Studies of BM samples by various methods have indicated that the presence or absence of BMM is associated with the clinical outcome of patients with esophageal carcinoma [15, 16]. We currently investigated the DTCs in PB and BM by nested RT-PCR, to further confirm their clinical significance in ESCC. Because PB and BM are mesenchymal tissues that do not learn more normally express epithelial cell markers, detection of the expression of specific epithelial markers

in the PB and BM implies the presence of metastatic cancer cells. Although many epithelial markers have been used previously, such as carcinoma embryonic antigen, cytokeratins and survivin, it is important to identify new potential biomarkers [14, 15, 17]. STC-1 is a kind of glycoprotein hormone, first found in bony fish and later in humans and mammals, with a highly conserved homology. Its primary function in fish is prevention of hypercalcemia and stimulation of phosphate reabsorption [18]. In mammals, STC-1 appears to play multiple roles in a series of biological processes, including pregnancy, lactation, angiogenesis,

cerebral ischemia, oxidative stress and apoptosis [19–22]. Moreover, there is growing evidences suggesting that STC-1 is involved in carcinogenesis CHIR-99021 order [23]. STC-1 expression levels are universally much higher in tumor tissues and cancer cell lines, such as hepatocellular, colorectal, ovarian, breast cancer and medullary thyroid cancer, than those in corresponding normal tissues [7, 24–29]. Recently, Shirakawa et al[8] found that STC-1 mRNA and protein are overexpressed in ESCC tumors, compared with those in corresponding normal tissues, which significantly correlates with an advanced T status and poor prognosis for ESCC patients. This observation suggests that STC-1 may be useful as a tumor marker for ESCC. In fact, use of the STC-1 expression level as a diagnostic or prognostic biomarker in the blood has been validated in breast, lung, colorectal cancer, as well as hepatocellular

carcinoma and leukemia [11, 25, 30–33]. The detection of STC-1 mRNA in BM has also been reported in breast cancer, which correlates with multiple histopathological prognostic factors, including primary tumor size, the selleck chemical number of positive lymph nodes and TNM stage [33]. In concordance with previous studies, we Celastrol found that the level of STC-1 protein expression in ESCC was much higher than that in matched normal tissues, which further confirmed STC-1 as a promising tumor marker for ESCC. Moreover, STC-1 mRNA detection in PB and BM showed good sensitivity and specificity, the frequencies in PB and BM were 37.6% and 21.2%, respectively, which was comparable with other epithelial markers reported in ESCC. A previous study has indicated that DTCs detected in PB of breast cancer could not be an alternative to detect it in BM, because there are some different characters with each other [34].

12. Iwen PC, Kelly DM, Linder J, Hinrichs SH, Dominguez EA, Rupp ME, Patil KD: Change in prevalence and antibiotic resistance of Enterococcus species isolated from blood cultures over an 8-year period. Antimicrob Agents Chemother 1997, 41:494–495.PubMed

13. Top J, Willems RJ, Blok H, de Regt MJ, Jalink K, Troelstra A, Goorhuis B, Bonten MJ: Ecological replacement of Enterococcus faecalis by multiresistant clonal complex 17 Enterococcus faecium. Clin Microbiol Infect 2007, 13:316–319.CrossRefPubMed 14. Treitman AN, Yarnold PR, Warren J, Noskin GA: Emerging incidence of Enterococcus faecium among hospital isolates (1993 to 2002). J Clin Microbiol 2005, 43:462–463.CrossRefPubMed 15. de Regt MJ, Wagen LE, Top J, Blok HE, Hopmans TE, #PFT�� clinical trial randurls[1|1|,|CHEM1|]# Dekker AW, Hene RJ, Siersema PD, Willems RJ, Bonten MJ: High acquisition and environmental contamination rates of CC17 ampicillin-resistant Enterococcus faecium in a Dutch hospital. J Antimicrob Chemother 2008, 62:1401–1406.CrossRefPubMed 16. Willems RJ, Top J, van Santen M, Robinson DA, Coque TM, Baquero F, Grundmann H, Bonten MJ: Global spread

