RC341 Islet-3. Phylogeny of Vibrio sp. RC341 Islet-3 as determined by reconstructing a neighbor-joining tree using the Kimura-2 parameter as a nucleotide substitution model. (TIFF 7 KB) References 1. Pacha RE, Kiehn ED: Characterization and relatedness of marine vibrios pathogenic to fish: physiology, AZD1080 serology, and epidemiology. Journal of Bacteriology 1969,100(3):1242–1247.PubMed 2. Kushmaro A, Banin E, Loya Y, Stackebrandt E, Rosenberg E: Vibrio shiloi sp. nov., the causative agent of bleaching of the coral Oculina patagonica

. Int J Syst Evol Microbiol 2001, 51:1383–1388.PubMed 3. Guerinot ML, West PA, Lee JV, Colwell RR: Vibrio diazotrophicus sp. nov., a marine nitrogen-fixing bacterium. International Emricasan nmr Journal of Systematic and Evolutionary Microbiology 1982,32(3):350–357. 4. Hada HS, West PA, Lee JV, Stemmler J, Colwell RR: Vibrio tubiashii sp. nov., a pathogen of bivalve mollusks. International Journal of Systematic and Evolutionary Microbiology 1984,34(1):1–4. 5. Hedlund BP, Staley JT: Vibrio cyclotrophicus sp. nov., a polycyclic aromatic hydrocarbon (PAH)-degrading marine bacterium. Int J Syst Evol Microbiol 2001, 51:61–66.PubMed 6. Thompson CCVA, Souza RC, Vasconcelos ATR, Vesth T, Alves N, Ussery DW, Iida T, Thompson FL: Genomic Taxonomy of the Vibrios. In Vibrio2009. Rio de Janeiro, Brasil; 2009. 7. Thompson FL, Iida

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Nakagawa T, Shi L, Bi K, Kanoh Y, Tomochika K, Miyoshi S, Shimada T: Distribution of virulence-associated genes in Vibrio mimicus isolates from clinical and environmental origins. Microbiol Immunol 2004,48(7):547–551.PubMed 12. Boyd EF, Moyer KE, Shi L, Waldor MK: Infectious CTXΦ and the Vibrio pathogenicity island prophage in Vibrio mimicus : learn more evidence for recent horizontal transfer between V. mimicus and V. cholerae . Infection and Immunity 2000,68(3):1507–1513.PubMedCrossRef 13. Thompson FL, Swings J: Taxonomy of the Vibrios. In Biology of the Vibrios. Edited by: Thompson FL, Austin B, Swings J. Washington, D.C: ASM Press; 2006:29–43. 14. Choopun N: The population structure of Vibrio cholerae in Chesapeake Bay. In PhD Thesis.

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P. Trouillas     HQ692605 HQ692490 SACOO1 E. leptoplaca Populus nigra ‘C646 order italica’ Coonawarra, South Australia F.P. Trouillas     HQ692596 HQ692486 SACOO2 E. leptoplaca Populus nigra

‘italica’ Coonawarra, South Australia F.P. Thiazovivin nmr Trouillas     HQ692597 HQ692487 TUQU01 E. leptoplaca Quercus sp. Tumbarumba, New South Wales F.P. Trouillas     HQ692598 HQ692491 TUPN02 E. leptoplaca Populus nigra ‘italica’ Tumbarumba, New South Wales F.P. Trouillas     HQ692607 HQ692492 CNP03 Eutypella australiensis Acacia longifolia subsp. sophorae Coorong, South Australia F.P. Trouillas   DAR80712 HM581945 HQ692479 ADEL100 Eutypella citricola Ulmus procera Adelaide, South Australia F.P. Trouillas     HQ692580 HQ692520 ADSC100 E. citricola Schinus molle var. areira Adelaide, South Australia F.P. Trouillas     HQ692577 HQ692510 T10R4S7 ª E. citricola Vitis vinifera Hunter Valley, New South Wales W.M. Pitt     HQ692578   T2R3S3 ª E. citricola Vitis vinifera Hunter Valley, New South Wales W.M. Pitt     HQ692575   T3R2S2 ª E. citricola Vitis vinifera Hunter Valley, New South Wales W.M. Pitt     HQ692576 HQ692519

