Figure 1 A schematic representation of the cadF gene and its adja

Figure 1 A schematic representation of the cadF gene and its adjacent genetic loci for C. lari RM2100, including locations of the novel primers designed in silico (A). Nucleotide sequences of the primers are also shown (B). Table 1 C. lari isolates and other thermophilic Campylobacter reference strains analyzed in the present study and their accession numbers of the nucleotide sequence data accessible in DDBJ/EMBL/GenBank Isolate no. Source Country

Accession number C. lari JCM2530T Seagull Japan AB465344 C. lari 298 Human Canada AB465345 C. lari 300 Seagull USA AB465346 C. lari 84C-1 Human N. Ireland AB465347 UPTC 99 Sea water N. Ireland AB465348 UPTC NCTC12892 River water ABT-888 datasheet England AB295430 UPTC NCTC12893 River water England AB295431 UPTC NCTC12894 Sea water England AB295432 UPTC NCTC12895 Mussel England AB295433 UPTC NCTC12896 Mussel Salubrinal solubility dmso England AB295434 UPTC CF89-12 River water Japan AB295435 UPTC A1 Seagull N. Ireland AB295436 UPTC A2 Seagull N. Ireland AB295437 UPTC A3 Seagull N. Ireland AB295438 UPTC 89049 Human France AB295439 UPTC 92251 Human France AB295440 C. lari RM2100 Human USA AAFK01000002 C. jejuni NCTC11168 Human USA NC_002163 C. jejuni RM1221 Chicken USA NC_003912 C. jejuni 81-176 Human USA NC_008787 C. jejuni 260.94 Human South

Africa AANK01000004 C. jejuni CF93-6 Human Japan AAFJ01000005 C. jejuni HB93-13 Human China AANQ01000001 C. jejuni 84-25 Human Unknown AANT02000001 C. jejuni ss doylei

269.97 Human Unknown AARB01000000 C. coli RM2228 Chicken GSK1904529A supplier USA AAFL01000008 C. upsaliensis RM3195 Human USA AAFJ01000005 The combined sequences of an approximately 2.3 kbp region encoding a partial and putative ribosomal protein SI rpsI open reading frame (ORF) (165 bp), a NC region downstream of the ORF (approximately 250 bp), a putative cadF (-like) ORF (984 bp), a Cla_0387 ORF (642 bp), a NC region (approximately 120 bp) and a partial and putative Cla_0388 ORF (126 or 128 bp) were identified with all 16 C. lari isolates examined. The present sequence analyses identified the putative ORF for cadF (-like) gene to be 984 bp [nucleotide position (np) 414-1,397 bp for the C. lari JCM2530T] with all 16 C. selleck chemicals llc lari isolates (n = 4 UN C. lari; n = 12 UPTC) and UN C. lari RM 2100. With regard to the cadF-like gene, the sequence commenced with an ATG start codon for all isolates and terminated with a TAA for 13 isolates and with a TGA for the other three isolates (NCTC12894, 12895 and 99). Regarding putative ORFs for cadF (-like) gene, apparent size differences occurred amongst the four thermophilic Campylobacter species examined, 984 bp (328 amino acid residues) for 16 C. lari isolates and C. lari RM2100 strain, 957 (319) for C. jejuni RM1221 and NCTC11168, 996 (332) for C. coli RM2228, and 948 (316) for C. upsaliensis RM3195, as shown in Table 2, although in this limited study a small number of reference strains of C. jejuni, C. coli and C.