of vancomycin-resistant Enterococcus faecium from distinct nosocomial genetic complex. Emerg Selleckchem Talazoparib Infect Dis 2005, 11:821–828.PubMed 17. Moreno F, Grota P, Crisp C, Magnon K, Melcher GP, Jorgensen JH, Patterson JE: Clinical and molecular epidemiology of vancomycin-resistant Enterococcus faecium during its emergence in a City in southern Texas. Clin Infect Dis 1995, 21:1234–1237.PubMed 18. Wells CL, Juni BA, Cameron SB, Mason KR, Dunn DL, Ferrieri P, Rhame FS: Stool carriage, clinical isolation, and mortality during an outbreak of vancomycin-resistant enterococci in hospitalized medical and/or surgical patients. Clin Infect Dis 1995, 21:45–50.PubMed 19. Leavis H, Top J, Shankar N, Borgen K, Bonten M, van Embden JD, Willems RJ: A novel putative enterococcal pathogeniCity island linked to the esp

virulence gene of Enterococcus faecium and associated with epidemiCity. J Bacteriol 2004, 186:672–682.CrossRefPubMed 20. Willems RJ, Homan W, Top J, van Santen-Verheuvel M, Tribe D, Manzioros X, Gaillard C, Vandenbroucke-Grauls CM, Mascini EM, van Kregten E, van Embden JD, Bonten MJ: Variant esp gene as a marker of a distinct genetic lineage of vancomycin-resistant Enterococcus many faecium spreading in hospitals. Lancet 2001, 357:853–855.CrossRefPubMed 21. Heikens E, Bonten MJ, Willems RJ: Enterococcal Surface Protein Esp is Important for Biofilm Formation of Enterococcus faecium E1162. J Bacteriol 2007, 189:8233–8240.CrossRefPubMed 22. Van Wamel WJ, Hendrickx AP, Bonten MJ, Top J, Posthuma G, Willems RJ: Growth condition-dependent Esp expression by Enterococcus faecium affects initial adherence and biofilm formation. Infect Immun 2007, 75:924–931.CrossRefPubMed 23. Lund B, Edlund C: Bloodstream isolates of Enterococcus faecium enriched with the enterococcal surface protein gene, esp , show increased adhesion to eukaryotic cells. J Clin Microbiol 2003, 41:5183–5185.

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Given the young age of our survivor population and the rarity of other diseases in young patients, the increased values of NTproBNP found in GANT61 survivors may provide an useful information on late ANT subclinical cardiotoxicity. Conclusions Higher levels of NTproBNP detected in childhood leukemia survivors after low anthracycline cumulative doses might reflect an initial stage of ANT cardiotoxicity before the development of echocardiographic abnormalities. Although the

current studies support NTproBNP as one of the best available biochemical markers of late anthracycline cardiotoxicity, a possible strategy toward further improvement and combination with other cardiac biomarkers and novel echocardiographic methods should be explored in additional studies. Acknowledgments The authors thank Katarina Ondrejkovicova, M.Sc., for assistance with the analyses Selleck BIX 1294 of biomarkers. This work was supported by a grant of the Scientific Agency of the Ministry of Health 2007/42-UK-18, Slovak Republic. References 1. Mulrooney DA, Yeazel MW, Kawashima T, Mertens AC, Mitby P, Stovall M, Donaldson SS, Green DM, Sklar CA, Robison LL, Leisenring WM: Cardiac outcomes in a cohort of adult survivors of childhood and adolescent cancer: retrospective analysis of the Childhood Cancer Survivor Study cohort. BMJ 2009, 339:b4606.PubMedCrossRef 2. Lipshultz