HVIT03 E. citricola Vitis vinifera Hunter Valley, New South Wales F.P. Trouillas/W.M. Pitt     HQ692582 HQ692511 HVIT07 click here E. citricola Vitis vinifera Hunter Valley, New South Wales F.P. Trouillas/W.M. Pitt CBS128330 DAR81033 HQ692579 HQ692512 HVIT08 E. citricola Vitis vinifera Hunter Valley, New South Wales F.P. Trouillas/W.M. Pitt     HQ692583 HQ692513 HVOT01 E. citricola Citru sinensis Hunter Valley, New South Wales F.P. Trouillas/W.M. Pitt CBS128331 DAR81034 HQ692581 HQ692509 HVGRF01 E. citricola Citrus paradisi Hunter Valley, New South Wales F.P. Trouillas/W.M. Pitt CBS128334 DAR81037 HQ692589 HQ692521 WA02BO E. citricola Vitis vinifera Western Australia F.P. Trouillas     HQ692584 HQ692514 WA03LE E. citricola Citrus limon Swan Valley, Western Australia F.P. Trouillas     HQ692585 HQ692515 WA04LE E. citricola Citrus limon Swan Valley, Western Australia F.P. Trouillas CBS128332 DAR81035 HQ692586 HQ692516 WA05SV E. citricola Vitis vinifera Swan Valley, Western Australia F.P. Trouillas CBS128333 DAR81036

HQ692587 HQ692517 WA06FH E. citricola Vitis vinifera Western Urocanase Australia F.P. Trouillas     HQ692588 HQ692518 HVFIG02 Eutypella cryptovalsoidea Ficus carica Hunter Valley, New South Wales F.P. Trouillas/W.M. Pitt CBS128335 DAR81038 HQ692573 HQ692524 HVFIG05 E. cryptovalsoidea Ficus carica Hunter Valley, New South Wales F.P. Trouillas/W.M. Pitt     HQ692574 HQ692525 ADEL200 Eutypella microtheca Ulmus procera Adelaide, South Australia F. P. Trouillas     HQ692559 HQ692527 ADEL300 E. microtheca Ulmus procera Adelaide, South Australia F. P Trouillas     HQ692560 HQ692528 YC16 ª E. microtheca Vitis vinifera Hunter Valley, New South Wales W.M. Pitt     HQ692561 HQ692529 YC17 ª E. microtheca Vitis vinifera Hunter Valley, New South Wales W.M. Pitt     HQ692562 HQ692537 YC18 ª E.

Discussion Due to the anticipated importance of membrane- and membrane-associated NCT-501 price proteins of M. tuberculosis in bacterial virulence, it is essential to map these proteins. Therefore, the aim of this study was to characterize the repertoire of membrane and membrane associated proteins from the two widely used M. tuberculosis strains H37Rv (virulent) and H37Ra (avirulent). As the M. tuberculosis H37Ra genome has recently been sequenced, there is currently great interest

in investigating the differences between the two strains in more detail [34–36]. The protein profile data of the two strains were further analysed with the aim of finding relative quantitative differences of the observed proteins. Using proteomic data to quantify proteins gives a more realistic impression about the protein content and hence the physiological state of the bacilli, rather than mRNA measurement, as mRNA levels do not necessarily reflect the amount of proteins expressed. High-throughput proteomics using state-of-art instruments is well suited for providing more detailed information of the differences in expressed proteins between the two strains, complementing and adding to prior studies that have mainly focussed on gene expression by mRNA measurements [10, 36]. We observed that the vast majority of the proteins were present in both strains

and had similar relative abundance (Figure 2). This was expected as the two strains are closely related. However, a small group of proteins had a different relative abundance in the two strains. Among the differently abundant proteins, a member check details of the general secretory (Sec) pathway (Rv2586c, SecF) was identified with over 6 fold higher relative abundance in M. tuberculosis before H37Rv compared to M. tuberculosis H37Ra (Table 1). In bacteria, the bulk of protein export across the cytoplasmic membrane is carried out by this pathway [37–39]. The final destination of Sec exported proteins can be the cell envelope or the extracellular space. The

Sec pathway is well-characterized in Escherichia coli [37, 38, 40]. At the core of the Sec pathway is a membrane-spanning translocation channel composed of the integral membrane proteins: Rv0638 (SecE1), Rv0379 (SecE2), Rv2586c (SecF), Rv1440 (SecG), AG-881 Rv0732 (SecY) [41]. SecA binds to cytoplasmic precursor proteins destined for export and delivers them to the translocation machinery through its ability to bind to membrane phospholipids [42]. The three subunits with predicted transmembrane regions that comprise the core of the Sec translocation and export machinery are all identified in both strains. The two other components, Rv0732 (SecY) and Rv2587c (SecD), also have higher relative abundance in M. tuberculosis H37Rv. Since we restricted the analysis only to the ones with 5 fold difference or more, these were not included in the Table 1. Nevertheless, our data indicates a trend of higher expression of these subunits.