Figure 1 Dose–response curve of PPI treatment in esophageal cance

Figure 1 Dose–response curve of PPI treatment in esophageal cancer cell lines. The figure presents an overview of the impact of PPI treatment with esomeprazole on tumour cell survival in SCC (A) and EAC (B) cells. PPI: proton pump inhibitor esomeprazole. Esomeprazole suppresses the metastatic potential of esophageal cancer cell lines Adhesion and migration are key determinants of the ability of tumour cells

to metastasize into distant organs, as metastasis includes invasion of see more circulating tumour cells into distant organs where the tumour cells have to adhere and migrate through the endothelium of the vessels. We therefore investigated the impact of esomeprazole treatment on adhesion and migration in esophageal cancer cell lines. Figure 2 presents an overview of the results of adhesion and migration assays performed on SCC (A) and SNS-032 mouse EAC (B) cell lines after PPI treatment with esomeprazole. After 15, 30, 60 and 90 minutes of PPI treatment, the ability of tumour cells to adhere

to coated wells under the stimulation of TGF-β2 was significantly reduced in both tumour entities compared to untreated controls (p ≤ 0.025). Furthermore, the ability of tumour cells (SCC and EAC) to migrate through 8-μm pores in a coated Boyden Chamber was significantly reduced after PPI treatment compared to controls (p < 0.0001). Figure 2 Effect of PPI treatment on metastatic potential of esophageal cancer cell lines. The figure presents an overview about the effect of PPI treatment on cell adhesion (1) and migration (2) in SCC (A) and EAC (B) cell lines. Negative controls (i.e. adhesion and migration assays with uncoated wells) were performed though for visual clarity they are not included in the figures. PPI treatment: treatment with proton pump

inhibitor esomeprazole. Control: untreated Roflumilast control cells. *: statistically significant different compared to control (p ≤ 0.025). Esomeprazole augments the cytotoxic effect of cisplatin and 5-FU in esophageal cancer cell lines Given the suppressive effect of esomeprazole on the survival and metastatic potential of esophageal cancer cells, we were interested if esomeprazole might affect the sensitivity of esophageal cancer cells towards commonly used chemotherapeutic drugs such as cisplatin and 5-FU. We therefore treated tumour cells with either esomeprazole alone at different concentrations, or with cisplatin or 5-FU at the respective LD50 concentrations, or with esomeprazole and chemotherapeutics together. Figure 3 presents an overview of the impact of esomeprazole treatment on otherwise untreated cells or on cells that were treated simultaneously with chemotherapeutics. Esomeprazole in „sub-lethal dose“ did not impact on survival of untreated or simultaneously chemotherapy treated SCC or EAC cancer cells. Applied in „lethal“ or „highly lethal doses“, however, esomeprazole reduced the survival of otherwise untreated cells of both tumour entities (p < 0.05) as expected.

Provided that the corresponding oligonucleotides were included on

Provided that the corresponding oligonucleotides were included on the array, all Pritelivir clinical trial species that were detected by cloning-sequencing could also be

identified with the phylochip. As the corresponding oligonucleotides were lacking on the phylochip, species belonging to the Atheliaceae, Sebacinaceae or Pezizales selleck kinase inhibitor were not detected. Furthermore, the comparison of array signal intensity with ITS sequence frequency in the ITS clone library revealed the potential of the phylochip to detect taxa that were represented by approx. 2% of DNA types in the amplified DNA sample. However, the quantitative potential of this custom phylochip remains to be further accessed as bias linked to the PCR amplification could take place. The phylochip also detected species that were not expected

according to the results obtained from the use of the other two approaches. This could be due to cross-hybridisations and/or to the fact that these under-represented species in the community could not be detected by the other Ralimetinib mw approaches as the rarefaction curves of the ITS library sequencing method did not reach a plateau (Additional file 1). When compared to each other, both of the other approaches provided similar, but not identical, profiles of the ECM communities. Approximately 70% of the species were detected using either method individually (Table 1). For the beech sample, three species were detected only by morphotyping as the PCR amplification of their DNA using ITS1F/ITS4 and/or NSI1/NLB4 primer pairs failed. Tedersoo et al. [35] showed that PCR of ITS from several ECM species failed using these universal fungal rDNA primers, and they stressed the need for additional taxon-specific PCR

primers to be used for comprehensive genotyping of ECM communities. One of the morphotypes detected in the beech sample was a Lactarius species. In the same root sample, a Pezizales species was found by ITS-sequencing and cloning/sequencing; this suggests a possible co-colonisation of the ECM root tip [36]. ECM root tips can be colonised by more Tyrosine-protein kinase BLK than one fungal taxon, by two different ECM species, or by one ECM species and an endophytic or parasitic species. Typically, these species are overlooked by the use of only morphotyping, but they can be detected by molecular biological approaches. Conclusion In this study, we demonstrated that identification of ECM fungi in environmental studies is possible using a custom phylochip. The detection of most of the species by the phylochip was confirmed by two other widely used detection methods. Although the possible application of the phylochip technique to other study areas is dependent on the fungal species to be analysed, high-quality sequence support for several temperate and boreal forest ecosystems is found in databases such as UNITE [11].