SE, Miller TL, Scully RE, Lipsitz SR, Rifai N, Silverman LB, Colan SD, Neuberg DS, Dahlberg SE, Henkel JM, Asselin BL, Athale UH, Clavell LA, Laverdière C, Michon B, Schorin MA, Sallan SE: Changes in cardiac biomarkers during doxorubicin treatment of pediatric patients with Selleckchem LDN-193189 high-risk acute lymphoblastic leukemia: associations with long-term echocardiographic outcomes. J Clin Oncol 2012,30(10):1042–1049.PubMedCrossRef 3. Paulides M, Kremers A, Stöhr W, Bielack S, Jürgens H, Treuner J, Beck JD, Langer T, German Late Effects Working Group in the Society of Pediatric Oncology and Haematology (GPOH): Prospective longitudinal evaluation of doxorubicin-induced

cardiomyopathy in sarcoma patients: a report of the Late Effects Surveillance System (LESS). Pediatr Blood Cancer 2006, 46:489–495.PubMedCrossRef 4. Mladosievicova B, Foltinova A, Luptak I, Petrasova H, Hulin I: Frequency-domain analysis of the QRS complex after treatment Oxaprozin of childhood cancer with anthracycline cytostatics. Pediatr Cardiol 2001, 22:478–482.PubMedCrossRef 5. Kremer LC, van Dalen EC, Offringa M, Ottenkamp J, Voute PA: Anthracycline-induced clinical heart failure in a cohort of 607 children: long-term follow-up study. J Clin Oncol 2001, 19:191–196.PubMed 6. Salzer WL, Devidas M, Carroll WL, Winick N, Pullen J, Hunger SP, Camitta BA: Long-term results of the Pediatric Ooncology Group studies for childhood acute lymphoblastic leukemia 1984–2001: a report from the Children´s Oncology Group. Leukemia 2010,24(2):355–370.

On the other hand inhibition of PGE2 by celecoxib enhanced necrosis in cells infected by both isolates. It has been reported that PGE2-preventing necrosis is due to PGE2 involvement in the synthesis of the lysossomal Ca2+ sensor SYT7, which is essential for prevention of mitochondrial damage, enabling repair of plasma membrane disruption [14]. Although virulent mycobacteria sabotage of PGE2 to induce necrosis has been associated with increased production of LXA4[12, 13, 41], we did not detect LXA4 in the supernatant

of Mtb-infected alveolar macrophages (data not shown). TSA HDAC mw Nevertheless, the potential relationship click here between mycobacterial PLCs and host-cell necrosis through down-regulation of PGE2 production shown in this study is new evidence of the relevance of this virulence factor. Indeed, despite the described plc gene polymorphism [10], there is no genome or proteome characterised for PF-3084014 chemical structure either Mtb isolate, and further studies are necessary to better understand the differences between 97-1505 and 97-1200,

and the role of PLC in Mtb virulence. However, our data make a valuable demonstration of subversion of lipid mediator synthesis and its association with cell necrosis. Furthermore, our data are consistent with the recent finding of Bakala N’Goma and colleagues [7], who showed for the first time the cytotoxic effect of mycobacterial PLCs on macrophages. Finally, the relevance of PLCs as determinants Sirolimus chemical structure of virulence in Mtb expands our understanding of how these virulence factors can act to the detriment of the host, and highlights eicosanoids, such as PGE2 and LTB4, as mediators with functions that extend beyond innate immune mechanisms. Conclusion We found that the Mycobacterium tuberculosis bearing PLCs genes is more resistant to microbicidal activity of alveolar macrophages and induces cell necrosis, which is associated with subversion of PGE2