Pooled samples did conceivably result in an enrichment of the more shared taxa possibly

preventing the detection of taxa associated only to a few individual samples. DNA was used as template to construct three 16 S rRNA libraries; a total of 276 clones (from 78 to 116 per library) were sequenced. Sequence analysis revealed, as expected, that the soil selleck kinase inhibitor community was the most diverse (Shannon H’ = 4.63; Chao1 = 168), while the nodule-associated community was less diverse (Shannon H’ = 1.98; Chao1 = 30), (Additional file 3: Table S3). As a consequence, the library of nodules showed a coverage (85.9%) higher than those of stems + leaves (74.1%) and soil (47.1%). The percentages of taxonomic classes detected in the sequences https://www.selleckchem.com/products/epz-6438.html of the clone libraries are reported in Figure

2. Seven classes were represented in both soil and stem + leaf communities, and 4 of them were also found in nodules. Alphaproteobacteria were dominant in nodules (as expected, due to the presence of high LGX818 ic50 titres of the symbiotic alphaproteobacterium S. meliloti) and in stems + leaves. Also in soil Alphaproteobacteria were highly prevalent, but Acidobacteria and Crenarchaeota were also abundant. Flavobacteria were found only in nodules, however a low presence in the other environments cannot be excluded, especially in relation to the lower coverage of the respective libraries. Beta- and Gammaproteobacteria and Actinobacteria were found in all three libraries. Figure 2 Representation of bacterial divisions in the 16 S rRNA gene clone libraries. The percentage of clones accounting for each division with respect to its origin (nodule, stems + leaves, soil) is reported. Concerning Alphaproteobacteria, only members of the Rhizobiaceae family were found in nodules, with all sequences assigned, as expected, to the Sinorhizobium/Ensifer genus (Figure 3). Flavopiridol (Alvocidib) Alphaproteobacteria present in soil belonged to the Rhizobiaceae, Bradyrhizobiaceae,

Methylocystaceae, Hypomicrobiaceae and Caulobacteraceae families. Rhizobiaceae, Aurantimonadaceae and Methylobacteriaceae, all belonging to the Rhizobiales, plus taxa of the order Sphingomonadales, were found in the stem + leaf library. The absence of sequences assigned to the Sinorhizobium/Ensifer genus from stem + leaves and soil libraries, though this species was found by qPCR in both these environments (see the following paragraph), could be due to its low abundance and to the relatively low coverage of clone libraries. Figure 3 Distribution of the recovered families in Alphaproteobacteria with respect to their origin (nodule, stems + leaves, soil). The percentage of clones present in the libraries for each family is reported.

Subsequently, she appeared infectious symptoms on July 20, with the highest body https://www.selleckchem.com/products/tideglusib.html temperature of 39.5°C. Urine and blood of the patient were collected on July 20 and 21 for microbiological culture. A carbapenem-susceptible E. coli isolate with only resistance to ampicillin, gentamycin, tobramycin and trimethoprim/sulfamethoxazole was isolated from urine sample, while another carbapenem-susceptible E. coli isolate with

same resistance profiling as that of the isolate from urine sample was isolated from blood sample. The patient’s symptoms improved following the treatment with cefuroxime and ceftazidime via intravenous drip. On August 6, urine sample was collected for microbiological culture again. Surprisingly, a carbapenem-resistant E. coli isolate with pure growth,

named E. coliWZ33, was isolated from urine sample. After subjected to be treated with antimicrobials for 5 days, the symptoms of the patient disappeared and she was discharged from the hospital. The other carbapenem-resistant isolate E. coliWZ51 was isolated from the sputum of a 66-year-old male patients with pulmonary infection at FAHWMU. Before admitted to FAHWMU, the patient was hospitalized at another comprehensive hospital away from FAHWMU about SHP099 research buy 30 kilometers for anti-infection therapy using levofloxacin. After hospitalization at FAHWMU on March 19, the patient was subjected to treatment of pulmonary infection using ceftazidime via intravenous drip. On March 20, sputum sample was collected for bacterial culture and carbapenem-resistant isolate, E. coliWZ51, was identified later. After subjected to be treated with ceftazidime for 4 days, the symptoms of the patient disappeared. Antimicrobial resistance determinants As both E. coli WZ33 and WZ51 were resistant to third-generation mafosfamide cephalosporin