The survival of HCC patients after

resection remains poor

The survival of HCC patients after

resection remains poor, mainly attributing to frequent metastases and recurrence [2]. Recently, plenty of researches have performed to explore mechanisms underlying the initiation, propagation and development of HCC [3,4]. However, the complexity of HCC need further hypothesis-drove researches to be exerted. Dysfunction of the cellular transport machinery is commonly observed in multiple cancers including HCC [5]. Although some molecules are able to diffuse through the large Nucleus Pore Complexes (NPCs) in the nucleus membrane, factors larger than 45 kDa including that associated with malignant diseases need to be mediated by karyopherin to import into the nucleus [6]. Karyopherin alpha 2 (KPNA2) is one of A-1155463 order karyopherin a family, and could form heterodimer with Karyopherin 1 to promote nucleus protein import as an adapter protein [7]. Recent studies have illustrated that KPNA2 might be a critical oncogene and a potential prognostic biomarker in malignant diseases including HCC [8–11]. Furthermore, Barasertib in vitro KPNA2 knock-down could significantly inhibit HCC proliferation [12]. But till now, the mechanistic evidence of KPNA2 in HCC

was obscure and deserved to be explored. Transcriptional factors are widely involved in cancers and are bound to be enriched in nucleus. It raised the hypothesis that KPNA2 might affect cancer cells through the translocation of cancer-associated transcriptional factors. Previous report has indicated the direct association of KPNA2 with a zinc-finger transcription factors pleomorphic adenoma gene 1 (PLAG1) by the yeast two-hybrid system [13], suggesting PLAG1 might be one of critical mediators of KPNA2 effects in malignant diseases. PLAG1 was identified as a candidate oncogene in various malignant cancers. Recent report illustrated the over-expression of PLAG1 in hepatoblastoma, suggesting a potential role of PLAG1 in liver malignant disease [14]. Besides, insulin-like Montelukast Sodium growth factor

2 (IGF-II), cellular retinoic acid binding protein (CRABP2) and cytokine receptor-like factor 1 (CRLF1), which are confirmed targets of PLAG1, might be involved in pathological process of HCC [15,16]. However, whether KPNA2 might associate with PLAG1 and assist PLAG1 nucleus import to activate downstream effectors in HCC remains unclassified. Here, we explored the functional interaction of KPNA2 with PLAG1 and the clinical significance of the mechanism in HCC. Methods Clinical specimens and follow-up The study SNX-5422 nmr protocol was approved by the clinical research ethics committee of Second Military Medical University (Shanghai, China). Written informed consent was obtained from all patients according to the policies of the committee. Information that could identify the patients was not included in this article. The tissue microarray (TMA) were constructed as described previously [17]. Tumoral and corresponding non-tumoral tissues are separately deposed in different slices.

The spoke model was used to derive binary interactions from the c

The spoke model was used to derive binary interactions from the copurification data. Only proteins discussed in the text are shown. The complete network is depicted in Additional file 6. The prefixes “Che” and