production. Methods Mycobacterium tuberculosis isolates The clinical isolates 97-1505 and 97-1200 were obtained from patients with active tuberculosis in 1998 and belong to a collection of 790 strains from RIVM (Bilthoven, The Netherlands). Both isolates were characterised regarding the polymorphisms in plc genes. The former has the entire plc-A and plc-B genes and an insertion of a copy of IS6110 at plc-C and the latter has all plc genes deleted. Also, analysis of the RFLP (Restriction fragment length polymorphism) pattern revealed similarities greater than 70% in the IS6110-RFLP profiles between the isolates [10]. Cultures were grown on Lowenstein-Jensen (LJ) solid medium then transferred to Middlebrook 7H9 (Difco, Detroit, MI) liquid medium supplemented with OADC (Difco). The culture was harvested by centrifugation, and the cell pellet was resuspended in sterile phosphate-buffered saline (PBS) and the number of bacteria was adjusted to 1 × 107 bacteria/ mL by absorbance in DO600nm.

Appl Environ Microbiol 2002, 68:2453–2460.PubMedCrossRef 42. Schibli DJ, Hwang PM, Vogel HJ: The structure of the antimicrobial

active center of lactoferricin B bound to sodium dodecyl sulfate micelles. FEBS Lett 1999, 446:213–217.PubMedCrossRef 43. Monk BC, Niimi K, Lin S, Knight A, Kardos TB, Cannon RD, et al.: Surface-active fungicidal D-peptide inhibitors of the plasma membrane proton pump that block azole resistance. Antimicrob Agents Chemother 2005, 49:57–70.PubMedCrossRef 44. Wessolowski A, Bienert M, Dathe M: Antimicrobial activity of arginine- and tryptophan-rich hexapeptides: the effects of aromatic clusters, D-amino acid substitution and cyclization. J Pept Res 2004, 64:159–169.PubMedCrossRef 45. López-García B, Ubhayasekera W, Gallo MK5108 RL, Marcos JF: Parallel evaluation of antimicrobial peptides derived from the synthetic PAF26 and the human LL37. Biochem Biophys Res Commun

selleckchem 2007, 356:107–113.PubMedCrossRef 46. Muñoz A, López-García B, Marcos JF: Studies on the mode of action of the antifungal hexapeptide PAF26. Antimicrob Agents Chemother 2006, 50:3847–3855.PubMedCrossRef 47. Yun DJ, Zhao Y, Pardo JM, Narasimhan ML, Damsz B, Lee H, et al.: Stress proteins on the yeast cell surface determine resistance to osmotin, a plant antifungal protein. Proc Natl Acad Sci USA 1997, 94:7082–7087.PubMedCrossRef 48. Toh-e A, Yasunaga S, Nisogi H, Tanaka K, Oguchi T, Matsui Y: Three yeast genes, Pir1 , Pir2 and Pir3 , containing internal tandem repeats, are related to each other, and Pir1 PAK6 and Pir2 are required for tolerance to heat-shock. Yeast 1993, 9:481–494.PubMedCrossRef 49. Stephens C, Harrison SJ, Kazan K, Smith FWN, Goulter KC, selleck inhibitor Maclean DJ, et al.: Altered fungal sensitivity to a plant antimicrobial peptide through over-expression of yeast cDNAs. Curr Genet 2005, 47:194–201.PubMedCrossRef 50. Levin DE: Cell wall integrity signaling in Saccharomyces cerevisiae . Microbiol Mol Biol Rev 2005, 69:262–291.PubMedCrossRef 51. Chen RE, Thorner J: Function and regulation in MAPK signaling pathways: Lessons learned from the yeast Saccharomyces cerevisiae. BBA-Mol Cell Res 2007, 1773:1311–1340.

