound Library supplier|Antiinfection Compound Library in vitro|Antiinfection Compound Library cell line|Antiinfection Compound Library concentration|Antiinfection Compound Library clinical trial|Antiinfection Compound Library cell assay|Antiinfection Compound Library screening|Antiinfection Compound Library high throughput|Anti-infection Compound high throughput screening| ifoxetine plasmids harboring hybrid genes encoding the first 10, 15, 20, or 40 amino acids of each protein (C. trachomatis

proteins, SycT and YopE) and the mature form of TEM-1 β-lactamase (TEM-1) [47]. These hybrids were made using as vector pLJM3, a low-copy plasmid which enables expression of the cloned genes driven by the promoter of the Y. enterocolitica yopE gene [48], either by overlapping PCR or by using a cloning strategy previously described for the construction of plasmids encoding Inc-TEM-1 hybrid proteins [45]. To analyze secretion of full-length C. trachomatis proteins, we constructed plasmids Temsirolimus mouse expressing the proteins C-terminally tagged with a haemagglutinin (HA) epitope. For this, the genes were amplified by PCR from chromosomal DNA of strain L2/434/Bu using a reverse primer with a sequence complementary to the transcribed strand of the DNA encoding the HA-epitope. PCR products digested with the appropriate enzymes were ligated into pLJM3 [48]. The accuracy of the nucleotide sequence of all the inserts in the constructed plasmids was checked by DNA sequencing. Y. enterocolitica T3S assays T3S assays were done as previously described [46]. We used Y. enterocolitica ΔHOPEMT or ΔHOPEMT ΔYscU strains carrying the plasmids described in Additional file 1: Table S1.