“Htr” were omitted from the protein labels. The core signaling proteins CheA, CheW1 and CheY are highlighted by red shading. The weak binding of CheW2 to the core signaling complexes (see text) is indicated by red and white stripes. The gray areas delineate different groups of Htrs that can be distinguished by their interactions with CheA, CheR, CheW1, CheW2 and GW-572016 in vivo CheY (see text). For clarity, interactions identified with these baits are shown in different colors. The interactions detected in this study were compared to interactions between the Che proteins in other prokaryotic organisms (Additional file 7). However, the comparability of the datasets is rather low because the only other protein-protein interaction (PPI) study in an archaeal organism (P.horikoshii, [66]) reported just one interaction between Che proteins (CheC-CheD). The large-scale studies in bacteria (Escherichia coli[67, 68], Helicobacter pylori[69], Campylobacter jejuni[70], Treponema pallidum[71]) as well as a dedicated PPI YAP-TEAD Inhibitor 1 study of the E.coli taxis signaling

system [72] were performed in organisms with quite different taxis signaling systems compared to that of Hbt.salinarum. For example, none of these organisms contains CheC and CheD proteins, which together account for a substantial part of the interactions described in the present study. Figure 4 presents a general interaction network for enough prokaryotic taxis signaling systems. Figure 4 Physical and functional interactions in prokaryotic taxis signaling systems. The interactions of the core signaling

proteins are generally in agreement between Hbt.salinarum and the data of the other organisms. The Hbt.salinarum dataset probably contains indirect interactions (e. g. CheY-CheW, CheY-Htr) because it was generated by AP-MS. The interactions of the other Che proteins have, with the exception of CheC-CheD, not been described in other organisms. References for literature data are given in Additional file 7. The core signaling structure The centerpiece of the chemotaxis signal transduction system is the histidine kinase CheA, which is bound to the Htrs together with the coupling protein CheW. It check details phosphorylates the response regulator CheY to generate the output signal CheY-P [19, 73]. Bait fishing experiments with the core signaling proteins confirmed this assumed organization of the core structure (Figure 3) and also led to the identification of novel protein complexes around the core signaling proteins (described below). CheA was found to strongly interact with CheW1, and 6 of the 18 Htrs were found to interact with both CheA and CheW1.

Despite the lack of any biophysical mechanism which would be able

Despite the lack of any biophysical mechanism which would be able to explain such interactions, the results not only confirm the group’s previous findings, but they apparently extend them to another frequency range (UMTS, around 1,950 MHz) and to lower SAR levels which are well below internationally accepted exposure limits for the general public (ICNIRP 1998). The arguments given in Cytoskeletal Signaling inhibitor this paper, focusing on the

effects seen on DNA damage of fibroblasts, question the validity and the origin of the data published by Schwarz et al. (2008). Many of the arguments listed here, though, would be valid for the analysis of the micronuclei (MN), too (e.g., low standard deviations, low standard deviations at high MN numbers, low inter-individual differences, lack of random effects, etc.). For several reasons, the extremely low standard deviations are far too low for this kind of experiment in living cells with respect to the cells’ status in many independently performed experiments, methodological variations (e.g., variations in the SAR levels), random effects of cells counted,

and estimation errors due to microscopical inspection and manual classification. The statistical analysis was done inappropriately and several calculation errors are irritating. As long as no convincing evidence is provided rebutting all arguments as listed here, the paper of Schwarz et al. must be treated with extreme caution. SGC-CBP30 Open Access This article is distributed under ADAMTS5 the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. References Diem

E, Ivancsits S, Rüdiger HW (2002) Basal levels of DNA strand breaks in human leukocytes determined by comet assay. J Toxicol Environ Health A 65:641–648PubMedCrossRef Diem E, Schwarz C, Adlkofer F, Jahn O, Rüdiger H (2005) Non-thermal DNA breakage by mobile-phone radiation (1800 MHz) in human fibroblasts and in transformed GFSH-R17 rat granulosa cells in vitro. Mutat Res 583:178–183PubMed ICNIRP (1998) Guidelines for limiting exposure to time-varying electric, magnetic, and electromagnetic fields (up to 300 GHz). Health Phys 74:494–522 Ivancsits S, Pilger A, Diem E, Jahn O, Rüdiger HW (2005) Cell type-specific Tozasertib mouse genotoxic effects of intermittent extremely low-frequency electromagnetic fields. Mutat Res 583:184–188PubMed Oberto G, Rolfo K, Yu P, Carbonatto M, Peano S, Kuster N, Ebert S, Tofani S (2007) Carcinogenicity study of 217 Hz pulsed 900 MHz electromagnetic fields in Pim1 transgenic mice. Radiat Res 168:316–326PubMedCrossRef Repacholi MH, Basten A, Gebski V, Noonan D, Finnie J, Harris AW (1997) Lymphomas in E mu-Pim1 transgenic mice exposed to pulsed 900 MHZ electromagnetic fields.