52. Hohmann S: Osmotic stress signaling and osmoadaptation in Yeasts. Microbiol Mol Biol Rev 2002, 66:300–372.PubMedCrossRef 53. Al-Shahrour F, Díaz-Uriarte R, Dopazo J: FatiGO: a web tool for finding significant associations of Gene Ontology terms with groups of genes. Bioinformatics 2004, 20:578–580.PubMedCrossRef 54. Vaquerizas JM, Conde L, Yankilevich P, Cabezon A, Minguez P, Diaz-Uriarte JRS, et al.: GEPAS, an experiment-oriented pipeline for the analysis of microarray gene expression data. Nucleic Acids Res 2005, 33:W616-W620.PubMedCrossRef 55. Giaever G, Chu AM, Ni L, Connelly C, Riles L, Veronneau S, et al.: Functional profiling of the Saccharomyces cerevisiae genome. Nature 2002, 418:387–391.PubMedCrossRef 56.

aureus adhesion to and OSI-027 ic50 invasion of human osteoblasts. MG-63 osteoblastic cells were infected for 2 h at approximately 50 bacteria/cell with S. aureus strain 8325-4, pre-treated or not (untreated control) with 1/2 MIC linezolid, oxacillin or rifampicin, and S. aureus strain DU5883 Torin 2 solubility dmso lacking

fnbA and fnbB (negative control). To enumerate cell-associated bacteria, infected cells were washed twice to discard unbound bacteria and analysed by osmotic shock in pure water, and then, suitable dilutions of the lysates were plated on agar. The same procedure was used to quantify intracellular bacteria, except that the cells were incubated for 1 h with 200 mg/L gentamicin before the lysis step to kill extracellular bacteria. Adherent bacteria were calculated by subtracting intracellular bacteria from cell-associated bacteria. The results were expressed as the means +/- standard deviation of the percentage of recovered internalised (a) or adherent (b) bacteria with respect to inoculated bacteria derived from four independent experiments performed in duplicate. Asterisk = significantly different from the control (corresponding isolate grown without antibiotic), with a P value

of 0.05 by one-way analysis of variance followed by a posteriori Dunnett’s test. Discussion Several check details major findings emerge from this investigation of the impact of sub-inhibitory concentrations of anti-staphylococcal drugs on S. aureus adhesion and invasion phenotypes. S. aureus binding to human fibronectin and the transcriptional levels of the fnbA/B genes encoding the fibronectin-binding proteins were differentially modulated by antimicrobial agents. Oxacillin, moxifloxacin and linezolid treatment led to the development of a hyper-adhesive phenotype, along with an increase in fnbA/B mRNA levels relative to the gyrB 3-mercaptopyruvate sulfurtransferase internal standard. The same hyper-adhesive phenotype was induced by clindamycin treatment, although no significant change in fnbA/B mRNA levels was observed. Rifampin was the only antimicrobial agent among

those tested that significantly inhibited S. aureus binding to fibronectin without affecting relative fnbA/B transcription profiles. Vancomycin and gentamicin induced no change in either the adhesion phenotype or the fnbA/B transcription. S. aureus adhesion to and invasion of live eukaryotic cells was also assessed after oxacillin, linezolid or rifampin treatment in an ex vivo infection model of cultured human osteoblasts. Oxacillin treatment significantly increased S. aureus adhesion but not invasion, while no significant change in adhesion or invasion levels was observed after linezolid or rifampin treatment. Several recent studies have focused on the influences of sub-inhibitory concentrations of antimicrobial agents on the expression of various virulence factors produced by S. aureus and on the various regulation mechanisms involved in this modulation [6, 8, 17].

Cell flocculation also occurred when either arabinose or glycerol were added to M9/sup media instead of glucose (data not shown). Figure 1 Cell aggregation and adhesion by E . coli C PNPase-defective strain. A. Growth curves of E. coli C-1a (pnp +; solid symbols) and E. coli C-5691 (Δpnp-751; open symbols) in different media