and carbapenems, MHT was performed to determine the production of carbapenemases. Unexpectedly, both tested isolates were MHT negative. For further investigation on carbapenemase production, a double-disc synergy test was used for detecting the MBL production. As expected, both tested isolates were found to produce MBLs. The genes encoding carbapenemases, including bla VIM, bla IMP, bla SPM-1, bla GIM-1, bla SIM-1 and bla NDM-1, were further investigated by PCR and DNA sequencing. Two carbapenem-resistant isolates with carbapenemase production, E. coli WZ33 and WZ51, were positive for bla NDM-1. The MHT has an excellent selleck inhibitor sensitivity for detecting enterobacterial isolates producing KPC- and OXA-48-type carbapenemases, but has low sensitivity for the detection of NDM-1 producers [26]. Previous study reported that negative or weakly positive MHT results were observed for 11 of 15 NDM-1-producing strains [27]. Two NDM-1-producing K. pneumonia clinical isolates reported by our previous study were also MHT negative [16]. In the present study, two NDM-1-producing E. coli isolates were also negative for MHT.

The deconvolution of emission band allows to put in evidence two different signals: the first one, with a maximum at 420 nm, due to the emission from band edge, and the second one, in the range 520 to 560 nm, due to ‘shallow defect’. These reticular defects, mainly localized on the NCs selleck surface, can Momelotinib be attributed to anionic insaturation [26, 27]. In the literature, many examples of CdS NCs in which shallow defects play an important role are reported [28, 29]. In our case, the intensity of

emission from shallow defects is very low with respect to the emission band edge, indicating a good optical quality of synthesized CdS NCs. Figure 4 PL spectra of CdS NCs. In MEH-PPV (a) and in PMMA (b) grown at 175°C and 185°C (excitation wavelength 330 nm), respectively. Microstructural analysis: X-ray scattering and transmission electron microscopy The X-ray diffraction (wide angle X-ray scattering (WAXS)) measurements of CdS/MEH-PPV nanocomposites obtained at 185°C for the samples with a weight/weight ratio

of 1:4 and 4:1 are shown in Figure 5. Curve A shows the WAXS pattern of the pristine MEH-PPV polymer (without of [Cd(SBz)2]2·MI precursors) exhibiting the broad polymer peak (labelled as P) and the characteristic weak Bragg peaks (denoted by asterisk ‘*’) that are related to the presence of nanodomains of mesomorphic order, i.e. crystallites of orthorhombic structure (local packing chains of MEH-PPV chains), as observed and reported in the literature [30, 31]. ML323 In particular, the broad peak P corresponds to the interbackbone spacing (0.43 nm) in the direction normal to the

coplanar phenylene rings, while the periodic angular peak distribution yields a lattice spacing of about 2.5 nm, and is in very good agreement with the bilayer spacing of the two neighbouring MEH-PPV chains (2.47 nm), i.e. MEH-PPV ethylhexyloxy side groups are interdigitated [32]. Figure 5 X-ray scattering Astemizole measurements (WAXS) of CdS/MEH-PPV nanocomposites. Obtained at 185°C for samples with precursor/polymer weight/weight ratio of 1:4 (curve B) and 4:1 (curve C). For reference and comparison, the WAXS pattern of pristine MEH-PPV is also shown (curve A). The diffraction peaks labelled as ‘P’ and asterisk ‘*’are due to the crystalline nanodomains of the conjugated polymer. Curve B in Figure 5 shows the WAXS pattern of the CdS/MEH-PPV nanocomposites obtained after annealing at 185°C for the samples with a weight/weight ratio of 1:4. Here, besides the MEH-PPV diffraction peaks, broad X-ray peaks attributed to the formation of CdS nanocrystals are also observed. Also, curve C obtained for the samples with a weight/weight ratio of 4:1 shows the CdS nanocrystal peaks. However, in this case, the polymer peaks (P and the weak peaks of the polymer superstructure) are not observed or are too low to be experimentally observed due to the low polymer content.