Although strictly anaerobic, P gingivalis, which is phylogenetic

Although strictly anaerobic, P. gingivalis, which is phylogenetically close to B. fragilis, can also survive in the presence

of atmospheric oxygen [29]. Significantly, two known virulence factors encoded by this organism, haemolysin (hem) and the cysteine protease gingipain A (rgpA), display elevated expression levels, 3.66-fold and 2-fold respectively, in the presence of atmospheric oxygen [30]. Thus it appears RGFP966 datasheet that cysteine protease gene expression in a related Bacteroidetes P. gingivalis is sensitive to environmental cues including oxygen. This study investigates how the expression of B. fragilis C10 protease genes responds to key changes in environmental stimuli, and thus indicates their potential involvement in pathogenesis and survival in the non-gut environs. In addition, expression analysis data is presented for a set of genes encoding newly identified and described C10 paralogues in B. thetaiotaomicron. Results Identification of a family of paralogous C10 protease genes in B. thetaiotaomicron By a combination of global buy Entospletinib homology-based approaches, supplemented by searching for active site motifs associated with cysteine protease activity, we identified 4 genes encoding homologues of the streptococcal C10 protease SpeB in the genome sequence of B. thetaiotaomicron strain VPI-5482. The genes were named btpA (BT2450), btpB (BT2219), btpC (BT2217)

and btpZ (BT2220) for B acteroides t hetaiotaomicron protease. Unlike btpA, the btpB

btpC and btpZ genes were found clustered together in the genome (Figure 1). The btp gene APR-246 supplier products ranged from 20.0% to 22.6% residue identity to SpeB, and 38.4% to 42.3% similarity (Table 1). The btp gene products were also found to share significant homology with the recently described [9] Bfp proteases of B. fragilis (18.3% to 27.6% identity and 38.4% to 49.8% similarity) (Table 1). Among the protein set, BtpA displayed the highest level of residue identity to Bfp1 and Bfp2, while BtpB, BtpC and BtpZ formed a separate cluster of related proteins (Figure 2(a)). Within this cluster, the most similar pair-wise alignment was between BtpB and BtpC, which were 54.3% identical and 2.5% similar (Figure 2(a) and Table 1). Figure 1 Schematic Osimertinib mouse diagram of two C10 protease loci in B. thetaiotaomicron VPI-5482. The upper diagram represents the genomic region that includes btpA, the lower diagram the genomic region associated with the btp cluster. The proteases are represented by the larger open arrows. The propeptide region is represented by pale grey shading and the mature protease region by the darker grey. The white open arrows represent the stapostatin-like inhibitors. The black region at the 5’ end of each gene corresponds to the leader peptide encoding region of the gene. The co-ordinates for the region of the VPI-5482 are given by the numbers in italics above the DNA, the numbers in italics below the DNA are the intergenic distances.

For cDNA synthesis 1 μg of total RNA was transcribed with the

For cDNA synthesis 1 μg of total RNA was transcribed with the Bindarit price iScript™ Select cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA), using the random primers supplied, and following the manufacturer’s instructions. The PCR amplifications were performed using the primer pairs BDhoxHF1-BDhoxHR1, VNhoxWF1-VNhoxWR1, BDhupLF1-BDhupLR1, BDhupWF1- BDhupWR1, BD16SF1- BD16SR1 for hoxH, hoxW, hupL, hupW, and 16S rDNA detection, respectively (Table 2). For each analysis 16S rRNA gene was used for normalization. The PCRs (for Real-time analysis) were performed using 0.25 μM of each primer, 10 μl of iQ™ SYBR® Green Supermix