(M9Glu/sup, diamonds; M9Glu, triangles) (left panel). Cell clumping by the C-5691 (Δpnp) strain led to deposition of ring-like aggregates on the flask walls (ABT-888 purchase indicated by the arrow; right panel). The picture was taken in the late exponential phase (OD600 = 5–6). B. Cultures of strains carrying pBAD24 derivatives grown up to OD600 = 0.6-0.8 in M9Glu/sup at 37°C with aeration were harvested by centrifugation, AR-13324 mouse resuspended in 0.04 vol M9 and diluted 25 fold in pre-warmed M9/sup with either 0.4% glucose (solid symbols) or 1% arabinose (empty symbols). Incubation at 37°C was resumed and growth monitored spectrophotometrically. Left panel: PNPase complementation. Right panel: suppression by RNase II. The aggregative phenotype of the C-5691 (Δpnp) strain was complemented by basal expression from a multicopy plasmid of the pnp gene under araBp promoter, indicating that low PNPase expression GSK2118436 nmr is sufficient to restore planktonic growth. Conversely, arabinose addition did not completely restore a wild type

phenotype (Figure 1B, left panel), suggesting that PNPase overexpression may also cause aggregation. Ectopic expression of RNase II suppressed the aggregative phenotype of the

pnp mutant (Figure 1B, right panel), thus suggesting that such a phenotype is controlled by the RNA degrading activity of PNPase. In contrast, however, RNase R overexpression did not compensate for lack of PNPase, indicating that different ribonucleases are not fully interchangeable in this process. Inactivation of the pnp gene induces poly-N-acetylglucosamine (PNAG) production In addition to macroscopic cell aggregation (Figures 1 and 2A), deletion of pnp stimulated adhesion to polystyrene microtiter Atazanavir plates in a standard biofilm formation assay [33] (Figure 2B) and resulted in red phenotype on solid medium supplemented with Congo red, a dye binding to polymeric extracellular structures such as amyloid fibers and polysaccharides (Figure 2C). Cell aggregation was also observed by phase contrast microscopy (Figure 2D). Altogether, these observations strongly suggest that inactivation of pnp triggers the expression of one or more extracellular factors implicated in cell aggregation and adhesion to solid surfaces. In order to identify such factor(s), we searched for deletion mutants in genes encoding known adhesion factors and biofilm determinants that could suppress the aggregative phenotype of the C-5691 (Δpnp) mutant strain.

Control siRNA (#MRT67307 purchase 4390843, Ambion, Inc., Austin, TX, USA or #6568 s, Cell Signaling Technology or sc-37007, Santa Cruz Biotechnology), RB siRNA (Silencer Select ID:s523, Ambion or sc-29468, Santa Cruz Biotechnology), and P53

siRNA (#6231 s, Cell Signaling Technology, or sc-29435, Santa Cruz Biotechnology) were employed. The sequences of these control siRNAs are detailed in the manufacturer websites. Quantitative real-time RT-PCR Total RNA was isolated with Quick-RNA miniPrep (Zymo Research, Irvine, CA, USA). Reverse Selleck LY2603618 transcription and quantitative real-time PCR was performed on ABI Prism 7500 (PE Applied Biosystems, TX, USA) using the One-Step SYBR ExTaq qRT-PCR kit (Takara, Shiga, Japan) according to manufacturer’s instructions. The following primers were used: for GAPDH 5′-GGTTTACATGTTCCAATATGATTCCA-3′

(forward), and 5′-ATGGGATTTCCATTGATGACAAG -3′ (reverse); for RB 5′-GCAGTATGCTTCCACCAGGC-3′ (forward), and 5′-AAGGGCTTCGAGGAATGTGAG-3′ (reverse); and for P53 5′-GCCCCCAGGGAGCACTA-3′ (forward), and 5′-GGGAGAGGAGCTGGTGTTG-3′ (reverse). Gene expression in clinical samples–data from databases NDC80 (Hec1) gene expression data in non-small cell AZD0156 datasheet lung cancer (NSCLC) were retrieved from publicly available database (Gene Expression Omnibus-GSE8894, GSE3141 and GSE37745). Gene expression intensities were normalized with quantile normalization. NDC80 expression between adenocarcinoma and squamous carcinoma was compared for all three different datasets. Eight genes known to associate with NDC80 were identified (18, 27). One way hierarchical clustering analysis for adenocarcinoma and squamous carcinoma of NSCLC was conducted by using R package software (http://​www.​r-project.​org/​). Results Hec1 inhibitor TAI-1 is highly potent with a wide anti-cancer spectrum The initial small molecule hits identified by Drs. Chen in Dr. WH Lee’s laboratory, INH1 and INH2, had micromolar