Outwardly, the N1 spectra of the catalysts synthesized

with cobalt acetate and cobalt nitrate are apparently different from that with cobalt oxalate and cobalt chloride. The peak at about 401 eV is obviously higher than that at about 398 eV for the former, while the height of these peaks check details is almost the same for the latter. The spectra in Figure 7 have been deconvoluted into various types of nitrogen as shown and the specific concentration of each state of nitrogen is listed in Table 3. The nitrogen distribution in the studied catalysts can be classified into two groups. Similar results have been obtained in the catalysts prepared from cobalt acetate and cobalt nitrate, and closely similar distributions have been exhibited in the catalysts synthesized from cobalt oxalate and cobalt chloride. This is probably

because of the fact that the reconfiguration of the catalyst, especially the decomposition of PPy and the insertion of nitrogen into carbon, during high-temperature pyrolysis could be interfered by the transforming process of cobalt ion in the used precursor into metallic cobalt. When cobalt acetate and cobalt nitrate are used, they thermally decompose under inert atmosphere into cobalt oxide and then metallic cobalt [42–45]. When cobalt oxalate is employed, C646 However, it thermally Mdm2 inhibitor decomposes into metallic cobalt directly [46–48], and the cobalt ion in cobalt chloride is reduced by carbon directly into metallic cobalt [49, 50]. Thus, different states and the corresponding content of nitrogen in the final catalysts have been achieved. As to the correlation selleck chemicals between the ORR performance of the catalysts and the concentration of each type of nitrogen in the catalysts, neither positive nor negative trend could be found. Therefore, it is difficult at present to expatiate the specific contribution of each type of nitrogen to the ORR catalytic performance of the Co-PPy-TsOH/C catalysts, maybe synergistic

effects exist among them. Figure 7 XPS spectra for N1s core-level peaks in Co-PPy-TsOH/C catalysts prepared from various cobalt precursors. (a) Cobalt acetate; (b) cobalt nitrate; (c) cobalt oxalate; (d) cobalt chloride. Table 3 Surface atomic concentration of different types of nitrogen in Co-PPy-TsOH/C catalysts prepared from various cobalt precursors Cobalt precursor Pyridinic-N Pyrrolic-N Graphitic-N Oxidized-N Cobalt acetate 0.308 0.225 0.279 0.188 Cobalt nitrate 0.297 0.204 0.293 0.207 Cobalt oxalate 0.345 0.305 0.197 0.153 Cobalt chloride 0.355 0.311 0.175 0.159 Figure 8 exhibits content of diverse elements in the Co-PPy-TsOH/C catalysts prepared with various precursors. Comparable carbon contents have been achieved in the studied catalysts. However, the content of other elements differs greatly from each other. Cobalt content in the catalysts prepared with cobalt acetate, cobalt nitrate, and cobalt chloride is obviously higher than the designed value of 10.

1%). 12 patients (4.7%) underwent gastro-duodenal resection and 6 patients (2.4%) received conservative treatment. The remaining patients underwent alternative procedures. Of the 145 patients with small bowel perforations, 98 underwent open small bowel resection (85.2%) and 3 (2%) underwent laparoscopic small bowel resection. 28 patients (19.3%) were treated by stoma. Among the 115 patients with colonic non-diverticular perforation, 42 (36.5%) underwent Hartmann resection, 26 (22.6%) underwent open resection with anastomosis and without stoma protection, and 26 underwent open resection with stoma protection (22.6%). 170 cases (8.9%) were attributable to post-operative

infections. Source control was successfully implemented for 1,735 patients (91.4%). Microbiology Intraperitoneal

specimens were collected BVD-523 in vitro from 1,190 patients (62.7%). These specimens Selleck PD 332991 were obtained from 977 of the 1,645 patients presenting with community-acquired intra-abdominal infections (59.4%). Intraperitoneal specimens were collected from 213 (84.2%) of the remaining 253 patients with nosocomial intra-abdominal infections. The aerobic bacteria identified in intraoperative samples are reported In Table 4, 5. Table 4 Aerobic bacteria identified from intra-operative peritoneal fluid Total 1.330 (100%) Aerobic Gram-negative bacteria 957 (71.9%) Escherichia coli 548 (41.2%) (Escherichia coli resistant to third generation cephalosporins) 75 (5.6%) Klebsiella pneuumoniae 140 (10.5%) (Klebsiella pneumoniae resistant to third generation cephalosporins) 26 (1.4%) Klebsiella oxytoca 11 (0.8%) (Klebsiella oxytoca resistant to third generation cephalosporins) 2 (0.1) Enterobacter 64 (4.8%) Proteus 47 (3.5%) Pseudomonas 74 (5.6%) Others 73 (5.6%) Aerobic Gram-positive bacteria 373 (29.1%) Z-VAD-FMK chemical structure Enterococcus faecalis 153 Rho (11.5%) Enterococcus faecium 58 (4.4%) Staphylococcus