(Bio-Rad Laboratories, Inc., Hercules, CA) and 2 μl of template cDNA, while the PCRs for the RT-PCR assays were performed as described previously [48]. The PCR profile was: 3 min at 95°C followed by 50 cycles (Real-time RT-PCR) or 30 and 40 cycles (RT-PCR) of 30 s at 95°C, 30 s at 51°C and 30 s at 72°C. Standard dilutions of the cDNA were used to check the relative efficiency and quality of primers. Negative controls (no template cDNA) were included in all Real-time PCR and RT-PCR assays. A melting curve analysis was performed at the end of each Real-time PCR assay to exclude the formation of nonspecific

products. Real-time PCRs were carried out in the ICycler iQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., Hercules, CA). The data obtained were analyzed using the method described in Pfaffl [51]. Acknowledgements This work was financially supported by FCT (SFRH/BD/1695/2004,

SFRH/BPD/20255/2004), POCI 2010 (III Quadro Comunitário de Apoio), Instituto selleck chemical de Emprego e Formação Profissional (008/EP/06), and EU FP6-NEST-2005-Path-SYN project BioModularH2 (contract n° 043340). We thank Elsa Leitão for the preliminary studies on L. majuscula hox genes. References 1. Ferreira D, Leitão E, Sjöholm J, Oliveira P, Lindblad P, Moradas-Ferreira P, Tamagnini P: Transcription and regulation of the hydrogenase(s) accessory genes, hypFCDEAB Dichloromethane dehalogenase , in the cyanobacterium Lyngbya majuscula CCAP 1446/4. Arch Microbiol 2007, 188:609–617.PubMedCrossRef 2. Leitão E, Oxelfelt F, Oliveira P, Moradas-Ferreira P, Tamagnini P: Analysis of the hupSL operon of the nonheterocystous cyanobacterium Lyngbya majuscula CCAP 1446/4: Regulation of transcription and expression under a light-dark PLX3397 research buy regime. Appl Environ Microbiol 2005, 71:4567–4576.PubMedCrossRef 3. Leitão E, Pereira S, Bondoso J, Ferreira D, Pinto F, Moradas-Ferreira P, Tamagnini P: Genes involved in the maturation of hydrogenase(s) in the nonheterocystous cyanobacterium Lyngbya majuscula CCAP 1446/4. Int J Hydrogen Energy 2006, 31:1469–1477.CrossRef 4. Schütz K, Happe T, Troshina O, Lindblad P, Leitão E, Oliveira P, Tamagnini P: Cyanobacterial H 2 production – a comparative analysis. Planta 2004, 218:350–359.PubMedCrossRef 5. Böck A, King PW, Blokesch M, Posewitz MC: Maturation of hydrogenases. Adv Microb Physiol 2006, 51:1–71.PubMedCrossRef 6.

pestis travels from the site of infection to draining lymph nodes

pestis travels from the site of infection to draining lymph nodes (LN) prior to disseminating throughout the rest of the body [15, 16]. Bacterial burden data from these experiments give a snapshot of a very narrow window (a specific organ at a specific time) through the course of infection. Furthermore, the approach is invasive, requires a large number of animals, and animals must be sacrificed at each

time point making it impossible to keep track of the progression of infection BI 2536 purchase on the same group of individuals. In vivo bioluminescence imaging (BLI) is an approach that has been used to detect light-emitting cells inside of small mammals [17]. Using BLI, researchers have described and studied dissemination of viral, parasitic and bacterial pathogens within a host in a non-invasive manner [18–21]. Thus, the same group of animals can be imaged for as long as desired over the course of infection. The system requires that the pathogen produce luminescence, and infected animals are then imaged with a high-sensitivity camera that detects very small amounts of light. Non-luminescent bacteria can be genetically modified to express