potency on cancer cell lines [3, 11, 12]. Leukotriene-A4 hydrolase Through medicinal chemical efforts to modify the hit structure, we have significantly improved the potency of the Hec1-targeted compound to low nanomolar level. The new compound, TAI-1, has a GI50 of 13.48 nM (K562 cells), which is close to 1000 times improvement in potency compared to INH1 (GI50 = 11.7 μM) (14). To characterize the potency of the new compound, TAI-1 (Figure 1), a series of cancer cell lines were tested. The screen includes 31 cancer cell lines, is comprise of 12 cell lines from the NCI-60 panel, and includes breast cancer, leukemia, liver, lung, colon cancer, cervical cancer, prostate cancer and bone cancer with various cellular characteristics. Growth inhibition was quantitated with established MTS assay. As summarized in Table 1, TAI-1 inhibits cellular growth at nM levels for the majority of cancer cell lines screened. Figure 1 Structure of Hec1 Inhibitor TAI-1.

With respect to flagellum biogenesis, an uncleaved form of FlhB (a YscU homologue) was demonstrated to selectively export only rod/hook-type Compound C nmr protein substrates but not filament type substrates [32]. This observation is in line with a modulatory

or substrate switching role for FlhB auto-cleavage. From all these studies, it appears that the context of the YscU homologue and its interactions with other secretory components influence T3SS function. It remains that auto-cleavage function is likely contextual and may have specific secretory consequences in different bacteria. In this study, we provide experimental evidence that EscU auto-cleavage Trichostatin A in vivo in EPEC promotes effector protein translocation into host cells during infection. In the absence of EscU auto-cleavage, very low levels of effector proteins were secreted as non-functional and abnormal forms.

EscU auto-cleavage also promoted efficient membrane association of the multicargo type III chaperone CesT, which has implications for effector delivery into cells during infection. Results Uncleaved forms of EscU support low levels of translocator and effector protein secretion learn more into culture supernatants Based on previous protein crystallography studies [26], we generated three recombinant plasmids that encode auto-cleaved or uncleaved histidine Interleukin-2 receptor tagged forms of EscU (39 kDa) (see Materials and Methods). EscU-HIS (pJLT21), EscU(N262A)-HIS (pJLT22) and EscU(P263A)-HIS (pJLT23) were created

for initial characterization studies. Unlike Shigella species where Congo Red is used to ‘induce’ in vitro type III secretion [33], culturing EPEC in DMEM ‘induces’ type III secretion [34, 35]. After culturing for 6 hours in DMEM, whole cell lysates and culture supernatants were collected from ΔescU strains harbouring pJLT21, pJLT22 and pJLT23. EscU auto-cleavage at the NPTH catalytic site is predicted to produce an 89 amino acid C-terminal product of 10.3 kDa. Immunoblotting whole cell lysates indicated that EscU-HIS was auto-cleaved due to the detection of an approximately 10 kDa species with anti-HIS antibodies (Figure 1A). A longer immunoblot exposure did not reveal any uncleaved EscU (39 kDa) suggesting complete auto-cleavage. In contrast, ΔescU whole cell lysates containing EscU(N262A) or EscU(P263A) produced a 39 kDa species detected by anti-HIS antibodies, a molecular weight consistent with uncleaved (intact) EscU. Figure 1 Efficient translocon and effector secretion is dependent on EscU auto-cleavage. (A): Immunoblot demonstrating EscU variant cleavage status within whole cell lysates. The blots were imaged separately to get representative signals for the auto-cleavage products. A longer exposure was used for the 39 kDa protein species.