Aureus 38 (2.8%) Streptococcus spp. 85 (6,4%) Others 39 (2.9%) Table 5 Aerobic bacteria from intra-operative samples in both community-acquired and healthcare-associated IAIs Community-acquired IAIs Isolates n° Healthcare-associated (nosocomial) IAIs Isolates n° Aerobic bacteria 1030 (100%) Aerobic bacteria 300 (100%) Escherichia coli 456 (44.3%) Escherichia coli 92 (21%) (Escherichia coli resistant to third generation cephalosporins) 56 (5.4%) (Escherichia coli resistant to third generation cephalosporins) 19 (6.3%) Klebsiella pneumoniae 105 (10.1%) Klebsiella pneumoniae 35 (11.7%) (Klebsiella pneumoniae resistant to third generation cephalosporins) 11 (0.1%) (Klebsiella pneumoniae resistant to third generation cephalosporins) 15 (5%) Pseudomonas 56 (5.4%) Pseudomonas 18 (5.7%) Enterococcus faecalis 106 (10.3%) Enterococcus faecalis 47 (15.7%) Enterococcus faecium 38 (3.7%) Enterococcus faecium 20 (6.7%) The microorganisms isolated in subsequent samples from peritoneal fluid are reported in Table 6.

Samples were incubated in the presence (+) or absence (-) of trypsin Selleckchem Ruxolitinib and analyzed by immunoblot analysis using polyclonal anti-VacA serum #958. To analyze potential differences in folding properties of the VacA mutant proteins compared to wild-type VacA, we analyzed the susceptibility of these proteins to proteolytic cleavage. Lysates of H. pylori strains were generated by sonication, and the solubilized proteins

were treated with trypsin as described in Methods. Trypsin digestion of two of the mutant proteins (Δ511-536 and Δ517-544) yielded proteolytic digest patterns that were identical to each other and similar to that of trypsin-digested wild-type VacA (Fig. 3B). Trypsin digestion of two other mutant proteins (Δ433-461 and Δ484-504) yielded different digest patterns, but these mutant proteins were not completely degraded (Fig. 3B). Four mutant proteins (Δ462-483, Δ559-579, Δ580-607, and Δ608-628) were completely degraded by trypsin (Fig. 3B). In general, the four mutant proteins that exhibited SB203580 supplier Relative resistance to trypsin digestion were secreted at relatively high levels compared to mutant proteins that were completely degraded by trypsin (compare Fig. 2 and Fig. 3B). The observed variation among mutant VacA proteins in susceptibility to trypsin-mediated proteolysis suggested that the individual mutant proteins differed Selleck SN-38 in

folding properties. The proteins that were highly susceptible to trypsin digestion and secreted at very

low levels (Δ462-483, Δ559-579, Δ580-607, and Δ608-628) were probably misfolded. Due to the very low Cetuximab cost concentrations of these four proteins in the broth culture supernatants, these mutant VacA proteins were not studied further. To evaluate whether the four mutant proteins exhibiting relative resistance to trypsin-mediated proteolysis (i.e. VacA Δ433-461, Δ484-504, Δ511-536, and Δ517-544) shared other features with wild-type VacA, we analyzed the reactivity of these proteins with an anti-VacA monoclonal antibody (5E4) that recognizes a conformational epitope [35]. Each of the four mutant VacA proteins was recognized by the 5E4 antibody (Fig. 4), which provided additional evidence that these mutant proteins were folded in a manner similar to that of wild-type VacA. Figure 4 Reactivity of VacA mutant proteins with a monoclonal anti-VacA antibody. Wild-type H. pylori strain 60190 and strains expressing mutant VacA proteins were grown in broth culture, and secreted VacA proteins were normalized as described in Methods. Wells of ELISA plates were coated with broth culture supernatants, and reactivity of the proteins with an anti-VacA monoclonal antibody (5E4) that recognizes a conformational epitope was determined by ELISA. Reactivity of a vacA null mutant was subtracted as background. Relative VacA concentrations are indicated. Values represent the mean ± SD from triplicate samples.