the lux genes (luxCDABE), which encode a bacterial luciferase and other enzymes that are necessary to generate substrate for luciferase [22]. In the presence of oxygen, luciferase catalyzes a reaction that produces light as a byproduct [23]. We transformed Y. pestis CO92 with plasmid pGEN-luxCDABE that contains the luxCDABE genes [24]. Using this strain of Y. pestis expressing the lux genes we determined that it is suitable for in vivo BLI after subcutaneous, intradermal and intranasal inoculation. Selleck TSA HDAC In addition, we determined that BLI is suitable for the study of mutant strains that are attenuated or defective in dissemination or colonization during infection. This extends the findings of a recent report demonstrating

the suitability of BLI to study Y. pestis infections by the subcutaneous route of inoculation [25]. BLI technology offers a new perspective to study the spread of Y. pestis in the host. This technology could be adopted in the future as an alternative to experiments that measured bacterial burdens in specific organs, facilitating the discovery Cyclin-dependent kinase 3 and study of genes that are important in pathogenesis. Results The pGEN-luxCDABE vector is stable in Y. pestis during infection Bacteria carrying a reporter plasmid could potentially lose it at a specific site or time point during infection. A subpopulation lacking the plasmid could result in false negatives or decreases in signal detection that are not necessarily related to lower numbers of bacteria. To determine if pGEN-luxCDABE (pGEN-lux) was maintained during Y. pestis infections, we performed a kinetic study with mice infected with CO92 carrying pGEN-lux. Mice were inoculated subcutaneously (SC) and LN this website harvested at 24 hours post inoculation (hpi), LN and spleens harvested at 48 and 72 hpi, and LN, spleens and lungs harvested at 96 hpi.

aeruginosa strains [25, 26] By contrast, LES phages may allow LE

aeruginosa strains [25, 26]. By contrast, LES phages may allow LES to displace other P. aeruginosa strains during superinfection in the CF lung [11] by lysing susceptible resident strains [39]. LES phage infection is Type IV pilus-dependent We demonstrate that LES phage infection is dependent on the type IV pilus, which is required by P. aeruginosa for adhesion, biofilm formation and twitching motility [40–42]. This important surface structure is commonly used as a receptor by diverse Pseudomonas phages [43]. Both non-piliated (pilA -

) and hyper-piliated (pilT – ) PAO1 mutants were resistant to infection by all three LES phages. However, a different hyper-piliated mutant (pilU – ) remained susceptible. These findings mirror other pilin-dependent P. aeruginosa phage studies [43–45]. Hyper-piliated mutants are incapable of twitching motility due to abrogated pili retraction. These data suggest that retraction is involved in the infection process by LESφ2 LESφ3 and LESφ4. Despite infecting via an important and common selleck chemical surface structure, all three LES phages exhibited narrow host ranges and each showed strain specificities. For example, LESφ4 was able to infect PA14 and several keratitis isolates that were resistant to infection by the other LES phages. It is likely that many clinical strains of P. aeruginosa harbour

prophages that may belong to the same immunity group and therefore exclude super-infection by one or more of the LES phages [20]. Alternatively, resistance could be achieved by loss or modification of the type IV pili receptor [44, 45]. Conclusion In summary,

we demonstrate that the LES phages exhibit differential sensitivities to induction, narrow host ranges and divergent infection behaviour in the model host selleck inhibitor PAO1 compared with the native LESB58 host background. Extensive genotypic and phenotypic variation has been observed in clinical LES populations [46], including changes in the number of resident LES prophages [25]. These phages may, therefore, be important contributors to diversity of the LES populations. Methods Bacterial strains and growth conditions All bacterial strains used in this study and their sources are listed in Table 3. LES phages were induced from the sequenced CF P. aeruginosa isolate, LESB58 [16]. Strain PAO1 was susceptible to infection by all three LES phages and was therefore used as a model host to purify and study the PD0325901 chemical structure characteristics of each phage. Successive infection of PAO1 with purified LES phages yielded single, double and triple PAO1 LES Phage Lysogens (PLPLs) each harbouring single copies of one, two or three LES phages simultaneously. All lysogens were confirmed by PCR amplification of specific prophage sequences and Southern blot analysis. Non-piliated (pilA – ) or hyperpiliated (pilT – and pilU – ) PAO1 mutants [47] were used to determine whether LES phages infect via the type IV pili.